Western blot banding pattern in early Lyme borreliosis among patients from an endemic region of north-eastern Poland.

Science.gov (United States)

Flisiak, R; Wierzbicka, I; Prokopowicz, D

1998-01-01

Aim of this study was evaluation of Western blot banding patterns in different clinical forms of early Lyme borreliosis diagnosed in patients from north-eastern Poland, recognized as endemic for tick-borne diseases. Study was performed on serum samples of 48 patients with Lyme borreliosis and 26 healthy volunteers, as controls. Samples tested routinely for total antibody with enzyme immunoassay were subsequently analysed for specific antibodies with Western blot based on antigen extract of European strain of Borrelia burgdorferi. In patients, IgM antibodies were the most frequently directed against 41 kDa and 58 kDa antigens, whereas in control group only antibodies against 45 kDa and 58 kDa were present. Similar response was observed in respect to IgG antibodies. Evaluation of banding pattern in respect to clinical form of the disease revealed the highest prevalence of IgM and IgG anti-41 kDa antibodies in patients with erythema migrans and Lyme arthritis, and anti-58 kDa in neuroborreliosis patients, who had no anti-21 kDa antibodies. Relatively high frequency of IgG antibodies against 21, 30 and 93 kDa antigens was typical for neuroborreliosis. Bands count was significantly higher in different clinical forms of the disease than in controls, and it was the highest in neuroborreliosis. Combined analysis of Western blot results (IgM/IgG) enabled to achieve higher sensitivity (84%) and specificity (100%) than available with the most recommended EIA kits.

Multiplexed Western Blotting Using Microchip Electrophoresis.

Science.gov (United States)

Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

2016-07-05

Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus.

Science.gov (United States)

Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young-Hwan; Yu, Jae-Hyuk

2013-07-11

FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus. Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion

Multistrip western blotting to increase quantitative data output.

Science.gov (United States)

Kiyatkin, Anatoly; Aksamitiene, Edita

2009-01-01

The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip western blotting increases the data output per single blotting cycle up to tenfold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology, and cell signaling research.

Analysis of differentially expressed genes in two immunologically distinct strains of Eimeria maxima using suppression subtractive hybridization and dot-blot hybridization

Science.gov (United States)

2014-01-01

Background It is well known that different Eimeria maxima strains exhibit significant antigenic variation. However, the genetic basis of these phenotypes remains unclear. Methods Total RNA and mRNA were isolated from unsporulated oocysts of E. maxima strains SH and NT, which were found to have significant differences in immunogenicity in our previous research. Two subtractive cDNA libraries were constructed using suppression subtractive hybridization (SSH) and specific genes were further analyzed by dot-blot hybridization and qRT-PCR analysis. Results A total of 561 clones were selected from both cDNA libraries and the length of the inserted fragments was 0.25–1.0 kb. Dot-blot hybridization revealed a total of 86 differentially expressed clones (63 from strain SH and 23 from strain NT). Nucleotide sequencing analysis of these clones revealed ten specific contigs (six from strain SH and four from strain NT). Further analysis found that six contigs from strain SH and three from strain NT shared significant identities with previously reported proteins, and one contig was presumed to be novel. The specific differentially expressed genes were finally verified by RT-PCR and qRT-PCR analyses. Conclusions The data presented here suggest that specific genes identified between the two strains may be important molecules in the immunogenicity of E. maxima that may present potential new drug targets or vaccine candidates for coccidiosis. PMID:24894832

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

Science.gov (United States)

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Detection of mutations related to drug resistance in M. tuberculosis by dot blot hybridization and spoligotyping using specific radiolabelled probes

International Nuclear Information System (INIS)

El-Maghraby, T.K.; Abdelazeim, O.

2002-01-01

The present work has been conducted to determine the mutations related to drug resistance in M. tuberculosis in 63 Egyptian isolates using dot blot hybridization and spoligotyping. The PCR was done for amplification rpoB and katG genes in isolates. Dot blot hybridization were done to PCR products by using specific radiolabelled probes. Moreover, spoligotyping was done to know about the different strains found in Egypt. The results revealed that 58% from isolates had drug resistance to one or more of antituberculosis drugs. The results of spoligotyping have revealed that some Egyptian isolates are identical with the international code while the rest has not been identified yet. DNA sequencing was done to identify the mutation that not clear in dot blot hybridization. Early diagnosis of geno typing resistance to antituberculosis drugs is important as well as allow appropriate early patients management with few days of TB diagnosis. Using such strategy for early diagnosis of TB drug resistance allow and fast and potent patient's management

A streamlined Western blot exercise: An efficient and greener approach in the laboratory classroom.

Science.gov (United States)

Ness, Traci L; Robinson, Rebekah L; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H

2015-01-01

SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment. © 2015 The International Union of Biochemistry and Molecular Biology.

Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

Directory of Open Access Journals (Sweden)

Samantha L Eaton

Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

Other notable protein blotting methods: a brief review.

Science.gov (United States)

Kurien, Biji T; Scofield, R Hal

2015-01-01

Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

Western blotting using chemiluminescent substrates.

Science.gov (United States)

Alegria-Schaffer, Alice

2014-01-01

Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Copyright © 2014 Elsevier Inc. All rights reserved.

Multistrip Western blotting to increase quantitative data output

OpenAIRE

Kiyatkin, Anatoly; Aksamitiene, Edita

2009-01-01

The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single mem...

Western Blot of Stained Proteins from Dried Polyacrylamide Gels

Science.gov (United States)

Gruber, Claudia; Stan-Lotter, Helga

1996-01-01

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

Post-staining electroblotting for efficient and reliable peptide blotting.

Science.gov (United States)

Lee, Der-Yen; Chang, Geen-Dong

2015-01-01

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

International Nuclear Information System (INIS)

Bodkin, D.K.

1985-01-01

RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

Automated design of genomic Southern blot probes

Directory of Open Access Journals (Sweden)

Komiyama Noboru H

2010-01-01

Full Text Available Abstract Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to

Determination of Diagnostic Antigens in Cattle Amphistomiasis Using Western Blotting

Directory of Open Access Journals (Sweden)

A Halajian

2009-05-01

Full Text Available "nBackground: Mixed infection with amphistomes seems common in native cattle of Iran. The aim of this study was to determine diagnostic antigens in cattle mixed amphistomiasis."nMethods: Specific antigens of Cotylophoron cotylophorum, Gastrothylax crumenifer and Paramphisto­mum cervi (mixed infection, the most common species, were collected from cattle was deter­mined. Adult trematodes were collected from the rumen of naturally infected cattle at meat inspec­tion. After their homogenization and centrifugation, somatic antigens were prepared and ana­lyzed by SDS-PAGE. Specific antigens were determinated by western blot with homologous and heterolo­gous sera. SDS-PAGE of whole worms extract was performed at different concentrations and subse­quent gels staining. Immunoblotting analysis using sera from cattle naturally infected with am­phistomes, Dicrocoelium dendriticum, Fasciola spp. and hydatid cyst was performed."nResults: Electrophorese analysis of somatic antigens revealed the presence of 10 and 21 protein bands at 4 µgr/ml and 8 µgr/ml with molecular weights ranging from 25-120 and 25-150 kDa, respectively. The best result was taken at 8 mg/ml concentration. Although western blot of these proteins demon­strate 5 major antigenic polypeptides ranging from 50 to 100 kDa which were recognized by serum of cat­tle naturally infected with mixed amphistomes.

Ultrasensitive Detection of Proteins on Western Blots with Semiconducting Polymer Dots

OpenAIRE

Ye, Fangmao; Smith, Polina B.; Wu, Changfeng; Chiu, Daniel T.

2013-01-01

We demonstrate ultrasensitive fluorescence imaging of proteins on Western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CN-PPV). We achieved a detection limit at the single-picogram level in dot blots; with conventional Western blotting, we detected 50 pg of transferrin and trypsin inhibitor after SDS-PAGE and transfer onto a PVDF membrane. Our method does not require any additional equipment or time compared to the conventional procedure with traditional fluo...

A brief review of other notable protein blotting methods.

Science.gov (United States)

Kurien, Biji T; Scofield, R Hal

2009-01-01

A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

In Situ Blotting : A Novel Method for Direct Transfer of Native Proteins from Sectioned Tissue to Blotting Membrane

NARCIS (Netherlands)

Okabe, Masashi; Nyakas, Csaba; Buwalda, Bauke; Luiten, Paul G.M.

1993-01-01

We describe a novel technique for direct transfer of native proteins from unfixed frozen tissue sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility

Detection of alien genetic introgressions in bread wheat using dot-blot genomic hybridisation.

Science.gov (United States)

Rey, María-Dolores; Prieto, Pilar

2017-01-01

Simple, reliable methods for the identification of alien genetic introgressions are required in plant breeding programmes. The use of genomic dot-blot hybridisation allows the detection of small Hordeum chilense genomic introgressions in the descendants of genetic crosses between wheat and H. chilense addition or substitution lines in wheat when molecular markers are difficult to use. Based on genomic in situ hybridisation, DNA samples from wheat lines carrying putatively H. chilense introgressions were immobilised on a membrane, blocked with wheat genomic DNA and hybridised with biotin-labelled H. chilense genomic DNA as a probe. This dot-blot screening reduced the number of plants necessary to be analysed by molecular markers or in situ hybridisation, saving time and money. The technique was sensitive enough to detect a minimum of 5 ng of total genomic DNA immobilised on the membrane or about 1/420 dilution of H. chilense genomic DNA in the wheat background. The robustness of the technique was verified by in situ hybridisation. In addition, the detection of other wheat relative species such as Hordeum vulgare , Secale cereale and Agropyron cristatum in the wheat background was also reported .

An alternative method for processing northern blots after capillary transfer.

Science.gov (United States)

Nilsen, Timothy W

2015-03-02

Different laboratories use different methods for the prehybridization, hybridization, and washing steps of the northern blotting procedure. In this protocol, a northern blot is pretreated with Church and Gilbert hybridization buffer to block nonspecific probe-binding sites. The immobilized RNA is then hybridized to a DNA probe specific for the RNA of interest. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. The solutions and conditions described here may be ideal for those who prefer to use fewer ingredients in their solutions. This protocol is designed to achieve the same goals as other northern blotting approaches. It minimizes background (nonspecific adherence of probe to membrane and nonspecific hybridization) and maximizes specific hybridization to RNAs immobilized on a membrane. © 2015 Cold Spring Harbor Laboratory Press.

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

Science.gov (United States)

Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

2008-09-01

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

SDS-Polyacrylamide Electrophoresis and Western Blotting Applied to the Study of Asthma.

Science.gov (United States)

García-Solaesa, Virginia; Abad, Sara Ciria

2016-01-01

Western blotting is used to analyze proteins after being separated by electrophoresis and subsequently electro-transferred to a membrane. Once immobilized, a specific protein can be identified through its reaction with a labeled antibody or antigen. It is a methodology commonly used in biomedical research such as asthma studies, to assess the pathways of inflammatory mediators involved in the disease.Here, we describe an example of western blotting to determine the factors involved in asthma. In this chapter, the methodology of western blotting is reviewed, paying attention on potential problems and giving interesting recommendations.

Multistrip Western blotting: a tool for comparative quantitative analysis of multiple proteins.

Science.gov (United States)

Aksamitiene, Edita; Hoek, Jan B; Kiyatkin, Anatoly

2015-01-01

The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western blotting increases data output per single blotting cycle up to tenfold; allows concurrent measurement of up to nine different total and/or posttranslationally modified protein expression obtained from the same loading of the sample; and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data and therefore is advantageous to apply in biomedical diagnostics, systems biology, and cell signaling research.

Should we ignore western blots when selecting antibodies for other applications?

DEFF Research Database (Denmark)

Uhlén, Mathias

2017-01-01

.In the Human Protein Atlas (HPA) program, we have validated more than 24,000 in-house-generated antibodies directed to 17,000 human target proteins2. Although there is often a correlation between performance in different applications, we have observed many examples of antibodies that show strong support...... applications and that this influences the epitopes exposed on the target protein, which might have profound consequences for the ability of a given antibody to bind specifically to its target. As an example, proteins that are analyzed by immunohistochemistry (IHC) are normally first cross-linked with formalin.......In conclusion, western blot and protein array analyses can indeed be useful tools when selecting specific antibodies for other applications. The use of these methods is encouraged both for antibody providers and users, and antibodies with signs of cross-reactivity in these applications should be treated...

A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

Science.gov (United States)

Chang, Ming-Mei; Lovett, Janice

2011-01-01

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

Zinc blotting assay for detection of zinc binding prolamin in barley (Hordeum vulgare) grain

DEFF Research Database (Denmark)

Uddin, Mohammad Nasir; Nielsen, Ane Langkilde-Lauesen; Vincze, Eva

2014-01-01

In plants, zinc is commonly found bound to proteins. In barley (Hordeum vulgare), major storage proteins are alcohol-soluble prolamins known as hordeins, and some of them have the potential to bind or store zinc. 65Zn overlay and blotting techniques have been widely used for detecting zinc......-binding protein. However, to our knowledge so far this zinc blotting assay has never been applied to detect a prolamin fraction in barley grains. A radioactive zinc (65ZnCl2) blotting technique was optimized to detect zinc-binding prolamins, followed by development of an easy-to-follow nonradioactive colorimetric...... zinc blotting method with a zinc-sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc-binding specificity of certain proteins was detected either by autoradiography or color...

INSITU BLOTTING - A NOVEL METHOD FOR DIRECT TRANSFER OF NATIVE PROTEINS FROM SECTIONED TISSUE TO BLOTTING MEMBRANE - PROCEDURE AND SOME APPLICATIONS

NARCIS (Netherlands)

OKABE, M; NYAKAS, C; BUWALDA, B; LUITEN, PGM

We describe a novel technique for direct transfer of native proteins from unfixed frozen tissue sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility

Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

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Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

2016-09-01

SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

OpenAIRE

Silva, Jillian M.; McMahon, Martin

2014-01-01

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the convent...

HIV‑2 antibody detection after indeterminate or negative HIV‑1 Western blot in Cuba, 2005-2008.

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Díaz, Dervel F; Ortiz, Eva; Martín, Dayamí; Nibot, Carmen; Rizo, Adis; Silva, Eladio

2012-01-01

INTRODUCTION Differentiating between HIV-1 and HIV-2 infection is the first step to understanding HIV transmission, epidemiology and pathogenesis in geographical areas where both viruses circulate. In Cuba, positive results in mixed HIV-1/2 screening assays are confirmed by HIV-1 Western blot. Indeterminate results constitute the main limitation of this test and HIV-2 infection is among their possible causes; hence the importance of second-stage screening and confirmatory tests for HIV-2 infection. OBJECTIVE Investigate the contribution of HIV-2 antibodies to negative or indeterminate HIV-1 Western blot results in serum samples from 2005 through 2008 in Cuba. METHODS HIV-2 reactivity was studied using the ELISA DAVIH-VIH-2 diagnostic kit (Cuba) in 1723 serum samples with negative or indeterminate results for HIV-1 Western blot from January 2005 through December 2008. Duplicate sera reactive by ELISA were confirmed by HIV-2 Western blot, results interpreted according to WHO criteria. The epidemiological interview established by Cuba's National Program for Prevention and Control Sexually-Transmitted Diseases and HIV/AIDS was applied to HIV-2 Western blot-positive patients. RESULTS Among all sera studied, HIV-2 ELISA identified 12 reactive serum samples (0.70%) and 1711 non-reactive (99.30%). Western blot analysis of the 12 ELISA-reactive samples confirmed two positive samples (16.67%), 4 negative (33.33%) and 6 indeterminate (50%). Positive samples reacted against the p16, p26, gp36, p53, p56, p68 and gp105 proteins. All 12 ELISA-reactive samples belonged to the HIV-1 Western blot indeterminate group. The two HIV-2-positive samples showed well defined reactivity to gp160, p53, p55 and p34 of HIV-1. HIV-1 seroconversion was observed in all 10 remaining samples during serological followup. CONCLUSIONS Two new HIV-2 seropositive cases were diagnosed using DAVIH-VIH-2 and HIV-2 Western blot in indeterminate HIV-1 Western blot samples. Results support the recommendation

Lectin-Array Blotting.

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Pazos, Raquel; Echevarria, Juan; Hernandez, Alvaro; Reichardt, Niels-Christian

2017-09-01

Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Positive IgG Western Blot for Borrelia burgdorferi in Colombia

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Palacios Ricardo

1999-01-01

Full Text Available In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma, the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.

Genomic Analyses Reveal the Influence of Geographic Origin, Migration, and Hybridization on Modern Dog Breed Development

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Heidi G. Parker

2017-04-01

Full Text Available There are nearly 400 modern domestic dog breeds with a unique histories and genetic profiles. To track the genetic signatures of breed development, we have assembled the most diverse dataset of dog breeds, reflecting their extensive phenotypic variation and heritage. Combining genetic distance, migration, and genome-wide haplotype sharing analyses, we uncover geographic patterns of development and independent origins of common traits. Our analyses reveal the hybrid history of breeds and elucidate the effects of immigration, revealing for the first time a suggestion of New World dog within some modern breeds. Finally, we used cladistics and haplotype sharing to show that some common traits have arisen more than once in the history of the dog. These analyses characterize the complexities of breed development, resolving longstanding questions regarding individual breed origination, the effect of migration on geographically distinct breeds, and, by inference, transfer of trait and disease alleles among dog breeds.

Comparative genomics analyses revealed two virulent Listeria monocytogenes strains isolated from ready-to-eat food.

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Lim, Shu Yong; Yap, Kien-Pong; Thong, Kwai Lin

2016-01-01

Listeria monocytogenes is an important foodborne pathogen that causes considerable morbidity in humans with high mortality rates. In this study, we have sequenced the genomes and performed comparative genomics analyses on two strains, LM115 and LM41, isolated from ready-to-eat food in Malaysia. The genome size of LM115 and LM41 was 2,959,041 and 2,963,111 bp, respectively. These two strains shared approximately 90% homologous genes. Comparative genomics and phylogenomic analyses revealed that LM115 and LM41 were more closely related to the reference strains F2365 and EGD-e, respectively. Our virulence profiling indicated a total of 31 virulence genes shared by both analysed strains. These shared genes included those that encode for internalins and L. monocytogenes pathogenicity island 1 (LIPI-1). Both the Malaysian L. monocytogenes strains also harboured several genes associated with stress tolerance to counter the adverse conditions. Seven antibiotic and efflux pump related genes which may confer resistance against lincomycin, erythromycin, fosfomycin, quinolone, tetracycline, and penicillin, and macrolides were identified in the genomes of both strains. Whole genome sequencing and comparative genomics analyses revealed two virulent L. monocytogenes strains isolated from ready-to-eat foods in Malaysia. The identification of strains with pathogenic, persistent, and antibiotic resistant potentials from minimally processed food warrant close attention from both healthcare and food industry.

BLOTS AND ALL: A HISTORY OF THE RORSCHACH INK BLOT TEST IN BRITAIN.

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Hubbard, Katherine; Hegarty, Peter

2016-01-01

Despite the easily recognizable nature of the Rorschach ink blot test very little is known about the history of the test in Britain. We attend to the oft-ignored history of the Rorschach test in Britain and compare it to its history in the US. Prior to the Second World War, Rorschach testing in Britain had attracted advocates and critiques. Afterward, the British Rorschach Forum, a network with a high proportion of women, developed around the Tavistock Institute in London and The Rorschach Newsletter. In 1968, the International Rorschach Congress was held in London but soon after the group became less exclusive, and fell into decline. A comparative account of the Rorschach in Britain demonstrates how different national institutions invested in the 'projective hypothesis' according to the influence of psychoanalysis, the adoption of a nationalized health system, and the social positioning of 'others' throughout the twentieth century. In comparing and contrasting the history of the Rorschach in Britain and the US, we decentralize and particularize the history of North American Psychology. © 2016 Wiley Periodicals, Inc.

Blotting from PhastGel to Membranes by Ultrasound.

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Kost, Joseph; Azagury, Aharon

2015-01-01

Ultrasound based approach for enhanced protein blotting is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm(2)) was sufficient for a clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or Nylon 66 Biotrans membrane. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide standards (18,500-106,000 Da) and 14C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

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Rossano, M G; Mansfield, L S; Kaneene, J B; Murphy, A J; Brown, C M; Schott, H C; Fox, J C

2000-01-01

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (Pblot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.

Single-Cell Analyses of ESCs Reveal Alternative Pluripotent Cell States and Molecular Mechanisms that Control Self-Renewal

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Dmitri Papatsenko

2015-08-01

Full Text Available Analyses of gene expression in single mouse embryonic stem cells (mESCs cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM. In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal.

Use of a Western blot technique for the serodiagnosis of glanders

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de Souza Marcilia MA

2011-01-01

Full Text Available Abstract Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT. Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. Results The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. Conclusions The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees

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Khaled DJELOUAH

2015-01-01

Full Text Available A direct tissue blot immunoassay (DTBIA technique has been compared with ELISA and PCR for detection of Xylella fastidiosa in olive trees from Apulia (southern Italy. Fresh cross-sections of young twigs and leaf petioles were printed onto nitrocellulose membranes and analyzed in the laboratory. Analyses of a first group of 61 samples gave similar efficiency for the three diagnostic techniques for detection the bacterium (24 positive and 36 negative samples, except for a single sample which was positive only with DTBIA and PCR. Similar results were obtained by separately analyzing suckers and twigs collected from different sectors of tree canopies of a second group of 20 olive trees (ten symptomatic and ten symptomless. In this second test the three diagnostic techniques confirmed the irregular distribution of the bacterium in the tree canopies and erratic detectability of the pathogen in the young suckers. It is therefore necessary to analyse composite samples per tree which should be prepared with twigs collected from different sides of the canopy. The efficiency comparable to ELISA and PCR, combined with the advantages of easier handling, speed and cost, make DTBIA a valid alternative to ELISA in large-scale surveys for occurrence of X. fastidiosa. Moreover, the printing of membranes directly in the field prevents infections spreading to Xylella-free areas, through movement of plant material with pathogen vectors for laboratory testing.

Trial sequential analysis reveals insufficient information size and potentially false positive results in many meta-analyses

DEFF Research Database (Denmark)

Brok, J.; Thorlund, K.; Gluud, C.

2008-01-01

in 80% (insufficient information size). TSA(15%) and TSA(LBHIS) found that 95% and 91% had absence of evidence. The remaining nonsignificant meta-analyses had evidence of lack of effect. CONCLUSION: TSA reveals insufficient information size and potentially false positive results in many meta......OBJECTIVES: To evaluate meta-analyses with trial sequential analysis (TSA). TSA adjusts for random error risk and provides the required number of participants (information size) in a meta-analysis. Meta-analyses not reaching information size are analyzed with trial sequential monitoring boundaries...... analogous to interim monitoring boundaries in a single trial. STUDY DESIGN AND SETTING: We applied TSA on meta-analyses performed in Cochrane Neonatal reviews. We calculated information sizes and monitoring boundaries with three different anticipated intervention effects of 30% relative risk reduction (TSA...

Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola.

Science.gov (United States)

Wan, Huafang; Cui, Yixin; Ding, Yijuan; Mei, Jiaqin; Dong, Hongli; Zhang, Wenxin; Wu, Shiqi; Liang, Ying; Zhang, Chunyu; Li, Jiana; Xiong, Qing; Qian, Wei

2016-01-01

Understanding the regulation of lipid metabolism is vital for genetic engineering of canola ( Brassica napus L.) to increase oil yield or modify oil composition. We conducted time-series analyses of transcriptomes and proteomes to uncover the molecular networks associated with oil accumulation and dynamic changes in these networks in canola. The expression levels of genes and proteins were measured at 2, 4, 6, and 8 weeks after pollination (WAP). Our results show that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. We found that genes in almost every node of fatty acid synthesis pathway were significantly up-regulated during oil accumulation. Moreover, significant expression changes of two genes, acetyl-CoA carboxylase and acyl-ACP desaturase, were detected on both transcriptomic and proteomic levels. We confirmed the temporal expression patterns revealed by the transcriptomic analyses using quantitative real-time PCR experiments. The gene set association analysis show that the biosynthesis of fatty acids and unsaturated fatty acids are the most significant biological processes from 2-4 WAP and 4-6 WAP, respectively, which is consistent with the results of time-series analyses. These results not only provide insight into the mechanisms underlying lipid metabolism, but also reveal novel candidate genes that are worth further investigation for their values in the genetic engineering of canola.

Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism.

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Gareth D Westrop

Full Text Available Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

Individual-based analyses reveal limited functional overlap in a coral reef fish community.

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Brandl, Simon J; Bellwood, David R

2014-05-01

Detailed knowledge of a species' functional niche is crucial for the study of ecological communities and processes. The extent of niche overlap, functional redundancy and functional complementarity is of particular importance if we are to understand ecosystem processes and their vulnerability to disturbances. Coral reefs are among the most threatened marine systems, and anthropogenic activity is changing the functional composition of reefs. The loss of herbivorous fishes is particularly concerning as the removal of algae is crucial for the growth and survival of corals. Yet, the foraging patterns of the various herbivorous fish species are poorly understood. Using a multidimensional framework, we present novel individual-based analyses of species' realized functional niches, which we apply to a herbivorous coral reef fish community. In calculating niche volumes for 21 species, based on their microhabitat utilization patterns during foraging, and computing functional overlaps, we provide a measurement of functional redundancy or complementarity. Complementarity is the inverse of redundancy and is defined as less than 50% overlap in niche volumes. The analyses reveal extensive complementarity with an average functional overlap of just 15.2%. Furthermore, the analyses divide herbivorous reef fishes into two broad groups. The first group (predominantly surgeonfishes and parrotfishes) comprises species feeding on exposed surfaces and predominantly open reef matrix or sandy substrata, resulting in small niche volumes and extensive complementarity. In contrast, the second group consists of species (predominantly rabbitfishes) that feed over a wider range of microhabitats, penetrating the reef matrix to exploit concealed surfaces of various substratum types. These species show high variation among individuals, leading to large niche volumes, more overlap and less complementarity. These results may have crucial consequences for our understanding of herbivorous processes on

A bead-based western for high-throughput cellular signal transduction analyses

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Treindl, Fridolin; Ruprecht, Benjamin; Beiter, Yvonne; Schultz, Silke; Döttinger, Anette; Staebler, Annette; Joos, Thomas O.; Kling, Simon; Poetz, Oliver; Fehm, Tanja; Neubauer, Hans; Kuster, Bernhard; Templin, Markus F.

2016-01-01

Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes. PMID:27659302

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

Science.gov (United States)

Abeyrathne, Priyanka D; Lam, Joseph S

2007-04-01

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Banding pattern indicative of echinococcosis in a commercial cysticercosis western blot

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Tappe D

2009-09-01

Full Text Available Abstract Objective A commercial cysticercosis Western blot was evaluated for serological cross-reactivity of sera from patients with alveolar (AE and cystic echinococcosis (CE. Methods A total of 161 sera were examined, including 31 sera from AE-patients, 11 sera from CE-patients, 9 sera from patients with other parasitic diseases and 109 sera from patients with unrelated medical conditions. All AE-and CE-sera were also examined by the echinococcosis Western blot. Results More sera from patients with AE than with CE showed cross-reactivity in the form of ladder-like patterns ("Mikado aspect" and untypical bands at 6-8 kDa (71% and 77.4% versus 27.3% and 45.5%, respectively. In contrast, triplets of bands in the area above 50 kDa and between 24 and 39-42 kDa were more frequent in CE than in AE sera. The fuzzy band at 50-55 kDa typical for cysticercosis was absent in all AE and CE sera. Conclusions Atypical banding patterns in the cysticercosis Western blot should raise the suspicion of a metacestode infection different from Taenia solium, i.e. Echinococcus multilocularis or E. granulosus, especially when the Mikado aspect and an altered 6-8 kDa band is visible in the absence of a fuzzy 50-55 kDa band.

Characterization of the structure of the erythropoietin receptor by ligand blotting

International Nuclear Information System (INIS)

Atkins, H.L.; Broudy, V.C.; Papayannopoulou, T.

1991-01-01

Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with 125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd ± 4 Kd was sufficient to bind 125I Epo in both human (OCIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of 125I-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding

Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

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Yukihiro Shoyama

2013-01-01

Full Text Available To prepare the antiglycyrrhizin (GC monoclonal antibody (MAb, GC was treated with NaIO4 resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF membrane. The membrane was treated by NaIO4 and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4 treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

Routine Western blot to check autophagic flux : Cautions and recommendations

NARCIS (Netherlands)

Gomez-Sanchez, Ruben; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodriguez-Arribas, Mario; Bravo-San Pedro, Jose M.; Fuentes, Jose M.; Gonzalez-Polo, Rosa A.

2015-01-01

At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible

Modification of T-cell antigenic properties of tetanus toxoid by SDS-PAGE separation. Implications for T-cell blotting

DEFF Research Database (Denmark)

Christensen, C B; Theander, T G

1997-01-01

Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20% of the ......Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20......% of the PBMC cultures whereas proliferation was induced in 79% of the same cultures offered similar treated TT (except for the PAGE separation). When T-cell blotting was performed with TT separated in a SDS-agarose matrix, proliferation was induced in 80% of donors responding to soluble TT. The results show...... that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis...

Multi-strip Western blotting to increase quantitative data output

OpenAIRE

Aksamitiene, Edita; Hoek, Jan B.; Kholodenko, Boris; Kiyatkin, Anatoly

2007-01-01

The qualitative and quantitative measurement of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. We propose a modified immunoblotting procedure, which is based on simultaneous transfer of proteins from multiple gel-strips onto the same membrane, and is compatible wi...

[Clinical manifestation of Lyme borreliosis in children with positive and negatiwe western blot results].

Science.gov (United States)

Ołdak, Elzbieta; Rozkiewicz, Doroto; Sulik, Artur

2008-01-01

In the afforested area of North-Eastern Poland the risk of Borrelia burgdorferi infection seems to be higher compared to the other regions. Because of unspecific clinical manifestation of Lyme borreliosis in children the positive ELISA IgM results should be confirmed with Western blot IgM tests. Retrospective analysis of clinical signs and symptoms of Lyme borreliosis in children with positive ELISA IgM and positive Western blot IgM results and in children with positive ELISA IgM and negative Western blot IgM results. The study included 20 children reactive with ELISA IgM (Bellco Biomedica, Austria), hospitalized in Pediatric Infectious Diseases Clinic in 2007 due to probable diagnosis of Lyme disease. All children were tested with B. burgdorferi Western blot IgM and/or IgG assay (DRG, Diagnostics, Germany) as a second-step diagnosis. In 10 (50% females, 50% males) out of 20 children the results were positive (borreliosis) and in other 10 (80% females, 20% males) the results were negative (controls). In both groups of patients the retrospective analysis of signs and symptoms was done. The most often clinical manifestation of Lyme borreliosis in children was neuroborreliosis. Children presented Lyme meningitis (30%), facial nerve palsy (10%) and chronic or recurrent headaches (40%), associated with vertigo (20%), weakness (30%), fever (40%), and fatigue syndrome (30%). One patient presented Lyme arthritis. Children of control group presented with unspecific symptoms like isolated headaches (40%), arthralgias (70%), myalgias (10%) and abdomen pain (20%) (1) The most frequent clinical presentation of Lyme borreliosis in analyzed children was neuroborreliosis; (2) Isolated arthralgias in children reactive with B. burgdorferi ELISA IgM need to be confirmed with Western blot assay before implementing the antibiotic therapy.

Murine interleukin 1 receptor. Direct identification by ligand blotting and purification to homogeneity of an interleukin 1-binding glycoprotein

International Nuclear Information System (INIS)

Bird, T.A.; Gearing, A.J.; Saklatvala, J.

1988-01-01

Functional receptors (IL1-R) for the proinflammatory cytokine interleukin 1 (IL1) were solubilized from plasma membranes of the NOB-1 subclone of murine EL4 6.1 thymoma cells using the zwitterionic detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Membrane extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and ligand blotted with 125 I-labeled recombinant human IL1 alpha in order to reveal proteins capable of specifically binding IL1. A single polydisperse polypeptide of Mr approximately equal to 80,000 was identified in this way, which bound IL1 alpha and IL1 beta with the same affinity as the IL1-R on intact NOB-1 cells (approximately equal to 10(-10) M). The IL1-binding polypeptide was only seen in membranes from IL1-R-bearing cells and did not react with interleukin 2, tumor necrosis factor alpha, or interferon. IL1-R was purified to apparent homogeneity from solubilized NOB-1 membranes by affinity chromatography on wheat germ agglutinin-Sepharose and IL1 alpha-Sepharose. Gel electrophoresis and silver staining of purified preparations revealed a single protein of Mr approximately equal to 80,000 which reacted positively in the ligand-blotting procedure and which we identify as the ligand-binding moiety of the murine IL1-R. Purified IL1-R exhibited the same affinity and specificity as the receptor on intact cells. The relationship of this protein to proteins identified by covalent cross-linking studies is discussed

Profiling EGFR activity in head and neck squamous cell carcinoma by using a novel layered membrane Western blot technology.

Science.gov (United States)

Patel, Vyomesh; Ramesh, Arun; Traicoff, June L; Baibakov, Galina; Emmert-Buck, Michael R; Gutkind, J Silvio; Knezevic, Vladimir

2005-05-01

Given the role of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), several rational approaches have now been utilized to abrogate tyrosine kinase activity and its disengagement from downstream signal transducers. Monitoring the activity of these molecules could potentially be useful to determine not only drug efficacy but also to identify HNSCC patients most likely to benefit from this type of therapy. In this study we have used a novel high throughput multi-layered Western blotting (MLWestern) method that allows the detection of multiple proteins from a single experiment in order to characterize key components in the EGFR signaling pathway in HNSCC cells. Total and activated forms of EGFR and the downstream effectors, Erk and Akt were readily detected in HNSCC cells, where in the control cells (HaCaT) these proteins could only be detected in EGF stimulated cells. Results from conventional Western blot and MLWestern were comparable. Clustering analysis of protein expression revealed similarities in cellular response between some of the cell lines indicative of similarities in their biological response. The data indicate that MLWestern can be potentially applied to identify molecular targets that could be used for rational therapeutic intervention strategies.

Evaluation of two commercial systems for automated processing, reading, and interpretation of Lyme borreliosis Western blots.

Science.gov (United States)

Binnicker, M J; Jespersen, D J; Harring, J A; Rollins, L O; Bryant, S C; Beito, E M

2008-07-01

The diagnosis of Lyme borreliosis (LB) is commonly made by serologic testing with Western blot (WB) analysis serving as an important supplemental assay. Although specific, the interpretation of WBs for diagnosis of LB (i.e., Lyme WBs) is subjective, with considerable variability in results. In addition, the processing, reading, and interpretation of Lyme WBs are laborious and time-consuming procedures. With the need for rapid processing and more objective interpretation of Lyme WBs, we evaluated the performances of two automated interpretive systems, TrinBlot/BLOTrix (Trinity Biotech, Carlsbad, CA) and BeeBlot/ViraScan (Viramed Biotech AG, Munich, Germany), using 518 serum specimens submitted to our laboratory for Lyme WB analysis. The results of routine testing with visual interpretation were compared to those obtained by BLOTrix analysis of MarBlot immunoglobulin M (IgM) and IgG and by ViraScan analysis of ViraBlot and ViraStripe IgM and IgG assays. BLOTrix analysis demonstrated an agreement of 84.7% for IgM and 87.3% for IgG compared to visual reading and interpretation. ViraScan analysis of the ViraBlot assays demonstrated agreements of 85.7% for IgM and 94.2% for IgG, while ViraScan analysis of the ViraStripe IgM and IgG assays showed agreements of 87.1 and 93.1%, respectively. Testing by the automated systems yielded an average time savings of 64 min/run compared to processing, reading, and interpretation by our current procedure. Our findings demonstrated that automated processing and interpretive systems yield results comparable to those of visual interpretation, while reducing the subjectivity and time required for Lyme WB analysis.

Special analyses reveal coke-deposit structure

International Nuclear Information System (INIS)

Albright, L.F.

1988-01-01

A scanning electron microscope (SEM) and an energy dispersive X-ray analyzer (EDAX) have been used to obtain information that clarifies the three mechanisms of coke formation in ethylene furnaces, and to analyze the metal condition at the exit of furnace. The results can be used to examine furnace operations and develop improved ethylene plant practices. In this first of four articles on the analyses of coke and metal samples, the coking mechanisms and coke deposits in a section of tube from an actual ethylene furnace (Furnace A) from a plant on the Texas Gulf Coast are discussed. The second articles in the series will analyze the condition of the tube metal in the same furnace. To show how coke deposition and metal condition dependent on the operating parameters of an ethylene furnace, the third article in the series will show the coke deposition in a Texas Gulf Coast furnace tube (Furnace B) that operated at shorter residence time. The fourth article discusses the metal condition in that furnace. Some recommendations, based on the analyses and findings, are offered in the fourth article that could help extend the life of ethylene furnace tubes, and also improve overall ethylene plant operations

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

Science.gov (United States)

Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

Demonstration of functional low-density lipoprotein receptors by protein blotting in fibroblasts from a subject with homozygous receptor-negative familial hypercholesterolemia

International Nuclear Information System (INIS)

Semenkovich, C.F.; Ostlund, R.E. Jr.; Yang, J.; Reaban, M.E.

1985-01-01

We report the detection of low-density lipoprotein (LDL) receptors by the technique of receptor blotting in fibroblasts from a patient with homozygous familial hypercholesterolemia (FHC) previously classified as ''receptor negative.'' Solubilized receptors were electrophoresed, transferred to nitrocellulose paper, treated with LDL followed by radiolabeled antibody to LDL, and visualized by autoradiography. GM 2000 FHC fibroblasts revealed LDL receptors with an apparent molecular weight of approximately 140,000, the same as in normal cells. LDL receptor activity by blotting in GM 2000 cells was greatly diminished in comparison with normal cells, but was calcium dependent. Receptor activity was also detectable by conventional monolayer binding and degradation assays. Thus, GM 2000 cells have profoundly diminished LDL receptor activity, but retain the genetic capacity to make LDL receptor material of normal molecular weight that is capable of binding LDL. Previous studies have demonstrated the presence of trace amounts of immunoreactive LDL receptor protein in fibroblasts from some receptor-negative FHC homozygotes. These studies are extended by demonstrating the ability of this material to bind LDL

Genome-wide analyses reveal a role for peptide hormones in planarian germline development.

Directory of Open Access Journals (Sweden)

James J Collins

Full Text Available Bioactive peptides (i.e., neuropeptides or peptide hormones represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Lifestyle Features of Nematode Endoparasitic Fungus Hirsutella minnesotensis

Science.gov (United States)

Lai, Yiling; Liu, Keke; Zhang, Xinyu; Zhang, Xiaoling; Li, Kuan; Wang, Niuniu; Shu, Chi; Wu, Yunpeng; Wang, Chengshu; Bushley, Kathryn E.; Xiang, Meichun; Liu, Xingzhong

2014-01-01

Hirsutella minnesotensis [Ophiocordycipitaceae (Hypocreales, Ascomycota)] is a dominant endoparasitic fungus by using conidia that adhere to and penetrate the secondary stage juveniles of soybean cyst nematode. Its genome was de novo sequenced and compared with five entomopathogenic fungi in the Hypocreales and three nematode-trapping fungi in the Orbiliales (Ascomycota). The genome of H. minnesotensis is 51.4 Mb and encodes 12,702 genes enriched with transposable elements up to 32%. Phylogenomic analysis revealed that H. minnesotensis was diverged from entomopathogenic fungi in Hypocreales. Genome of H. minnesotensis is similar to those of entomopathogenic fungi to have fewer genes encoding lectins for adhesion and glycoside hydrolases for cellulose degradation, but is different from those of nematode-trapping fungi to possess more genes for protein degradation, signal transduction, and secondary metabolism. Those results indicate that H. minnesotensis has evolved different mechanism for nematode endoparasitism compared with nematode-trapping fungi. Transcriptomics analyses for the time-scale parasitism revealed the upregulations of lectins, secreted proteases and the genes for biosynthesis of secondary metabolites that could be putatively involved in host surface adhesion, cuticle degradation, and host manipulation. Genome and transcriptome analyses provided comprehensive understanding of the evolution and lifestyle of nematode endoparasitism. PMID:25359922

Phylogeographic analyses of the pampas cat (Leopardus colocola; Carnivora, Felidae) reveal a complex demographic history

Science.gov (United States)

da Silva Santos, Anelisie; Trigo, Tatiane Campos; de Oliveira, Tadeu Gomes; Silveira, Leandro

2018-01-01

Abstract The pampas cat is a small felid that occurs in open habitats throughout much of South America. Previous studies have revealed intriguing patterns of morphological differentiation and genetic structure among its populations, as well as molecular evidence for hybridization with the closely related L. tigrinus. Here we report phylogeographic analyses encompassing most of its distribution (focusing particularly on Brazilian specimens, which had been poorly sampled in previous studies), using a novel dataset comprising 2,143 bp of the mitogenome, along with previously reported mtDNA sequences. Our data revealed strong population strutucture and supported a west-to-east colonization process in this species’ history. We detected two population expansion events, one older (ca. 200 thousand years ago [kya]) in western South America and another more recent (ca. 60-50 kya) in eastern areas, coinciding with the expansion of savanna environments in Brazil. Analyses including L. tigrinus individuals bearing introgressed mtDNA from L. colocola showed a complete lack of shared haplotypes between species, indicating that their hybridization was ancient. Finally, we observed a close relationship between Brazilian/Uruguayan L. colocola haplotypes and those sampled in L. tigrinus, indicating that their hybridization was likely related to the demographic expansion of L. colocola into eastern South America. PMID:29668017

Differential Expression of Proteins Associated with the Hair Follicle Cycle - Proteomics and Bioinformatics Analyses.

Directory of Open Access Journals (Sweden)

Lei Wang

Full Text Available Hair follicle cycling can be divided into the following three stages: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still unknown. To better understand the detailed protein networks controlling this process, proteomics and bioinformatics analyses were performed to construct comparative protein profiles of mouse skin at specific time points (0, 8, and 20 days. Ninety-five differentially expressed protein spots were identified by MALDI-TOF/TOF as 44 proteins, which were found to change during hair follicle cycle transition. Proteomics analysis revealed that these changes in protein expression are involved in Ca2+-regulated biological processes, migration, and regulation of signal transduction, among other processes. Subsequently, three proteins were selected to validate the reliability of expression patterns using western blotting. Cluster analysis revealed three expression patterns, and each pattern correlated with specific cell processes that occur during the hair cycle. Furthermore, bioinformatics analysis indicated that the differentially expressed proteins impacted multiple biological networks, after which detailed functional analyses were performed. Taken together, the above data may provide insight into the three stages of mouse hair follicle morphogenesis and provide a solid basis for potential therapeutic molecular targets for this hair disease.

Solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

International Nuclear Information System (INIS)

Valtorta, F.; Schiebler, W.; Jahn, R.; Ceccarelli, B.; Greengard, P.

1986-01-01

A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phyosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides

COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

Science.gov (United States)

A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

Detection of KatG Gen Mutation on Mycobacterium Tuberculosis by Means of PCR-Dot Blot Hybridization with 32P Labeled Oligonucleotide Probe Methods

International Nuclear Information System (INIS)

Maria Lina R; Budiman Bela; Andi Yasmon

2009-01-01

Handling and controlling of tuberculosis, a disease caused by Mycobacterium tuberculosis (MTB), is now complicated since there are many MTBs that are resistant against anti-tuberculosis drugs such as isoniazid. The drug resistance could occurred due to the inadequate and un-regular drug utilization that cause gene mutation of the drug target such as katG gene for isoniazid. The molecular biology techniques such as the PCR- dot blot hybridization with radioisotope ( 32 P) labeled oligonucleotide probe, has been reported as a technique that is more sensitive and rapid for detection of gene mutations related with drug resistances. Hence, the aim of this study was to apply the PCR- dot blot hybridization technique using 32 P labeled oligonucleotide probe for detection of single mutation at codon 315 of katG gene of MTBs that rise the isoniazid resistance. In this study, we used 89 sputum specimens and a standard MTB (MTB H 37 RV) as a control. DNA extractions were performed by the BOOM method and the phenol chloroform for sputum samples and standard MTB, respectively. Primers used for PCR technique were Pt8 and Pt9 and RTB59 and RTB36 for detecting tuberculosis causing Mycobacterium and the existence of katG gene, respectively. Both of the primers are specific for IS6110 region and katG gene, respectively. PCR products were detected by an agarose gel electrophoresis technique. Dot blot hybridization with 32 P-oligonucleotide probe 315mu was performed to detect mutation at codon 315 of tested samples. Results of the PCR using primer Pt8 and Pt9 showed that all sputum specimens had positive results. Mutation detection by PCR- dot blot hybridization with 32 P-oligonucleotide probe 315mu, revealed that 11 of 89 tested samples had a mutation at their codon 315 of katG gene. Based upon these results, it is concluded that PCR-dot blot hybridization with 32 P-oligonucleotide probe is a technique that is rapid and highly specific and sensitive for detection of mutation at codon

Inheritance of resistance to barley yellow dwarf virus detected by northern blot analysis

International Nuclear Information System (INIS)

Lorens, G.F.; Falk, B.W.; Qualset, C.O.

1989-01-01

Development of wheat (Triticum aestivum L.) cultivars tolerant to the barley yellow dwarf virus disease (BYD) has been limited by lack of precision in rating plants for response to infection, usually done by visual scoring of plant symptoms under field conditions. Other methodologies have been developed to study the host/pathogen relationship and to assess resistance or susceptibility. In this study northern dot blot analysis was used to determine barley yellow dwarf virus (BYDV) RNA concentrations of six wheat cultivars that differed in visual BYD symptom expression. Plants were infected with the NYPAV (PAV) isolate of BYDV in the greenhouse. At several dates after inoculation crude plant extracts were blotted on nitrocellulose and hybridized with a 32 P-labeled probe of the pPA8 cDNA clone of BYDV. The distribution of PRC for the F 2 population was compared to the distribution of BYD visual symptom scores for 403 F 2 plants of a similar F 2 population of NS 879/4 x Seri 82 under field conditions. The results were qualitatively similar, suggesting that northern dot blot analysis to measure PRC may be useful in understanding the genetics of resistance to BYD. This technique, when incorporated into breeding programs, could be important in the development of highly tolerant wheat cultivars with reduced losses to BYD

Mitochondrial DNA analyses reveal low genetic diversity in Culex quinquefasciatus from residential areas in Malaysia.

Science.gov (United States)

Low, V L; Lim, P E; Chen, C D; Lim, Y A L; Tan, T K; Norma-Rashid, Y; Lee, H L; Sofian-Azirun, M

2014-06-01

The present study explored the intraspecific genetic diversity, dispersal patterns and phylogeographic relationships of Culex quinquefasciatus Say (Diptera: Culicidae) in Malaysia using reference data available in GenBank in order to reveal this species' phylogenetic relationships. A statistical parsimony network of 70 taxa aligned as 624 characters of the cytochrome c oxidase subunit I (COI) gene and 685 characters of the cytochrome c oxidase subunit II (COII) gene revealed three haplotypes (A1-A3) and four haplotypes (B1-B4), respectively. The concatenated sequences of both COI and COII genes with a total of 1309 characters revealed seven haplotypes (AB1-AB7). Analysis using tcs indicated that haplotype AB1 was the common ancestor and the most widespread haplotype in Malaysia. The genetic distance based on concatenated sequences of both COI and COII genes ranged from 0.00076 to 0.00229. Sequence alignment of Cx. quinquefasciatus from Malaysia and other countries revealed four haplotypes (AA1-AA4) by the COI gene and nine haplotypes (BB1-BB9) by the COII gene. Phylogenetic analyses demonstrated that Malaysian Cx. quinquefasciatus share the same genetic lineage as East African and Asian Cx. quinquefasciatus. This study has inferred the genetic lineages, dispersal patterns and hypothetical ancestral genotypes of Cx. quinquefasciatus. © 2013 The Royal Entomological Society.

Optimization of northern analysis by vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation

International Nuclear Information System (INIS)

Kroczek, R.A.; Siebert, E.

1990-01-01

We have optimized Northern analysis at several steps. Overnight electrophoresis was replaced by short gel runs and overnight capillary transfer by rapid vacuum-blotting adapted to Northern analysis. Short uv irradiation was used as a substitute for the usual RNA fixation by baking. Direct staining of RNA before electrophoresis made it possible to check RNA integrity and to evaluate the quality of the size separation immediately after electrophoresis. In this system, RNA transfer onto the membrane support could also be quickly assessed after the blotting step. The net result of all modifications was a doubling of the autoradiography signal compared with that obtained by modern Northern protocols. At the same time, the duration of the procedure was shortened drastically, allowing an autoradiography signal to be obtained within 24 h

Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen.

Science.gov (United States)

Arias, M; Yeargan, M; Francisco, I; Dangoudoubiyam, S; Becerra, P; Francisco, R; Sánchez-Andrade, R; Paz-Silva, A; Howe, D K

2012-04-30

Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs. Copyright © 2011 Elsevier B.V. All rights reserved.

Fossil-based comparative analyses reveal ancient marine ancestry erased by extinction in ray-finned fishes.

Science.gov (United States)

Betancur-R, Ricardo; Ortí, Guillermo; Pyron, Robert Alexander

2015-05-01

The marine-freshwater boundary is a major biodiversity gradient and few groups have colonised both systems successfully. Fishes have transitioned between habitats repeatedly, diversifying in rivers, lakes and oceans over evolutionary time. However, their history of habitat colonisation and diversification is unclear based on available fossil and phylogenetic data. We estimate ancestral habitats and diversification and transition rates using a large-scale phylogeny of extant fish taxa and one containing a massive number of extinct species. Extant-only phylogenetic analyses indicate freshwater ancestry, but inclusion of fossils reveal strong evidence of marine ancestry in lineages now restricted to freshwaters. Diversification and colonisation dynamics vary asymmetrically between habitats, as marine lineages colonise and flourish in rivers more frequently than the reverse. Our study highlights the importance of including fossils in comparative analyses, showing that freshwaters have played a role as refuges for ancient fish lineages, a signal erased by extinction in extant-only phylogenies. © 2015 John Wiley & Sons Ltd/CNRS.

Silver and gold nanoparticle coated membranes applied to protein dot blots

International Nuclear Information System (INIS)

Xie, F.; Drozdowicz-Tomsia, K.; Shtoyko, T.; Goldys, E. M.

2011-01-01

Detection and identification of low abundance biomarker proteins is frequently based on various types of membrane-based devices. Lowering of the protein detection limits is vital in commercial applications such as lateral flow assays and in Western blots widely used in proteomics. These currently suffer from insufficient detection sensitivity and low retention for small 2–5 kDa proteins. In this study, we report the deposition of two types of metal nanoparticles: gold colloids (50–95 nm diameter) and silver fractals onto a range of commonly used types of membranes including polyvinylidene fluoride (PVDF). Due to strong affinity of proteins to noble metals, such modified membranes have the potential to effectively capture trace proteins preventing their loss. The membranes modified by metal particles were characterized optically and by SEM. The membrane performance in protein dot blots was evaluated using the protein—fluorophore conjugates Deep Purple-bovine serum albumin and fluorescein—human serum albumin. We found that the metal nanoparticles increase light extinction by metals, which is balanced by increased fluorescence, so that the effective fluorescence signal is unchanged. This feature combined with the capture of proteins by the nanoparticles embedded in the membrane increases the detection limit of membrane assays.

Integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected placements for fossil taxa.

Directory of Open Access Journals (Sweden)

Tod W Reeder

Full Text Available Squamate reptiles (lizards and snakes are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa. Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia. These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses without considering the possible impact of molecular data on their placement.

Integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected placements for fossil taxa.

Science.gov (United States)

Reeder, Tod W; Townsend, Ted M; Mulcahy, Daniel G; Noonan, Brice P; Wood, Perry L; Sites, Jack W; Wiens, John J

2015-01-01

Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement.

Integrated Analyses Resolve Conflicts over Squamate Reptile Phylogeny and Reveal Unexpected Placements for Fossil Taxa

Science.gov (United States)

Reeder, Tod W.; Townsend, Ted M.; Mulcahy, Daniel G.; Noonan, Brice P.; Wood, Perry L.; Sites, Jack W.; Wiens, John J.

2015-01-01

Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement. PMID:25803280

Northern and Southern blot analysis of human RNA and DNA in autopsy material

DEFF Research Database (Denmark)

Larsen, S; Rygaard, K; Asnaes, S

1992-01-01

was obtained less than two days postmortem. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five...

Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

Science.gov (United States)

Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

2017-11-01

The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oridonin Targets Multiple Drug-Resistant Tumor Cells as Determined by in Silico and in Vitro Analyses

Directory of Open Access Journals (Sweden)

Onat Kadioglu

2018-04-01

Full Text Available Drug resistance is one of the main reasons of chemotherapy failure. Therefore, overcoming drug resistance is an invaluable approach to identify novel anticancer drugs that have the potential to bypass or overcome resistance to established drugs and to substantially increase life span of cancer patients for effective chemotherapy. Oridonin is a cytotoxic diterpenoid isolated from Rabdosia rubescens with in vivo anticancer activity. In the present study, we evaluated the cytotoxicity of oridonin toward a panel of drug-resistant cancer cells overexpressing ABCB1, ABCG2, or ΔEGFR or with a knockout deletion of TP53. Interestingly, oridonin revealed lower degree of resistance than the control drug, doxorubicin. Molecular docking analyses pointed out that oridonin can interact with Akt/EGFR pathway proteins with comparable binding energies and similar docking poses as the known inhibitors. Molecular dynamics results validated the stable conformation of oridonin docking pose on Akt kinase domain. Western blot experiments clearly revealed dose-dependent downregulation of Akt and STAT3. Pharmacogenomics analyses pointed to a mRNA signature that predicted sensitivity and resistance to oridonin. In conclusion, oridonin bypasses major drug resistance mechanisms and targets Akt pathway and might be effective toward drug refractory tumors. The identification of oridonin-specific gene expressions may be useful for the development of personalized treatment approaches.

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

Directory of Open Access Journals (Sweden)

N.S. Sato

2004-07-01

Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

Science.gov (United States)

Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

2012-12-27

L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

A 15-year-long Southern blotting analysis of FMR1 to detect female carriers and for prenatal diagnosis of fragile X syndrome in Taiwan.

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Tzeng, C-C; Tsai, L-P; Chang, Y-K; Hung, Y-J; Chang, Y-Y; Su, Y-P; Jiang, J-J; Liang, H-M

2017-08-01

Here, we review the results of Southern blotting analyses of the FMR1 gene performed in our reference laboratory in Taiwan over a 15-year period. In total, 725 high-risk women with a family history of fragile X syndrome (FXS) or idiopathic intellectual disability, 3911 low-risk pregnant women without such family history, and prenatal diagnosis data for 32 foetuses from 24 carrier mothers were included. Only 2 carriers were in the low-risk group, which indicated a prevalence of 1 of 1955 women (95% confidence interval: 1/7156-1/539). A total of 100 carriers were found to be in the high-risk group, thus revealing a significantly higher frequency than the low-risk group (100/725 vs 2/3911, P<0.0001). Eight of the 14 foetuses that inherited the maternal mutant allele were verified to have a full mutation, with the smallest maternal pre-mutation allele carrying 56 CGG repeats. The overall findings confirmed that the carrier prevalence among low-risk women in Taiwan is significantly lower than that reported in western countries. Therefore, the most important step for preventing FXS in Taiwan would be to focus on high-risk women by promoting general awareness of this disease and spreading knowledge regarding the benefits of carrier screening and prenatal testing. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

Science.gov (United States)

Hazzalin, Catherine A; Mahadevan, Louis C

2017-01-01

Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.

Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

Science.gov (United States)

Maccari, Francesca; Volpi, Nicola

2002-09-01

We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

[Western Blot diagnostic yield for simultaneous antibody-detection in patients with human cysticercosis, hydatidosis, and human fascioliasis].

Science.gov (United States)

Davelois, Kelly; Escalante, Hermes; Jara, César

2016-01-01

. To determine the diagnostic yield using western blotting to simultaneously detect antibodies in patients with human cysticercosis, hydatidosis, and human fascioliasis. Materials and methods . Cross-sectional study of diagnostic yield assessment. Excretory/secretory antigens were obtained from Taenia solium larvae, Echinococcus granulosus cysts, and the adult flukes of Fasciola hepática, which were then separated using the polyacrylamide gel electrophoresis technique, transferred, and attached to a nitrocellulose membrane to be probed with sera from the patient infected with the three parasites. The sensitivity of the technique was assessed using 300 individual serum samples, 60 pools of two parasites, and 20 pools of three parasites with 75 sera from patients with other parasites, 10 from patients with other diseases, and 15 from patients without parasites. Results . The technique revealed 13 glycoproteins (GP): GP 35, 31, 24, 23, 18, 17, 14, and 13 kDa for cysticercosis; GP 8, 16, and 21 kDa for hydatidosis; and GP 17 and 23 kDa for fascioliasis. The test detected the presence of antibodies with a sensitivity of 96% (95% confidence interval [CI] = 94.62-98.54%) in the detection of one or the thirteen bands, a specificity of 100% (95% CI = 99.50-100.00%); individually, there was a sensitivity for cysticercosis of 97% (95% CI = 93.16-100.00%), for hydatidosis of 94% (95% CI = 88.85-99.15%) and for fascioliasis of 96% (95% CI = 91.66-100.00%). Conclusions . Western blotting is effective in the simultaneous detection of antibodies in patients with human cysticercosis, hydatidosis, and fascioliasis, and it can be used as a diagnostic test to either rule out or confirm the presence of antibodies in endemic areas.

Functional assays and metagenomic analyses reveals differences between the microbial communities inhabiting the soil horizons of a Norway spruce plantation.

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Stéphane Uroz

Full Text Available In temperate ecosystems, acidic forest soils are among the most nutrient-poor terrestrial environments. In this context, the long-term differentiation of the forest soils into horizons may impact the assembly and the functions of the soil microbial communities. To gain a more comprehensive understanding of the ecology and functional potentials of these microbial communities, a suite of analyses including comparative metagenomics was applied on independent soil samples from a spruce plantation (Breuil-Chenue, France. The objectives were to assess whether the decreasing nutrient bioavailability and pH variations that naturally occurs between the organic and mineral horizons affects the soil microbial functional biodiversity. The 14 Gbp of pyrosequencing and Illumina sequences generated in this study revealed complex microbial communities dominated by bacteria. Detailed analyses showed that the organic soil horizon was significantly enriched in sequences related to Bacteria, Chordata, Arthropoda and Ascomycota. On the contrary the mineral horizon was significantly enriched in sequences related to Archaea. Our analyses also highlighted that the microbial communities inhabiting the two soil horizons differed significantly in their functional potentials according to functional assays and MG-RAST analyses, suggesting a functional specialisation of these microbial communities. Consistent with this specialisation, our shotgun metagenomic approach revealed a significant increase in the relative abundance of sequences related glycoside hydrolases in the organic horizon compared to the mineral horizon that was significantly enriched in glycoside transferases. This functional stratification according to the soil horizon was also confirmed by a significant correlation between the functional assays performed in this study and the functional metagenomic analyses. Together, our results suggest that the soil stratification and particularly the soil resource

Mammalian α-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots

International Nuclear Information System (INIS)

SenGupta, D.N.; Kumar, P.; Zmudzka, B.Z.; Coughlin, S.; Vishwanatha, J.K.; Robey, F.A.; Parrott, C.; Wilson, S.H.

1987-01-01

A new polyclonal antibody against the α-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cellα-polymerase. The antibody neutralized α-polymerase activity and was strong and specific for the α-polymerase catalytic polypeptide (M/sub r/ 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in λgt11. A positive phage was identified and plaque purified. This phage, designated λpolα1.2, also was found to be positive with an antibody against Drosophila α-polymerase. The insert in λpolα1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified α-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating α-polymerase. This indicated the λpolα1.2 insert encoded an α-polymerase epitope and suggested that the cDNA corresponded to an α-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding α-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is ∼5.4 kilobases

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

OpenAIRE

Penna, Aubin; Cahalan, Michael

2007-01-01

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After tr...

DISKRIMINASI KELAMIN PADA IKAN TUNA SIRIP KUNING, Yellowfin tuna MENGGUNAKAN ANALISIS DOT BLOT DAN ELISA

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Gusti Ngurah Permana

2014-08-01

Full Text Available Pemahaman tentang penentuan jenis kelamin dalam populasi induk merupakan hal yang sangat penting bagi keberhasilan program pembenihan. Pengukuran reaksi antibodi dan aktivitas hormon testosterone, serta estradiol adalah metode dengan potensi yang secara akurat dapat menentukan jenis kelamin ikan tanpa mematikan ikan. Tujuan penelitian ini adalah untuk mengetahui akurasi metode dot blot dan ELISA dengan 11-ketotestorsterone (11-KT yang tersedia secara komersial EIA-kit untuk membedakan jenis kelamin ikan tuna sirip kuning. Hasil analisis menunjukkan bahwa metode dot blot menghasilkan ekspresi vitelogenin tampak jelas pada individu betina dan efek plasma terlihat transparan, jika dibandingkan dengan individu jantan. Interpretasi dari metode ini memerlukan pengalaman dan keahlian dalam akurasi pembacaan hasil. Aktivitas hormon 11-KT dengan sampel klip sirip dan plasma memberikan hasil yang baik dengan aktivitas hormon terlihat jelas.

Molecular analyses reveal high species diversity of trematodes in a sub-Arctic lake

Science.gov (United States)

Soldánová, Miroslava; Georgieva, Simona; Roháčováa, Jana; Knudsen, Rune; Kuhn, Jesper A.; Henriksen, Eirik H.; Siwertsson, Anna; Shaw, Jenny C.; Kuris, Armand M.; Amundsen, Per-Arne; Scholz, Tomáš; Lafferty, Kevin D.; Kostadinova, Aneta

2017-01-01

To identify trematode diversity and life-cycles in the sub-Arctic Lake Takvatn, Norway, we characterised 120 trematode isolates from mollusc first intermediate hosts, metacercariae from second intermediate host fishes and invertebrates, and adults from fish and invertebrate definitive hosts, using molecular techniques. Phylogenies based on nuclear and/or mtDNA revealed high species richness (24 species or species-level genetic lineages), and uncovered trematode diversity (16 putative new species) from five families typical in lake ecosystems (Allocreadiidae, Diplostomidae, Plagiorchiidae, Schistosomatidae and Strigeidae). Sampling potential invertebrate hosts allowed matching of sequence data for different stages, thus achieving molecular elucidation of trematode life-cycles and exploration of host-parasite interactions. Phylogenetic analyses also helped identify three major mollusc intermediate hosts (Radix balthica, Pisidium casertanum and Sphaerium sp.) in the lake. Our findings increase the known trematode diversity at the sub-Arctic Lake Takvatn, showing that digenean diversity is high in this otherwise depauperate sub-Arctic freshwater ecosystem, and indicating that sub-Arctic and Arctic ecosystems may be characterised by unique trematode assemblages.

Genetic Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

Science.gov (United States)

Demezas, David H.; Reardon, Terry B.; Watson, John M.; Gibson, Alan H.

1991-01-01

Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships. PMID:16348600

Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

International Nuclear Information System (INIS)

Thompson, G.A.; Davies, H.M.; McDonald, N.

1985-01-01

A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses.

Science.gov (United States)

Matsuo, S; Sugiyama, T; Okuyama, T; Yoshikawa, K; Honda, K; Takahashi, R; Maeda, S

1999-05-01

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

Dot-blot immunoassay of Fasciola gigantica infection using 27 kDa and adult worm regurge antigens in Egyptian patients.

Science.gov (United States)

Kamel, Hanan H; Saad, Ghada A; Sarhan, Rania M

2013-04-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

Science.gov (United States)

Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

2016-01-01

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

Proteínas inmunodominantes de Brucella Melitensis evaluadas por Western Blot

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Elizabeth Anaya

1997-01-01

Full Text Available Se separaron extractos de proteínas totales de Brucella melitensis en gel 15% SDS-PAGE. Su seroreactividad fue analizada por Western Blot con resultados satisfactorios. Para éste propósito sueros controles negativos (n=03, sueros de pacientes con brucelosis (n=34, cólera (n=12, tifoidea (n=02 y tuberculosis (n=02 fueron usados. Esta prueba inmunodiagnóstica detectó bandas seroreactivas altamente específicas (100% correspondientes a 8,14,18, un complejo de 25-48 y 58kDa. La sensibilidad del test fue del 90% usando los sueros antes mencionados.

FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

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D.V. Pilonetto

2009-03-01

Full Text Available Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L- was not detected in 77 patients (91.7%. In 2 patients (2.4%, there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L- and 5 patients (5.9% had both isoforms (FANCD2S+/FANCD2L+. This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84 and specificity of 100% (98/98. This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L- or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-, to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+ require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot

DEFF Research Database (Denmark)

Albers, Eliene; Sbroggiò, Mauro; Martin Gonzalez, Javier

2017-01-01

The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific...

Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada

OpenAIRE

Ogden, Nicholas H.; Arsenault, Julie; Hatchette, Todd F.; Mechai, Samir; Lindsay, L. Robbin

2017-01-01

Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographi...

Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

Science.gov (United States)

Cabral, Elaine C.; Mirabelli, Mario F.; Perez, Consuelo J.; Ifa, Demian R.

2013-06-01

Imprints of potato sprout ( Solanum tuberosum L.), gingko leaves (Gingko biloba L. ) and strawberries (Fragaria x ananassa Duch. ) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

Comparative analyses reveal different consequences of two oxidative stress inducers, gamma irradiation and potassium tellurite, in the extremophile Deinococcus radiodurans

International Nuclear Information System (INIS)

Narasimha, Anaganti; Basu, Bhakti; Apte, Shree Kumar

2014-01-01

Proteomic and mass spectrometric analyses revealed differential responses of D. radiodurans to two oxidative stressors. While both elicited oxidative stress alleviation response, major divergence was observed at the level of DNA repair, metabolic pathways and protein homeostasis. Response to gamma irradiation was focused on DNA repair and ROS scavenging but supported metabolism as well as protein homeostasis. Tellurite, induced oxidative stress alleviation but decreased reducing affected and adversely affected metabolism and protein homeostasis

Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

International Nuclear Information System (INIS)

Lee, C.Y.G.; Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

1982-01-01

A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility

La técnica de Western Blot como criterio de identidad para la vacuna antimeningocócica Men B Western Blot technique as an identity criterion for Men B antimeningococcal vaccine

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R. Rosario Diéguez Castro

2009-12-01

Full Text Available Se desarrolló y validó la técnica de Western Blot aplicada a la vacuna antimeningocócica Men B producida en el Instituto Finlay con el objetivo de demostrar un criterio de identidad. En el estudio de las proteínas antigénicas de la vacuna, P1.15 y P1.4 en vesícula de membrana externa,monograneles y producto final se emplearon en la identificación anticuerpos monoclonales específicos para estas proteínas. Los parámetros desarrollados en la validación de la técnica fueron: especificidad, límite de detección, repetibilidad, precisión intermedia, reproducibilidad y robustez. El método cumplió con los parámetros señalados, por lo que se consideró validado.Western Blot technique was developed and validated, applied to Men B meningococcal vaccine produced in "Carlos J, Finlay" Institute to demonstrate an identity criterion. In study of antigenic proteins of the vaccine, we used P1.15 y P1.4 in vesicle of external membrane, monogranels, and end product to identify the monoclonal antibodies specific of these proteins. Parameters developed in technique validation included: specificity, detection limit, repetition, average accuracy, reproduction, and strength. Method fulfilled with specified parameters, thus considering its validation.

Evaluation of the radioimmunoassay, indirect enzyme linked immunosorbent assay, and dot blot assay for the identification of Xanthomonas campestris pv. phaseoli

Energy Technology Data Exchange (ETDEWEB)

Malin, E; Belden, E L; Roth, D

1985-09-01

A radioimmunoassay (RIA), an indirect competitive enzyme-linked immunosorbent assay (ELISA), and a dot-blot modification of the ELISA were evaluated for detection and identification of Xanthomonas campestris pv. phaseoli (X. c. pv. phaseoli). RIA and the dot blot tests were specific for X. c. pv. phaseoli; however, significant cross reactions occurred in the indirect competitive ELISA when using anti-X. c. pv. phaseoli antiserum against other closely related bacteria. The sensitivity level of all procedures for X. c. pv. phaseoli was approximately l0/sup 5/ colony forming unitsmL. All procedures were unsatisfactory in reliably detecting low levels of X. c. pv. phaseoli directly from extracts of bean seed. However when used in conjunction with ilution plating the dot blot assay and the RIA would be useful in specifically identifying X. c. pv. phaseoli. The relative merits of these tests for identification of X. c. pv. phaseoli are discussed.

Global terrestrial water storage connectivity revealed using complex climate network analyses

Science.gov (United States)

Sun, A. Y.; Chen, J.; Donges, J.

2015-07-01

Terrestrial water storage (TWS) exerts a key control in global water, energy, and biogeochemical cycles. Although certain causal relationship exists between precipitation and TWS, the latter quantity also reflects impacts of anthropogenic activities. Thus, quantification of the spatial patterns of TWS will not only help to understand feedbacks between climate dynamics and the hydrologic cycle, but also provide new insights and model calibration constraints for improving the current land surface models. This work is the first attempt to quantify the spatial connectivity of TWS using the complex network theory, which has received broad attention in the climate modeling community in recent years. Complex networks of TWS anomalies are built using two global TWS data sets, a remote sensing product that is obtained from the Gravity Recovery and Climate Experiment (GRACE) satellite mission, and a model-generated data set from the global land data assimilation system's NOAH model (GLDAS-NOAH). Both data sets have 1° × 1° grid resolutions and cover most global land areas except for permafrost regions. TWS networks are built by first quantifying pairwise correlation among all valid TWS anomaly time series, and then applying a cutoff threshold derived from the edge-density function to retain only the most important features in the network. Basinwise network connectivity maps are used to illuminate connectivity of individual river basins with other regions. The constructed network degree centrality maps show the TWS anomaly hotspots around the globe and the patterns are consistent with recent GRACE studies. Parallel analyses of networks constructed using the two data sets reveal that the GLDAS-NOAH model captures many of the spatial patterns shown by GRACE, although significant discrepancies exist in some regions. Thus, our results provide further measures for constraining the current land surface models, especially in data sparse regions.

Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures

Directory of Open Access Journals (Sweden)

Peter A. Bell

2013-06-01

Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.

Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

Energy Technology Data Exchange (ETDEWEB)

Kale, Sonia [Agharkar Research Institute (India); Kale, Anup [University of Alabama, Center for Materials for Information Technology (United States); Gholap, Haribhau; Rana, Abhimanyu [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Desai, Rama [National Centre for Cell Science (India); Banpurkar, Arun [University of Pune, Department of Physics (India); Ogale, Satishchandra, E-mail: sb.ogale@ncl.res.in [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Shastry, Padma, E-mail: padma@nccs.res.in [National Centre for Cell Science (India)

2012-03-15

In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and {beta} actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques Comparação dos parâmetros imunológicos de cães infectados com leishmaniose visceral usando as técnicas de Western blot e neutralização

Directory of Open Access Journals (Sweden)

Yeda L. Nogueira

2007-12-01

Full Text Available The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.A técnica de Western blot foi utilizada para demonstrar a presença de anticorpos do soro de cães, que apresentavam leishmaniose visceral canina, contra algumas moléculas específicas no lisado da forma promastigota de Leshmania chagasi.Através da associa

Conserved regulators of nucleolar size revealed by global phenotypic analyses.

Science.gov (United States)

Neumüller, Ralph A; Gross, Thomas; Samsonova, Anastasia A; Vinayagam, Arunachalam; Buckner, Michael; Founk, Karen; Hu, Yanhui; Sharifpoor, Sara; Rosebrock, Adam P; Andrews, Brenda; Winston, Fred; Perrimon, Norbert

2013-08-20

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.

Conserved Regulators of Nucleolar Size Revealed by Global Phenotypic Analyses

Science.gov (United States)

Neumüller, Ralph A.; Gross, Thomas; Samsonova, Anastasia A.; Vinayagam, Arunachalam; Buckner, Michael; Founk, Karen; Hu, Yanhui; Sharifpoor, Sara; Rosebrock, Adam P.; Andrews, Brenda; Winston, Fred; Perrimon, Norbert

2014-01-01

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I–mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I–mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry–based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules. PMID:23962978

Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability.

Science.gov (United States)

He, Dong; Luo, Wen; Wang, Zhiyuan; Lv, Pengmei; Yuan, Zhenhong; Huang, Shaowei; Xv, Jingliang

2017-07-01

Directed evolution has been proved an effective way to improve the stability of proteins, but high throughput screening assays for directed evolution with simultaneous improvement of two or more properties are still rare. In this study, we aimed to establish a membrane-blot assay for use in the high-throughput screening of Rhizomucor miehei lipases (RMLs). With the assistance of the membrane-blot screening assay, a mutant E47K named G10 that showed improved thermal stability was detected in the first round of error-prone PCR. Using G10 as the parent, two variants G10-11 and G10-20 that showed improved thermal stability and methanol tolerance without loss of activity compared to the wild type RML were obtained. The T 50 60 -value of G10-11 and G10-20 increased by 12°C and 6.5°C, respectively. After incubation for 1h, the remaining residual activity of G10-11 and G10-20 was 63.45% and 74.33%, respectively, in 50% methanol, and 15.98% and 30.22%, respectively, in 80% methanol. Thus, we successfully developed a membrane-blot assay that could be used for the high-throughput screening of RMLs with improved thermostability and methanol tolerance. Based on our findings, we believe that our newly developed membrane-blot assay will have potential applications in directed evolution in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Mitochondrial dysfunction, oxidative stress and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson’s disease

Energy Technology Data Exchange (ETDEWEB)

Chin, Mark H.; Qian, Weijun; Wang, Haixing; Petyuk, Vladislav A.; Bloom, Joshua S.; Sforza, Daniel M.; Lacan, Goran; Liu, Dahai; Khan, Arshad H.; Cantor, Rita M.; Bigelow, Diana J.; Melega, William P.; Camp, David G.; Smith, Richard D.; Smith, Desmond J.

2008-02-10

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson disease (PD) are not completely understood. Here we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 85 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA following neurotoxin treatment. The combined protein and gene list provides a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response and apoptosis. Additionally, codon usage and miRNAs may play an important role in translational control in the striatum. These results constitute one of the largest datasets integrating protein and transcript changes for these neurotoxin models with many similar endpoint phenotypes but distinct mechanisms.

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

Science.gov (United States)

Yu, Qin; Hu, Liyan; Yao, Qing; Zhu, Yongqun; Dong, Na; Wang, Da-Cheng; Shao, Feng

2013-06-01

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

Immunodiagnosis of Echinococcus Infections: Confirmatory Testing and Species Differentiation by a New Commercial Western Blot

Science.gov (United States)

Liance, Martine; Janin, Veronique; Bresson-Hadni, Solange; Vuitton, Dominique-Angele; Houin, Rene; Piarroux, Renaud

2000-01-01

The Echinococcus Western Blot IgG (LDBIO Diagnostics, Lyon, France), using a whole larval antigen from Echinococcus multilocularis, was evaluated for serodiagnosis and differentiation between two human parasitic infections of worldwide importance: cystic echinococcosis, due to Echinococcus granulosus, and alveolar echinococcosis, due to E. multilocularis. Fifty and 61 serum samples from patients with cystic and alveolar echinococcosis, respectively, were used for assessing diagnostic sensitivity. The sensitivity of the assay was compared with those of screening tests used for these applications. Sera used for assessing cross-reactivities were from 154 patients with other diseases, either parasitic or not. The assay allowed the detection of serum immunoglobulin G antibodies in 97% of Echinococcus-infected patients. It had a higher sensitivity than screening assays for the detection for each echinococcosis. The assay allowed us to correctly distinguish between E. granulosus- and E. multilocularis-infected patients in 76% of cases. It did not allow us to distinguish active from inactive forms of both echinococcoses. The occurrence of cross-reactivities with neurocysticercosis indicates the necessity for retesting sera with species-specific antigens, for rare patients with neurologic disorders. This study shows the usefulness of the commercially available Echinococcus Western Blot IgG for the serological confirmation of human echinococcosis. PMID:11015390

Multiple spectroscopic analyses reveal the fate and metabolism of sulfamide herbicide triafamone in agricultural environments

International Nuclear Information System (INIS)

Wang, Mengcen; Qian, Yuan; Liu, Xiaoyu; Wei, Peng; Deng, Man; Wang, Lei; Wu, Huiming; Zhu, Guonian

2017-01-01

Triafamone, a sulfamide herbicide, has been extensively utilized for weed control in rice paddies in Asia. However, its fate and transformation in the environment have not been established. Through a rice paddy microcosm-based simulation trial combined with multiple spectroscopic analyses, we isolated and identified three novel metabolites of triafamone, including hydroxyl triafamone (HTA), hydroxyl triafamone glycoside (HTAG), and oxazolidinedione triafamone (OTA). When triafamone was applied to rice paddies at a concentration of 34.2 g active ingredient/ha, this was predominantly distributed in the paddy soil and water, and then rapidly dissipated in accordance with the first-order rate model, with half-lives of 4.3–11.0 days. As the main transformation pathway, triafamone was assimilated by the rice plants and was detoxified into HTAG, whereas the rest was reduced into HTA with subsequent formation of OTA. At the senescence stage, brown rice had incurred triafamone at a concentration of 0.0016 mg/kg, but the hazard quotient was <1, suggesting that long-term consumption of the triafamone-containing brown rice is relatively safe. The findings of the present study indicate that triafamone is actively metabolized in the agricultural environment, and elucidation of the link between environmental exposure to these triazine or oxazolidinedione moieties that contain metabolites and their potential impacts is warranted. - Highlights: • Multiple spectroscopic analyses were applied to investigate agrochemicals transformation in environment. • Three novel compounds were isolated and identified as triafamone metabolites. • The fate and transformation pathway of triafamone in rice paddy were revealed. • Long-term consumption of the triafamone-containing brown rice is relatively safe. • Elucidation of environmental impacts by exposure to these triazine or oxazolidinedione metabolites is warranted. - Triafamone rapidly dissipates in agricultural environments

Mere end blot en bid af hverdagen- Måltidet i et leve- og bomiljø

DEFF Research Database (Denmark)

Bundgaard, Karen Marie

2005-01-01

. Datamaterialet bygger på deltagerobservationer og interviews. Undersøgelsen viste, at den måde måltiderne var organiseret på gav tid og rum til en hjemlig atmosfære, til et levende fællesskab, til det at være noget og at være sig selv og til at have værdifulde gøremål. Måltiderne var ikke blot en bid – men en...

A single-step simultaneous protein staining procedure for polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis.

Science.gov (United States)

Pal, Jayanta K; Berwal, Sunil K; Soni, Rupali N

2012-01-01

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS) polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water and viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining), and cost-effective.

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

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Eikan Mishima

Full Text Available The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A, N6-methyladenosine (m6A, pseudouridine, and 5-methylcytidine (m5C showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

Science.gov (United States)

Mishima, Eikan; Jinno, Daisuke; Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

2015-01-01

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots

NARCIS (Netherlands)

Maagd, de R.A.; Klei, van der H.; Bakker, P.L.; Stiekema, W.J.; Bosch, D.

1996-01-01

We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) δ-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild- type and CryIC-CryIA hybrid toxins was used for this purpose. As

A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

Science.gov (United States)

Hood-Degrenier, Jennifer K

2008-01-01

The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

Evaluación de las pruebas dot blot y aglutinación de látex para el diagnóstico de cisticercosis en Perú

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Eduardo Miranda-Ulloa

Full Text Available Con el objetivo de evaluar las pruebas dot blot y aglutinación de látex para la detección de cisticercosis humana con antígeno de líquido de cisticerco de Taenia solium, se usaron 125 sueros humanos, de los cuales 60 procedían de personas con cisticercosis confirmada por Western Blot, 45 de personas con otras enfermedades parasitarias y 20 de personas aparentemente sanas. La concentración óptima del antígeno para impregnar las tiras dot blot fue de 0,01 ug/uL, y para impregnar las partículas de látex fue de 0,092 ug/uL. Para la prueba dot blot se encontró una sensibilidad del 100% y especificidad del 87,7%; para la aglutinación de látex una sensibilidad del 93,3% y especificidad del 89,2%. Ambas pruebas podrían ser de utilidad y factibles de implementar como alternativas de diagnóstico serológico en laboratorios de áreas endémicas del Perú

Comparison by restriction fragment pattern analyses and molecular characterization of some European isolates of Suid herpesvirus 1: A contribution to strain differentiation of European isolates

DEFF Research Database (Denmark)

Christensen, Laurids Siig

1988-01-01

Eleven European isolates of Suid herpesvirus type 1 (SHV-1) were compared by restriction fragment pattern analyses and Southern blot hybridization using different genomic probes. The presence of strain discriminative 4 major genome types and several subtypes as well as the molecular distinctions...

Structure-Related Roles for the Conservation of the HIV-1 Fusion Peptide Sequence Revealed by Nuclear Magnetic Resonance.

Science.gov (United States)

Serrano, Soraya; Huarte, Nerea; Rujas, Edurne; Andreu, David; Nieva, José L; Jiménez, María Angeles

2017-10-17

Despite extensive characterization of the human immunodeficiency virus type 1 (HIV-1) hydrophobic fusion peptide (FP), the structure-function relationships underlying its extraordinary degree of conservation remain poorly understood. Specifically, the fact that the tandem repeat of the FLGFLG tripeptide is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the nuclear magnetic resonance (NMR) structures adopted in nonpolar media by two FP surrogates, wtFP-tag and scrFP-tag, which had equal hydrophobicity but contained wild-type and scrambled core sequences LFLGFLG and FGLLGFL, respectively. In addition, these peptides were tagged at their C-termini with an epitope sequence that folded independently, thereby allowing Western blot detection without interfering with FP structure. We observed similar α-helical FP conformations for both specimens dissolved in the low-polarity medium 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure upon being solubilized in DPC. Western blot analyses further revealed the capacity of wtFP-tag to assemble trimers in membranes, whereas membrane oligomers were not observed in the case of the scrFP-tag sequence. We conclude that, beyond hydrophobicity, preserving sequence order is an important feature for defining the secondary structures and oligomeric states adopted by the HIV FP in membranes.

Patterns and processes of Mycobacterium bovis evolution revealed by phylogenomic analyses

Science.gov (United States)

Mycobacterium bovis is an important animal pathogen worldwide that parasitizes wild and domesticated vertebrate livestock as well as humans. A comparison of the five M. bovis complete genomes from UK, South Korea, Brazil and USA revealed four novel large-scale structural variations of at least 2,000...

Integrated in silico Analyses of Regulatory and Metabolic Networks of Synechococcus sp. PCC 7002 Reveal Relationships between Gene Centrality and Essentiality

Directory of Open Access Journals (Sweden)

Hyun-Seob Song

2015-03-01

Full Text Available Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as “topologically important.” Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as “functionally important” genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.

ANCA-GBM dot-blot : Evaluation of an assay in the differential diagnosis of patients presenting with rapidly progressive glomerulonephritis

NARCIS (Netherlands)

Rutgers, Abraham; Damoiseaux, Jan; Roozendaal, Caroline; Limburg, Pieter C; Stegeman, Coen A; Tervaert, Jan Willem Cohen

Rapidly progressive glomerulonephritis (RPGN) is characterized by rapid and progressive loss of renal function and the presence of crescentic glomerulonephritis (CGN). Early diagnosis and appropriate treatment is mandatory to prevent death and/or renal failure. We have evaluated an ANCA-GBM dot-blot

Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis

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Nilton Thaumaturgo

2002-10-01

Full Text Available Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females, Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests.

Revealing climate modes in steric sea levels: lessons learned from satellite geodesy, objective analyses and ocean reanalyses

Science.gov (United States)

Pfeffer, J.; Tregoning, P.; Purcell, A. P.

2017-12-01

Due to increased greenhouse gases emissions, the oceans are accumulating heat. In response to the ocean circulation and atmospheric forcing, the heat is irregularly redistributed within the oceans, causing sea level to rise at variable rates in space and time. These rates of steric expansion are extremely difficult to assess because of the sparsity of in-situ hydrographic observations available within the course of the 20th century. We compare here three methods to reconstruct the steric sea levels over the past 13, 25 and 58 years based on satellite geodesy, objective analyses and ocean reanalyses. The interannual to decadal variability of each dataset is explored with a model merging six climate indices representative of the natural variability of the ocean and climate system. Consistent regional patterns are identified for the Pacific Decadal Oscillation (PDO) and El Niño Southern Oscillation (ENSO) in all datasets at all timescales. Despite the short time coverage (13 years), the combination of satellite geodetic data (altimetry and GRACE) also reveals significant steric responses to the North Pacific Gyre Oscillation (NPGO), Indian Dipole (IOD) and Indian ocean basinwide (IOBM) mode. The richer information content in the ocean reanalyses allows us to recover the regional fingerprints of the PDO, ENSO, NPGO, IOD and IOBM, but also of the Atlantic Multidecadal Oscillation (AMO) acting over longer time scales (40 to 60 years). Therefore, ocean reanalyses, coupled with climate mode analyses, constitute innovative and promising tools to investigate the mechanisms triggering the variability of sea level rise over the past decades.

Cy5 total protein normalization in Western blot analysis.

Science.gov (United States)

Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

2015-10-01

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

Fibroid explants reveal a higher sensitivity against MDM2-inhibitor nutlin-3 than matching myometrium

Directory of Open Access Journals (Sweden)

Markowski Dominique N

2012-01-01

Full Text Available Abstract Background Spontaneous cessation of growth is a frequent finding in uterine fibroids. Increasing evidence suggests an important role of cellular senescence in this growth control. Deciphering the underlying mechanisms of growth control that can be expected not only to shed light on the biology of the tumors but also to identify novel therapeutic targets. Methods We have analyzed uterine leiomyomas and matching normal tissue for the expression of p14Arf and used explants to see if reducing the MDM2 activity using the small-molecule inhibitor nutlin-3 can induce p53 and activate genes involved in senescence and/or apoptosis. For these studies quantitative real-time RT-PCR, Western blots, and immunohistochemistry were used. Statistical analyses were performed using the student's t test. Results An in depth analysis of 52 fibroids along with matching myometrium from 31 patients revealed in almost all cases a higher expression of p14Arf in the tumors than in the matching normal tissue. In tissue explants, treatment with the MDM2 inhibitor nutlin-3 induced apoptosis as well as senescence as revealed by a dose-dependent increase of the expression of BAX as well as of p21, respectively. Simultaneously, the expression of the proliferation marker Ki-67 drastically decreased. Western-blot analysis identified an increase of the p53 level as the most likely reason for the increased activity of its downstream markers BAX and p21. Because as a rule fibroids express much higher levels of p14Arf, a major negative regulator of MDM2, than matching myometrium it was then analyzed if fibroids are more sensitive against nutlin-3 treatment than matching myometrium. We were able to show that in most fibroids analyzed a higher sensibility than that of matching myometrium was noted with a corresponding increase of the p53 immunopositivity of the fibroid samples compared to those from myometrium. Conclusions The results show that uterine fibroids represent a cell

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

Genome-wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants

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Eunyoung Seo

2016-08-01

Full Text Available Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR immune receptors are known play critical roles in effector-triggered immunity (ETI plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analyses and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analyses of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding.

Bibliometric Analyses Reveal Patterns of Collaboration between ASMS Members

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Palmblad, Magnus; van Eck, Nees Jan

2018-03-01

We have explored the collaborative network of the current American Society for Mass Spectrometry (ASMS) membership using bibliometric methods. The analysis shows that 4249 members are connected in a single, large, co-authorship graph, including the majority of the most published authors in the field of mass spectrometry. The map reveals topographical differences between university groups and national laboratories, and that the co-authors with the strongest links have long worked together at the same location. We have collected and summarized information on the geographical distribution of members, showing a high coverage of active researchers in North America and Western Europe. Looking at research fields, we could also identify a number of new or `hot' topics among ASMS members. Interactive versions of the maps are available on-line at https://goo.gl/UBNFMQ (collaborative network) and https://goo.gl/WV25vm (research topics). [Figure not available: see fulltext.

Iron homeostasis in Arabidopsis thaliana: transcriptomic analyses reveal novel FIT-regulated genes, iron deficiency marker genes and functional gene networks.

Science.gov (United States)

Mai, Hans-Jörg; Pateyron, Stéphanie; Bauer, Petra

2016-10-03

FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) is the central regulator of iron uptake in Arabidopsis thaliana roots. We performed transcriptome analyses of six day-old seedlings and roots of six week-old plants using wild type, a fit knock-out mutant and a FIT over-expression line grown under iron-sufficient or iron-deficient conditions. We compared genes regulated in a FIT-dependent manner depending on the developmental stage of the plants. We assembled a high likelihood dataset which we used to perform co-expression and functional analysis of the most stably iron deficiency-induced genes. 448 genes were found FIT-regulated. Out of these, 34 genes were robustly FIT-regulated in root and seedling samples and included 13 novel FIT-dependent genes. Three hundred thirty-one genes showed differential regulation in response to the presence and absence of FIT only in the root samples, while this was the case for 83 genes in the seedling samples. We assembled a virtual dataset of iron-regulated genes based on a total of 14 transcriptomic analyses of iron-deficient and iron-sufficient wild-type plants to pinpoint the best marker genes for iron deficiency and analyzed this dataset in depth. Co-expression analysis of this dataset revealed 13 distinct regulons part of which predominantly contained functionally related genes. We could enlarge the list of FIT-dependent genes and discriminate between genes that are robustly FIT-regulated in roots and seedlings or only in one of those. FIT-regulated genes were mostly induced, few of them were repressed by FIT. With the analysis of a virtual dataset we could filter out and pinpoint new candidates among the most reliable marker genes for iron deficiency. Moreover, co-expression and functional analysis of this virtual dataset revealed iron deficiency-induced and functionally distinct regulons.

Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

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Aravalli, Rajagopal N., E-mail: aravalli@umn.edu [Department of Radiology, University of Minnesota Medical School, MMC 292, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Park, Chang W. [Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 (United States)

2016-08-26

The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

International Nuclear Information System (INIS)

Aravalli, Rajagopal N.; Park, Chang W.; Steer, Clifford J.

2016-01-01

The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

Note sur la présence de lames aménagées par technique de Kostienki dans les couches gravettiennes du Blot (Cerzat,Haute-Loire).

OpenAIRE

Klaric , Laurent

2000-01-01

International audience; The unprecedented presence of Kostienki-technique prepared blades (also called Kostienki knives) in the Gravettian layers at Le Blot leads to a new analysis of these artefacts. Thorough technological study has pointed to the possible role of these items as cores, in association with or complementary to burin-forms, in particular context of backed-bladelet production. Le Blot is the second French site yielding such artefacts, the other being Corbiac (Dordogne). The aim ...

Detection of human papilloma virus 16 and 18 DNA sequences by southern blot hybridization in oral leukoplakia and squamous cell carcinoma.

Science.gov (United States)

Khanna, Rahul; Rao, G R K; Tiwary, S K; Rai, Ashish; Khanna, Seema; Khanna, A K

2009-04-01

The etiopathological role of human papilloma virus (HPV) in the causation of oral cancer is till a subject of speculation. We used the technique of Southern blot hybridization to detect the presence of HPV types 16 & 18 in biopsy specimens from oral cancer and leukoplakia patients as well as normal oral mucosal biopsies. The prevalence of either HPV type 16 or 18 was found in 64.5% (29/45) of oral cancer, 40%(12/30) of leukoplakia and 20%(9/45) of normal oral mucosal biopsies. No association could be demonstrated between tobacco usage habits or a history of genital warts with HPV prevalence. A significant finding was that none of the oral cancer patients were negative for both: a history of tobacco usage as well as presence of HPV infection, on Southern blot hybridization.

Interspecies introgressive hybridization in spiny frogs Quasipaa (Family Dicroglossidae) revealed by analyses on multiple mitochondrial and nuclear genes.

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Zhang, Qi-Peng; Hu, Wen-Fang; Zhou, Ting-Ting; Kong, Shen-Shen; Liu, Zhi-Fang; Zheng, Rong-Quan

2018-01-01

Introgression may lead to discordant patterns of variation among loci and traits. For example, previous phylogeographic studies on the genus Quasipaa detected signs of genetic introgression from genetically and morphologically divergent Quasipaa shini or Quasipaa spinosa . In this study, we used mitochondrial and nuclear DNA sequence data to verify the widespread introgressive hybridization in the closely related species of the genus Quasipaa , evaluate the level of genetic diversity, and reveal the formation mechanism of introgressive hybridization. In Longsheng, Guangxi Province, signs of asymmetrical nuclear introgression were detected between Quasipaa boulengeri and Q. shini . Unidirectional mitochondrial introgression was revealed from Q. spinosa to Q. shini . By contrast, bidirectional mitochondrial gene introgression was detected between Q. spinosa and Q. shini in Lushan, Jiangxi Province. Our study also detected ancient hybridizations between a female Q. spinosa and a male Q. jiulongensis in Zhejiang Province. Analyses on mitochondrial and nuclear genes verified three candidate cryptic species in Q. spinosa , and a cryptic species may also exist in Q. boulengeri . However, no evidence of introgressive hybridization was found between Q. spinosa and Q. boulengeri . Quasipaa exilispinosa from all the sampling localities appeared to be deeply divergent from other communities. Our results suggest widespread introgressive hybridization in closely related species of Quasipaa and provide a fundamental basis for illumination of the forming mechanism of introgressive hybridization, classification of species, and biodiversity assessment in Quasipaa .

Comprehensive analyses of imprinted differentially methylated regions reveal epigenetic and genetic characteristics in hepatoblastoma

International Nuclear Information System (INIS)

Rumbajan, Janette Mareska; Aoki, Shigehisa; Kohashi, Kenichi; Oda, Yoshinao; Hata, Kenichiro; Saji, Tsutomu; Taguchi, Tomoaki; Tajiri, Tatsuro; Soejima, Hidenobu; Joh, Keiichiro; Maeda, Toshiyuki; Souzaki, Ryota; Mitsui, Kazumasa; Higashimoto, Ken; Nakabayashi, Kazuhiko; Yatsuki, Hitomi; Nishioka, Kenichi; Harada, Ryoko

2013-01-01

Aberrant methylation at imprinted differentially methylated regions (DMRs) in human 11p15.5 has been reported in many tumors including hepatoblastoma. However, the methylation status of imprinted DMRs in imprinted loci scattered through the human genome has not been analyzed yet in any tumors. The methylation statuses of 33 imprinted DMRs were analyzed in 12 hepatoblastomas and adjacent normal liver tissue by MALDI-TOF MS and pyrosequencing. Uniparental disomy (UPD) and copy number abnormalities were investigated with DNA polymorphisms. Among 33 DMRs analyzed, 18 showed aberrant methylation in at least 1 tumor. There was large deviation in the incidence of aberrant methylation among the DMRs. KvDMR1 and IGF2-DMR0 were the most frequently hypomethylated DMRs. INPP5Fv2-DMR and RB1-DMR were hypermethylated with high frequencies. Hypomethylation was observed at certain DMRs not only in tumors but also in a small number of adjacent histologically normal liver tissue, whereas hypermethylation was observed only in tumor samples. The methylation levels of long interspersed nuclear element-1 (LINE-1) did not show large differences between tumor tissue and normal liver controls. Chromosomal abnormalities were also found in some tumors. 11p15.5 and 20q13.3 loci showed the frequent occurrence of both genetic and epigenetic alterations. Our analyses revealed tumor-specific aberrant hypermethylation at some imprinted DMRs in 12 hepatoblastomas with additional suggestion for the possibility of hypomethylation prior to tumor development. Some loci showed both genetic and epigenetic alterations with high frequencies. These findings will aid in understanding the development of hepatoblastoma

Three genetic stocks of frigate tuna Auxis thazard thazard (Lacepede, 1800) along the Indian coast revealed from sequence analyses of mitochondrial DNA D-loop region

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GirishKumar; Kunal, S.P.; Menezes, M.R.; Meena, R.M.

revealed from sequence analyses of mitochondrial DNA D-loop region Name of authors: 1. Girish Kumar* Biological Oceanography Division (BOD) National Institute of Oceanography (NIO) Dona Paula, Goa 403004, India. Email: girishkumar....nio@gmail.com Tel: +919766548060 2. Swaraj Priyaranjan Kunal Biological Oceanography Division (BOD) National Institute of Oceanography (NIO) Dona Paula, Goa 403004, India. Email: swar.mbt@gmail.com 3. Maria Rosalia Menezes Biological Oceanography...

Genome and metagenome enabled analyses reveal new insight into the global biogeography and potential urea utilization in marine Thaumarchaeota.

Science.gov (United States)

Ahlgren, N.; Parada, A. E.; Fuhrman, J. A.

2016-02-01

Marine Thaumarchaea are an abundant, important group of marine microbial communities as they fix carbon, oxidize ammonium, and thus contribute to key N and C cycles in the oceans. From an enrichment culture, we have sequenced the complete genome of a new Thaumarchaeota strain, SPOT01. Analysis of this genome and other Thaumarchaeal genomes contributes new insight into its role in N cycling and clarifies the broader biogeography of marine Thaumarchaeal genera. Phylogenomics of Thaumarchaeota genomes reveal coherent separation into clusters roughly equivalent to the genus level, and SPOT01 represents a new genus of marine Thaumarchaea. Competitive fragment recruitment of globally distributed metagenomes from TARA, Ocean Sampling Day, and those generated from a station off California shows that the SPOT01 genus is often the most abundant genus, especially where total Thaumarchaea are most abundant in the overall community. The SPOT01 genome contains urease genes allowing it to use an alternative form of N. Genomic and metagenomic analysis also reveal that among planktonic genomes and populations, the urease genes in general are more frequently found in members of the SPOT01 genus and another genus dominant in deep waters, thus we predict these two genera contribute most significantly to urea utilization among marine Thaumarchaea. Recruitment also revealed broader biogeographic and ecological patterns of the putative genera. The SPOT01 genus was most abundant at colder temperatures (45 degrees). The genus containing Nitrosopumilus maritimus had the highest temperature range, and the genus containing Candidatus Nitrosopelagicus brevis was typically most abundant at intermediate temperatures and intermediate latitudes ( 35-45 degrees). Together these genome and metagenome enabled analyses provide significant new insight into the ecology and biogeochemical contributions of marine archaea.

Proteomic and physiological analyses reveal the role of exogenous spermidine on cucumber roots in response to Ca(NO3)2 stress.

Science.gov (United States)

Du, Jing; Guo, Shirong; Sun, Jin; Shu, Sheng

2018-05-01

The mechanism of exogenous Spd-induced Ca(NO 3 ) 2 stress tolerance in cucumber was studied by proteomics and physiological analyses. Protein-protein interaction network revealed 13 key proteins involved in Spd-induced Ca(NO 3 ) 2 stress resistance. Ca(NO 3 ) 2 stress is one of the major reasons for secondary salinization that limits cucumber plant development in greenhouse. The conferred protective role of exogenous Spd on cucumber in response to Ca(NO 3 ) 2 stress cues involves changes at the cellular and physiological levels. To investigate the molecular foundation of exogenous Spd in Ca(NO 3 ) 2 stress tolerance, a proteomic approach was performed in our work. After a 9 days period of Ca(NO 3 ) 2 stress and/or exogenous Spd, 71 differential protein spots were confidently identified. The resulting proteins were enriched in seven different categories of biological processes, including protein metabolism, carbohydrate and energy metabolism, ROS homeostasis and stress defense, cell wall related, transcription, others and unknown. Protein metabolism (31.2%), carbohydrate and energy metabolism (15.6%), ROS homeostasis and stress defense (32.5%) were the three largest functional categories in cucumber root and most of them were significantly increased by exogenous Spd. The Spd-responsive protein interaction network revealed 13 key proteins, whose accumulation changes could be critical for Spd-induced resistance; all 13 proteins were upregulated by Spd at transcriptional and protein levels in response to Ca(NO 3 ) 2 stress. Furthermore, accumulation of antioxidant enzymes, non-enzymatic antioxidant and polyamines, along with reduction of H 2 O 2 and MDA, were detected after exogenous Spd application during Ca(NO 3 ) 2 stress. The results of these proteomic and physiological analyses in cucumber root may facilitate a better understanding of the underlying mechanism of Ca(NO 3 ) 2 stress tolerance mediated by exogenous Spd.

TSE strain differentiation in mice by immunohistochemical PrP(Sc) profiles and triplex Western blot.

Science.gov (United States)

van Keulen, Lucien J M; Langeveld, Jan P M; Dolstra, Corry H; Jacobs, Jorg; Bossers, Alex; van Zijderveld, Fred G

2015-10-01

TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of mice infected with the same TSE strain and to variation between observers and laboratories in scoring vacuolation and determining the final incubation period. We investigated the potential of PrP(Sc) immunohistochemistry and triplex Western blotting as possible alternative methods to differentiate between TSE strains. TSE reference strains ME7, 87A/87V, 22A/22C, 79A/79V and 301C/301V were intracerebrally inoculated in RIII or VM inbred mice that differ in their PrP genotype. Immunohistochemical PrP(Sc) profiles were drawn up by scanning light microscopy both on coronal and sagittal sections. On the basis of the localization of PrP(Sc) in the cerebral cortex, hippocampus, and cerebellar cortex and the overall type of PrP(Sc) staining, all TSE strains could be well differentiated from each other through their typical strain dependent characteristics. In addition, Western blot showed that the combination of glycosylation profile and 12B2 epitope content of PrP(Sc) allowed to distinguish between all reference strains except for ME7 and 22A in VM mice. TSE strains in mice can be identified on the basis of their PrP(Sc) profile alone. The potential to identify TSE strains in ruminants with these PrP(Sc) profiles after a single primary passage in mice will be the topic of future studies. © 2014 British Neuropathological Society.

Comparative Analyses of Nonpathogenic, Opportunistic, and Totally Pathogenic Mycobacteria Reveal Genomic and Biochemical Variabilities and Highlight the Survival Attributes of Mycobacterium tuberculosis

Science.gov (United States)

Singh, Yadvir; Kohli, Sakshi; Ahmad, Javeed; Ehtesham, Nasreen Z.; Tyagi, Anil K.

2014-01-01

ABSTRACT Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size—their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. PMID:25370496

Bliss and Loewe interaction analyses of clinically relevant drug combinations in human colon cancer cell lines reveal complex patterns of synergy and antagonism.

Science.gov (United States)

Kashif, Muhammad; Andersson, Claes; Mansoori, Sharmineh; Larsson, Rolf; Nygren, Peter; Gustafsson, Mats G

2017-11-28

We analyzed survival effects for 15 different pairs of clinically relevant anti-cancer drugs in three iso-genic pairs of human colorectal cancer carcinoma cell lines, by applying for the first time our novel software (R package) called COMBIA. In our experiments iso-genic pairs of cell lines were used, differing only with respect to a single clinically important KRAS or BRAF mutation. Frequently, concentration dependent but mutation independent joint Bliss and Loewe synergy/antagonism was found statistically significant. Four combinations were found synergistic/antagonistic specifically to the parental (harboring KRAS or BRAF mutation) cell line of the corresponding iso-genic cell lines pair. COMBIA offers considerable improvements over established software for synergy analysis such as MacSynergy™ II as it includes both Bliss (independence) and Loewe (additivity) analyses, together with a tailored non-parametric statistical analysis employing heteroscedasticity, controlled resampling, and global (omnibus) testing. In many cases Loewe analyses found significant synergistic as well as antagonistic effects in a cell line at different concentrations of a tested drug combination. By contrast, Bliss analysis found only one type of significant effect per cell line. In conclusion, the integrated Bliss and Loewe interaction analysis based on non-parametric statistics may provide more robust interaction analyses and reveal complex patterns of synergy and antagonism.

Electron Microscopic, Genetic and Protein Expression Analyses of Helicobacter acinonychis Strains from a Bengal Tiger

Science.gov (United States)

Tegtmeyer, Nicole; Rivas Traverso, Francisco; Rohde, Manfred; Oyarzabal, Omar A.; Lehn, Norbert; Schneider-Brachert, Wulf; Ferrero, Richard L.; Fox, James G.; Berg, Douglas E.; Backert, Steffen

2013-01-01

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5–6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections. PMID:23940723

Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger.

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Nicole Tegtmeyer

Full Text Available Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5-6 µm and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.

Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger.

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Tegtmeyer, Nicole; Rivas Traverso, Francisco; Rohde, Manfred; Oyarzabal, Omar A; Lehn, Norbert; Schneider-Brachert, Wulf; Ferrero, Richard L; Fox, James G; Berg, Douglas E; Backert, Steffen

2013-01-01

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5-6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.

Prediction of the optimum hybridization conditions of dot-blot-SNP analysis using estimated melting temperature of oligonucleotide probes.

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Shiokai, Sachiko; Kitashiba, Hiroyasu; Nishio, Takeshi

2010-08-01

Although the dot-blot-SNP technique is a simple cost-saving technique suitable for genotyping of many plant individuals, optimization of hybridization and washing conditions for each SNP marker requires much time and labor. For prediction of the optimum hybridization conditions for each probe, we compared T (m) values estimated from nucleotide sequences using the DINAMelt web server, measured T (m) values, and hybridization conditions yielding allele-specific signals. The estimated T (m) values were comparable to the measured T (m) values with small differences of less than 3 degrees C for most of the probes. There were differences of approximately 14 degrees C between the specific signal detection conditions and estimated T (m) values. Change of one level of SSC concentrations of 0.1, 0.2, 0.5, and 1.0x SSC corresponded to a difference of approximately 5 degrees C in optimum signal detection temperature. Increasing the sensitivity of signal detection by shortening the exposure time to X-ray film changed the optimum hybridization condition for specific signal detection. Addition of competitive oligonucleotides to the hybridization mixture increased the suitable hybridization conditions by 1.8. Based on these results, optimum hybridization conditions for newly produced dot-blot-SNP markers will become predictable.

ATHENA/INTRA analyses for ITER, NSSR-2

International Nuclear Information System (INIS)

Shen, Kecheng; Eriksson, John; Sjoeberg, A.

1999-02-01

The present report is a summary report including thermal-hydraulic analyses made at Studsvik Eco and Safety AB for the ITER NSSR-2 safety documentation. The objective of the analyses was to reveal the safety characteristics of various heat transfer systems at specified operating conditions and to indicate the conditions for which there were obvious risks of jeopardising the structural integrity of the coolant systems. In the latter case also some analyses were made to indicate conceivable mitigating measures for maintaining the integrity.The analyses were primarily concerned with the First Wall and Divertor heat transfer systems. Several enveloping transients were analysed with associated specific flow and heat load boundary conditions. The analyses were performed with the ATHENA and INTRA codes

ATHENA/INTRA analyses for ITER, NSSR-2

Energy Technology Data Exchange (ETDEWEB)

Shen, Kecheng; Eriksson, John; Sjoeberg, A

1999-02-01

The present report is a summary report including thermal-hydraulic analyses made at Studsvik Eco and Safety AB for the ITER NSSR-2 safety documentation. The objective of the analyses was to reveal the safety characteristics of various heat transfer systems at specified operating conditions and to indicate the conditions for which there were obvious risks of jeopardising the structural integrity of the coolant systems. In the latter case also some analyses were made to indicate conceivable mitigating measures for maintaining the integrity.The analyses were primarily concerned with the First Wall and Divertor heat transfer systems. Several enveloping transients were analysed with associated specific flow and heat load boundary conditions. The analyses were performed with the ATHENA and INTRA codes 8 refs, 14 figs, 15 tabs

Underestimation of species richness in neotropical frogs revealed by mtDNA analyses.

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Antoine Fouquet

2007-10-01

Full Text Available Amphibians are rapidly vanishing. At the same time, it is most likely that the number of amphibian species is highly underestimated. Recent DNA barcoding work has attempted to define a threshold between intra- and inter-specific genetic distances to help identify candidate species. In groups with high extinction rates and poorly known species boundaries, like amphibians, such tools may provide a way to rapidly evaluate species richness.Here we analyse published and new 16S rDNA sequences from 60 frog species of Amazonia-Guianas to obtain a minimum estimate of the number of undescribed species in this region. We combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs.In most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contrary to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species.Using this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia

Metabarcoding and metabolome analyses of copepod grazing reveal feeding preference and linkage to metabolite classes in dynamic microbial plankton communities.

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Ray, Jessica L; Althammer, Julia; Skaar, Katrine S; Simonelli, Paolo; Larsen, Aud; Stoecker, Diane; Sazhin, Andrey; Ijaz, Umer Z; Quince, Christopher; Nejstgaard, Jens C; Frischer, Marc; Pohnert, Georg; Troedsson, Christofer

2016-11-01

In order to characterize copepod feeding in relation to microbial plankton community dynamics, we combined metabarcoding and metabolome analyses during a 22-day seawater mesocosm experiment. Nutrient amendment of mesocosms promoted the development of haptophyte (Phaeocystis pouchetii)- and diatom (Skeletonema marinoi)-dominated plankton communities in mesocosms, in which Calanus sp. copepods were incubated for 24 h in flow-through chambers to allow access to prey particles (<500 μm). Copepods and mesocosm water sampled six times spanning the experiment were analysed using metabarcoding, while intracellular metabolite profiles of mesocosm plankton communities were generated for all experimental days. Taxon-specific metabarcoding ratios (ratio of consumed prey to available prey in the surrounding seawater) revealed diverse and dynamic copepod feeding selection, with positive selection on large diatoms, heterotrophic nanoflagellates and fungi, while smaller phytoplankton, including P. pouchetii, were passively consumed or even negatively selected according to our indicator. Our analysis of the relationship between Calanus grazing ratios and intracellular metabolite profiles indicates the importance of carbohydrates and lipids in plankton succession and copepod-prey interactions. This molecular characterization of Calanus sp. grazing therefore provides new evidence for selective feeding in mixed plankton assemblages and corroborates previous findings that copepod grazing may be coupled to the developmental and metabolic stage of the entire prey community rather than to individual prey abundances. © 2016 John Wiley & Sons Ltd.

The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

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Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

2012-06-01

With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods

Contribution of dot-blot assay to the diagnosis and management of myositis: a three-year practice at a university hospital centre.

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Martel, Clothilde; Vignaud, Guillaume; Liozon, Eric; Magy, Laurent; Gallouedec, Gael; Ly, Kim; Bezanahary, Holly; Cypierre, Anne; Lapébie, François-Xavier; Palat, Sylvain; Gondran, Guillaume; Jauberteau, Marie-Odile; Fauchais, Anne-Laure

2016-01-01

Idiopathic inflammatory myopathies (IIM) are heterogeneous autoimmune diseases with wide clinical spectrum that may lead to delayed diagnosis. The aim of this study was to examine the impact of IIM-specific dot-blot assay on diagnostic process of patients presenting with muscular or systemic symptoms evocating of IIM. We collected all the prescriptions of an IIM specific dot-blot assay (8 autoantigens including Jo-1, PL-7, PL-12, SRP, Mi-2, Ku, PM/Scl and Scl-70) over a 38-month period. 316 myositis dot-blot assays (MSD) were performed in 274 patients (156 women, mean age 53±10.6 years) referring for muscular and/or systemic symptoms suggesting IIM. The timing of dot prescription through the diagnostic process was highly variable: without (35%), concomitantly (16%) or after electromyographic studies (35%). Fifty-nine patients (22%) had IIM according to Bohan and Peter's criteria. Among them, 29 (49%) had positive dot (8 Jo-1, 6 PM-Scl, 5 PL-12, 5 SRP, 2 Mi-2, 2 PL-7 and 1 Ku). Various other diagnoses were performed including 35 autoimmune disease or granulomatosis (12%), 19 inflammatory rheumatic disease (7%), 16 non inflammatory muscular disorders (6%), 10 drug-induced myalgia (4%), 11 infectious myositis (4%). Except 11 borderline SRP results and one transient PM-Scl, MSD was positive only in one case of IIM. Dot allowed clinicians to correct diagnosis in 4 cases and improved the diagnosis of IIM subtypes in 4 cases. This study reflects the interest of myositis dot in the rapid diagnosis process of patients with non-specific muscular symptoms leading to various diagnoses including IIM.

Herpes simplex virus type 1 strain KOS carries a defective US9 and a mutated US8A gene.

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Negatsch, Alexandra; Mettenleiter, Thomas C; Fuchs, Walter

2011-01-01

The membrane protein encoded by the US9 gene of alphaherpesviruses plays an important role during virion assembly and transport in neurons. Here, we demonstrate that in herpes simplex virus type 1 (HSV-1) strain KOS, due to base substitutions, the predicted TATA-box of US9 is mutated, and a premature stop is present at codon 58 of US9, which contains 91 codons in other HSV-1 strains. The TATA-box mutation also removes the native stop codon of the adjacent US8A gene, leading to extension of the coding region from 160 to 191 codons. Northern blot analyses revealed reduced transcription of US9 in cells infected with HSV-1 KOS. Moreover, a US9-specific antiserum did not detect any gene products in Western blot and immunofluorescence analyses of KOS-infected cells, indicating that the truncated protein is not stable. In contrast, Western blot reactions of a pUS8A-specific antiserum confirmed enlargement of this protein in HSV-1 KOS.

Proteomic analysis of pig (Sus scrofa olfactory soluble proteome reveals O-GlcNAcylation of secreted odorant-binding proteins

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Patricia eNAGNAN-LE MEILLOUR

2014-12-01

Full Text Available The diversity of olfactory binding proteins (OBPs is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

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Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

2016-01-01

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

Comparison of Multispot EIA with Western blot for confirmatory serodiagnosis of HIV.

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Torian, Lucia V; Forgione, Lisa A; Punsalang, Amado E; Pirillo, Robert E; Oleszko, William R

2011-12-01

Recent improvements in the sensitivity of immunoassays (IA) used for HIV screening, coupled with increasing recognition of the importance of rapid point-of-care testing, have led to proposals to adjust the algorithm for serodiagnosis of HIV so that screening and confirmation can be performed using a dual or triple IA sequence that does not require Western blotting for confirmation. One IA that has been proposed as a second or confirmatory test is the Bio-Rad Multispot(®) Rapid HIV-1/HIV-2 Test. This test would have the added advantage of differentiating between HIV-1 and HIV-2 antibodies. To compare the sensitivity and type-specificity of an algorithm combining a 3rd generation enzyme immunoassay (EIA) followed by a confirmatory Multispot with the conventional algorithm that combines a 3rd generation EIA (Bio-Rad GS HIV-1/HIV-2 Plus O EIA) followed by confirmatory Western blot (Bio-Rad GS HIV-1 WB). 8760 serum specimens submitted for HIV testing to the New York City Public Health Laboratory between May 22, 2007, and April 30, 2010, tested repeatedly positive on 3rd generation HIV-1-2+O EIA screening and received parallel confirmatory testing by WB and Multispot (MS). 8678/8760 (99.1%) specimens tested WB-positive; 82 (0.9%) tested WB-negative or indeterminate (IND). 8690/8760 specimens (99.2%) tested MS-positive, of which 14 (17.1%) had been classified as negative or IND by WB. Among the HIV-1 WB-positive specimens, MS classified 26 (0.29%) as HIV-2. Among the HIV-1 WB negative and IND, MS detected 12 HIV-2. MS detected an additional 14 HIV-1 infections among WB negative or IND specimens, differentiated 26 HIV-1 WB positives as HIV-2, and detected 12 additional HIV-2 infections among WB negative/IND. A dual 3rd generation EIA algorithm incorporating MS had equivalent HIV-1 sensitivity to the 3rd generation EIA-WB algorithm and had the added advantage of detecting 12 HIV-2 specimens that were not HIV-1 WB cross-reactors. In this series an algorithm using EIA

HIV-1/2 indeterminate Western blot results: follow-up of asymptomatic blood donors in Belo Horizonte, Minas Gerais, Brazil

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CARNEIRO-PROIETTI A.B.F.

1999-01-01

Full Text Available The clinical and public health importance of indeterminate results in HIV-1/2 testing is still difficult to evaluate in volunteer blood donors. At Fundação Hemominas, HIV-1/2 ELISA is used as the screening test and, if reactive, is followed by Western blot (WB. We have evaluated 84 blood donors who had repeatedly reactive ELISA tests for HIV-1/2, but indeterminate WB results. Sixteen of the 84 donors (19.0% had history of sexually transmitted diseases; 18/84 (21.4% informed receiving or paying for sex; 3/84 (3.6% had homosexual contact; 2/26 women (7.6% had past history of multiple illegal abortions and 3/84 (3.6% had been previously transfused. Four out of 62 donors (6.5% had positive anti-nuclear factor (Hep2, with titles up to 1:640. Parasitological examination of the stool revealed eggs of S. mansoni in 4/62 (6.4% donors and other parasites in 8/62 (12.9%. Five (5.9% of the subjects presented overt seroconversion for HIV-1/2, 43/84 (51.2% had negative results on the last visit, while 36/84 (42.9% remained WB indeterminate. Although some conditions could be found associated with the HIV-1/2 indeterminate WB results and many donors had past of risky behavior, the significance of the majority of the results remains to be determined.

Differential staining of Western blots of human secreted glycoproteins from serum, milk, saliva, and seminal fluid using lectins displaying diverse sugar specificities.

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Gilboa-Garber, Nechama; Lerrer, Batya; Lesman-Movshovich, Efrat; Dgani, Orly

2005-12-01

Human milk, serum, saliva, and seminal fluid glycoproteins (gps) nourish and protect newborn and adult tissues. Their saccharides, which resemble cell membrane components, may block pathogen adhesion and infection. In the present study, they were examined by a battery of lectins from plants, animals, and bacteria, using hemagglutination inhibition and Western blot analyses. The lectins included galactophilic ones from Aplysia gonad, Erythrina corallodendron, Maclura pomifera (MPL), peanut, and Pseudomonas aeruginosa (PA-IL); fucose-binding lectins from Pseudomonas aeruginosa (PA-IIL), Ralstonia solanacearum (RSL), and Ulex europaeus (UEA-I), and mannose/glucose-binding Con A. The results demonstrated the chosen lectin efficiency for differential analysis of human secreted gps as compared to CBB staining. They unveiled the diversity of these body fluid gp glycans (those of the milk and seminal fluid being highest): the milk gps interacted most strongly with PA-IIL, followed by RSL; the saliva gps with RSL, followed by PA-IIL and MPL; the serum gps with Con A and MPL, followed by PA-IIL and RSL, and the seminal plasma gps with RSL and MPL, followed by UEA-I and PA-IIL. The potential usage of these lectins as probes for scientific, industrial, and medical purposes, and for quality control of the desired gps is clearly indicated.

Mitochondrial DNA analyses revealed low genetic diversity in the endangered Indian wild ass Equus hemionus khur.

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Khaire, Devendra; Atkulwar, Ashwin; Farah, Sameera; Baig, Mumtaz

2017-09-01

The Indian wild ass Equus hemionus khur, belonging to ass-like equid branch, inhabits the dry and arid desert of the Little Rann of Kutch, Gujarat. The E. h. khur is the sole survivor of Asiatic wild ass species/subspecies in South Asia. To provide first ever insights into the genetic diversity, phylogeny, and demography of the endangered Indian wild ass, we sampled 52 free-ranging individuals from the Little Rann of Kutch by using a non-invasive methodology. The sequencing of 230 bp in cytochrome b (Cyt b) and displacement loop (D-loop) region revealed that current ∼4000 extant population of Indian wild ass harbours low genetic diversity. Phylogenetic analyses confirmed that E. h. khur, E. h. onager, and E. h. kulan belong to a single strict monophyletic clade. Therefore, we suggest the delimitation of the five E. hemionus subspecies in vogue to a single species E. hemionus. The application of molecular clock confirmed that the Asiatic wild ass had undergone diversification 0.65 Million years ago. Demographic measurements assessed using a Bayesian skyline plot demonstrated decline in the maternal effective population size of the Indian wild ass during different periods; these periods coincided with the origin and rise of the Indus civilization in the northwest of the Indian subcontinent during the Neolithic. In conclusion, maintaining high genetic diversity in the existing isolated population of 4000 Indian wild asses inhabiting the wild ass sanctuary is important compared with subspecies preservation alone.

Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes.

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Ball, Robyn L; Fujiwara, Yasuhiro; Sun, Fengyun; Hu, Jianjun; Hibbs, Matthew A; Handel, Mary Ann; Carter, Gregory W

2016-08-12

The continuous and non-synchronous nature of postnatal male germ-cell development has impeded stage-specific resolution of molecular events of mammalian meiotic prophase in the testis. Here the juvenile onset of spermatogenesis in mice is analyzed by combining cytological and transcriptomic data in a novel computational analysis that allows decomposition of the transcriptional programs of spermatogonia and meiotic prophase substages. Germ cells from testes of individual mice were obtained at two-day intervals from 8 to 18 days post-partum (dpp), prepared as surface-spread chromatin and immunolabeled for meiotic stage-specific protein markers (STRA8, SYCP3, phosphorylated H2AFX, and HISTH1T). Eight stages were discriminated cytologically by combinatorial antibody labeling, and RNA-seq was performed on the same samples. Independent principal component analyses of cytological and transcriptomic data yielded similar patterns for both data types, providing strong evidence for substage-specific gene expression signatures. A novel permutation-based maximum covariance analysis (PMCA) was developed to map co-expressed transcripts to one or more of the eight meiotic prophase substages, thereby linking distinct molecular programs to cytologically defined cell states. Expression of meiosis-specific genes is not substage-limited, suggesting regulation of substage transitions at other levels. This integrated analysis provides a general method for resolving complex cell populations. Here it revealed not only features of meiotic substage-specific gene expression, but also a network of substage-specific transcription factors and relationships to potential target genes.

Standardization of micro-enzyme-linked immunosorbent assay (ELISA) and Western blot for detection of Trypanosoma cruzi antibodies using extracts from Mexican strains as antigens.

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Sánchez, B; Monteón, V; Reyes, P A; Espinoza, B

2001-01-01

This report describes two assays for the detection of anti-Trypanosoma cruzi antibodies using Mexican strains of the parasite and the concordance with two assays previously evaluated at the Instituto Nacional de Cardiología Ignacio Chávez in Mexico City. Micro-enzyme-linked immunosorbent assay (ELISA) and Western blot were used for the detection of T. cruzi antibodies with a total extract of epimastigote from Ninoa and Queretaro, which are Mexican strains of T. cruzi. To standardize these methods, a total of 246 serum samples was used. In addition, sera from six confirmed Mexican chronic individuals in the asymptomatic phase were also used for comparison with the Argentinean antigen. ELISA was 100% specific in that no false positive results were found with sera of both healthy individuals and non-Chagasic cardiopaths. Sera from individuals infected with Leishmania sp. showed approximately 16% of cross-reaction with ELISA. The test showed a positive predictive value of 90% and a negative predictive value of 100%. Western blot was also a highly sensitive test for detecting chronic Chagasic symptomatic patients from Mexico because no false negative results were obtained. Furthermore, it was possible to use Western blot to detect seven immunodominant antigens of approximately 30, 32, 40, 42, 65, 70, and 83 kDa. Concordance with two previous standardized tests at the Instituto Nacional de Cardiología showed a Kappa index of 0.96, indicating high concordance between the results obtained at these two laboratories. Finally, ELISA using Ninoa antigen extract was more sensitive than ELISA with an Argentinean extract, which failed to detect individuals in the chronic asymptomatic phase (undetermined phase) of infection. This study indicates that ELISA and Western blot using Ninoa and/or Queretaro extracts of T. cruzi as antigens are useful tools in the detection of individuals who have been exposed to T. cruzi both in the undetermined/asymptomatic and symptomatic phases

Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53.

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Kristina Kirschner

2015-03-01

Full Text Available The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage and chronically activated (in senescent or pro-apoptotic conditions p53. Compared to the classical 'acute' p53 binding profile, 'chronic' p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory 'p53 hubs' where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the 'lipogenic phenotype', a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.

Big data analyses reveal patterns and drivers of the movements of southern elephant seals

KAUST Repository

Rodrí guez, J. P.; Ferná ndez-Gracia, Juan; Thums, Michele; Hindell, Mark A.; Sequeira, Ana M. M.; Meekan, Mark G.; Costa, Daniel P.; Guinet, Christophe; Harcourt, R.; McMahon, Clive R.; Muelbert, Monica; Duarte, Carlos M.; Eguí luz, V. M.

2017-01-01

The growing number of large databases of animal tracking provides an opportunity for analyses of movement patterns at the scales of populations and even species. We used analytical approaches, developed to cope with

Big data analyses reveal patterns and drivers of the movements of southern elephant seals

KAUST Repository

Rodríguez, Jorge P.

2017-03-02

The growing number of large databases of animal tracking provides an opportunity for analyses of movement patterns at the scales of populations and even species. We used analytical approaches, developed to cope with

Comparative physiological, metabolomic, and transcriptomic analyses reveal mechanisms of improved abiotic stress resistance in bermudagrass [Cynodon dactylon (L). Pers.] by exogenous melatonin

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Shi, Haitao; Jiang, Chuan; Ye, Tiantian; Tan, Dun-xian; Reiter, Russel J.; Zhang, Heng; Liu, Renyi; Chan, Zhulong

2015-01-01

Melatonin (N-acetyl-5-methoxytryptamine), a well-known animal hormone, is also involved in plant development and abiotic stress responses. In this study, it is shown that exogenous application of melatonin conferred improved salt, drought, and cold stress resistances in bermudagrass. Moreover, exogenous melatonin treatment alleviated reactive oxygen species (ROS) burst and cell damage induced by abiotic stress; this involved activation of several antioxidants. Additionally, melatonin-pre-treated plants exhibited higher concentrations of 54 metabolites, including amino acids, organic acids, sugars, and sugar alcohols, than non-treated plants under abiotic stress conditions. Genome-wide transcriptomic profiling identified 3933 transcripts (2361 up-regulated and 1572 down-regulated) that were differentially expressed in melatonin-treated plants versus controls. Pathway and gene ontology (GO) term enrichment analyses revealed that genes involved in nitrogen metabolism, major carbohydrate metabolism, tricarboxylic acid (TCA)/org transformation, transport, hormone metabolism, metal handling, redox, and secondary metabolism were over-represented after melatonin pre-treatment. Taken together, this study provides the first evidence of the protective roles of exogenous melatonin in the bermudagrass response to abiotic stresses, partially via activation of antioxidants and modulation of metabolic homeostasis. Notably, metabolic and transcriptomic analyses showed that the underlying mechanisms of melatonin could involve major reorientation of photorespiratory and carbohydrate and nitrogen metabolism. PMID:25225478

Integrative functional analyses using rainbow trout selected for tolerance to plant diets reveal nutrigenomic signatures for soy utilization without the concurrence of enteritis.

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Jason Abernathy

Full Text Available Finding suitable alternative protein sources for diets of carnivorous fish species remains a major concern for sustainable aquaculture. Through genetic selection, we created a strain of rainbow trout that outperforms parental lines in utilizing an all-plant protein diet and does not develop enteritis in the distal intestine, as is typical with salmonids on long-term plant protein-based feeds. By incorporating this strain into functional analyses, we set out to determine which genes are critical to plant protein utilization in the absence of gut inflammation. After a 12-week feeding trial with our selected strain and a control trout strain fed either a fishmeal-based diet or an all-plant protein diet, high-throughput RNA sequencing was completed on both liver and muscle tissues. Differential gene expression analyses, weighted correlation network analyses and further functional characterization were performed. A strain-by-diet design revealed differential expression ranging from a few dozen to over one thousand genes among the various comparisons and tissues. Major gene ontology groups identified between comparisons included those encompassing central, intermediary and foreign molecule metabolism, associated biosynthetic pathways as well as immunity. A systems approach indicated that genes involved in purine metabolism were highly perturbed. Systems analysis among the tissues tested further suggests the interplay between selection for growth, dietary utilization and protein tolerance may also have implications for nonspecific immunity. By combining data from differential gene expression and co-expression networks using selected trout, along with ontology and pathway analyses, a set of 63 candidate genes for plant diet tolerance was found. Risk loci in human inflammatory bowel diseases were also found in our datasets, indicating rainbow trout selected for plant-diet tolerance may have added utility as a potential biomedical model.

RNA-seq analyses reveal insights into the function of respiratory nitrate reductase of the diazotroph Herbaspirillum seropedicae.

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Bonato, Paloma; Batista, Marcelo B; Camilios-Neto, Doumit; Pankievicz, Vânia C S; Tadra-Sfeir, Michelle Z; Monteiro, Rose Adele; Pedrosa, Fabio O; Souza, Emanuel M; Chubatsu, Leda S; Wassem, Roseli; Rigo, Liu Un

2016-09-01

Herbaspirillum seropedicae is a nitrogen-fixing β-proteobacterium that associates with roots of gramineous plants. In silico analyses revealed that H. seropedicae genome has genes encoding a putative respiratory (NAR) and an assimilatory nitrate reductase (NAS). To date, little is known about nitrate metabolism in H. seropedicae, and, as this bacterium cannot respire nitrate, the function of NAR remains unknown. This study aimed to investigate the function of NAR in H. seropedicae and how it metabolizes nitrate in a low aerated-condition. RNA-seq transcriptional profiling in the presence of nitrate allowed us to pinpoint genes important for nitrate metabolism in H. seropedicae, including nitrate transporters and regulatory proteins. Additionally, both RNA-seq data and physiological characterization of a mutant in the catalytic subunit of NAR (narG mutant) showed that NAR is not required for nitrate assimilation but is required for: (i) production of high levels of nitrite, (ii) production of NO and (iii) dissipation of redox power, which in turn lead to an increase in carbon consumption. In addition, wheat plants showed an increase in shoot dry weight only when inoculated with H. seropedicae wild type, but not with the narG mutant, suggesting that NAR is important to H. seropedicae-wheat interaction. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

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A Rasul

2013-06-01

Full Text Available Background: The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp. in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan.Methods: Reverse line blot (RLB assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products.Results: Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa.Conclusion: Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001. Risk factor analysis revealed that male (P = 0.03, ani­mals infested by ticks (P = 0.03 and herd composed of sheep only (P = 0.001 were more infected by blood parasites.

DNA sequence analyses reveal abundant diversity, endemism and evidence for Asian origin of the porcini mushrooms.

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Bang Feng

Full Text Available The wild gourmet mushroom Boletus edulis and its close allies are of significant ecological and economic importance. They are found throughout the Northern Hemisphere, but despite their ubiquity there are still many unresolved issues with regard to the taxonomy, systematics and biogeography of this group of mushrooms. Most phylogenetic studies of Boletus so far have characterized samples from North America and Europe and little information is available on samples from other areas, including the ecologically and geographically diverse regions of China. Here we analyzed DNA sequence variation in three gene markers from samples of these mushrooms from across China and compared our findings with those from other representative regions. Our results revealed fifteen novel phylogenetic species (about one-third of the known species and a newly identified lineage represented by Boletus sp. HKAS71346 from tropical Asia. The phylogenetic analyses support eastern Asia as the center of diversity for the porcini sensu stricto clade. Within this clade, B. edulis is the only known holarctic species. The majority of the other phylogenetic species are geographically restricted in their distributions. Furthermore, molecular dating and geological evidence suggest that this group of mushrooms originated during the Eocene in eastern Asia, followed by dispersal to and subsequent speciation in other parts of Asia, Europe, and the Americas from the middle Miocene through the early Pliocene. In contrast to the ancient dispersal of porcini in the strict sense in the Northern Hemisphere, the occurrence of B. reticulatus and B. edulis sensu lato in the Southern Hemisphere was probably due to recent human-mediated introductions.

DNA Sequence Analyses Reveal Abundant Diversity, Endemism and Evidence for Asian Origin of the Porcini Mushrooms

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Feng, Bang; Xu, Jianping; Wu, Gang; Zeng, Nian-Kai; Li, Yan-Chun; Tolgor, Bau; Kost, Gerhard W.; Yang, Zhu L.

2012-01-01

The wild gourmet mushroom Boletus edulis and its close allies are of significant ecological and economic importance. They are found throughout the Northern Hemisphere, but despite their ubiquity there are still many unresolved issues with regard to the taxonomy, systematics and biogeography of this group of mushrooms. Most phylogenetic studies of Boletus so far have characterized samples from North America and Europe and little information is available on samples from other areas, including the ecologically and geographically diverse regions of China. Here we analyzed DNA sequence variation in three gene markers from samples of these mushrooms from across China and compared our findings with those from other representative regions. Our results revealed fifteen novel phylogenetic species (about one-third of the known species) and a newly identified lineage represented by Boletus sp. HKAS71346 from tropical Asia. The phylogenetic analyses support eastern Asia as the center of diversity for the porcini sensu stricto clade. Within this clade, B. edulis is the only known holarctic species. The majority of the other phylogenetic species are geographically restricted in their distributions. Furthermore, molecular dating and geological evidence suggest that this group of mushrooms originated during the Eocene in eastern Asia, followed by dispersal to and subsequent speciation in other parts of Asia, Europe, and the Americas from the middle Miocene through the early Pliocene. In contrast to the ancient dispersal of porcini in the strict sense in the Northern Hemisphere, the occurrence of B. reticulatus and B. edulis sensu lato in the Southern Hemisphere was probably due to recent human-mediated introductions. PMID:22629418

The 'revealed preferences' theory: Assumptions and conjectures

International Nuclear Information System (INIS)

Green, C.H.

1983-01-01

Being kind of intuitive psychology the 'Revealed-Preferences'- theory based approaches towards determining the acceptable risks are a useful method for the generation of hypotheses. In view of the fact that reliability engineering develops faster than methods for the determination of reliability aims the Revealed-Preferences approach is a necessary preliminary help. Some of the assumptions on which the 'Revealed-Preferences' theory is based will be identified and analysed and afterwards compared with experimentally obtained results. (orig./DG) [de

Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency

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Cacchiarelli, Davide; Trapnell, Cole; Ziller, Michael J.; Soumillon, Magali; Cesana, Marcella; Karnik, Rahul; Donaghey, Julie; Smith, Zachary D.; Ratanasirintrawoot, Sutheera; Zhang, Xiaolan; Ho Sui, Shannan J.; Wu, Zhaoting; Akopian, Veronika; Gifford, Casey A.; Doench, John; Rinn, John L.; Daley, George Q.; Meissner, Alexander; Lander, Eric S.; Mikkelsen, Tarjei S.

2015-01-01

Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PMID:26186193

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

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Yongbin Dong

Full Text Available The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

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Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

2012-11-02

HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

Comparative physiological, metabolomic, and transcriptomic analyses reveal mechanisms of improved abiotic stress resistance in bermudagrass [Cynodon dactylon (L). Pers.] by exogenous melatonin.

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Shi, Haitao; Jiang, Chuan; Ye, Tiantian; Tan, Dun-Xian; Reiter, Russel J; Zhang, Heng; Liu, Renyi; Chan, Zhulong

2015-02-01

Melatonin (N-acetyl-5-methoxytryptamine), a well-known animal hormone, is also involved in plant development and abiotic stress responses. In this study, it is shown that exogenous application of melatonin conferred improved salt, drought, and cold stress resistances in bermudagrass. Moreover, exogenous melatonin treatment alleviated reactive oxygen species (ROS) burst and cell damage induced by abiotic stress; this involved activation of several antioxidants. Additionally, melatonin-pre-treated plants exhibited higher concentrations of 54 metabolites, including amino acids, organic acids, sugars, and sugar alcohols, than non-treated plants under abiotic stress conditions. Genome-wide transcriptomic profiling identified 3933 transcripts (2361 up-regulated and 1572 down-regulated) that were differentially expressed in melatonin-treated plants versus controls. Pathway and gene ontology (GO) term enrichment analyses revealed that genes involved in nitrogen metabolism, major carbohydrate metabolism, tricarboxylic acid (TCA)/org transformation, transport, hormone metabolism, metal handling, redox, and secondary metabolism were over-represented after melatonin pre-treatment. Taken together, this study provides the first evidence of the protective roles of exogenous melatonin in the bermudagrass response to abiotic stresses, partially via activation of antioxidants and modulation of metabolic homeostasis. Notably, metabolic and transcriptomic analyses showed that the underlying mechanisms of melatonin could involve major reorientation of photorespiratory and carbohydrate and nitrogen metabolism. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Comparative analyses of population-scale phenomic data in electronic medical records reveal race-specific disease networks

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Glicksberg, Benjamin S.; Li, Li; Badgeley, Marcus A.; Shameer, Khader; Kosoy, Roman; Beckmann, Noam D.; Pho, Nam; Hakenberg, Jörg; Ma, Meng; Ayers, Kristin L.; Hoffman, Gabriel E.; Dan Li, Shuyu; Schadt, Eric E.; Patel, Chirag J.; Chen, Rong; Dudley, Joel T.

2016-01-01

Motivation: Underrepresentation of racial groups represents an important challenge and major gap in phenomics research. Most of the current human phenomics research is based primarily on European populations; hence it is an important challenge to expand it to consider other population groups. One approach is to utilize data from EMR databases that contain patient data from diverse demographics and ancestries. The implications of this racial underrepresentation of data can be profound regarding effects on the healthcare delivery and actionability. To the best of our knowledge, our work is the first attempt to perform comparative, population-scale analyses of disease networks across three different populations, namely Caucasian (EA), African American (AA) and Hispanic/Latino (HL). Results: We compared susceptibility profiles and temporal connectivity patterns for 1988 diseases and 37 282 disease pairs represented in a clinical population of 1 025 573 patients. Accordingly, we revealed appreciable differences in disease susceptibility, temporal patterns, network structure and underlying disease connections between EA, AA and HL populations. We found 2158 significantly comorbid diseases for the EA cohort, 3265 for AA and 672 for HL. We further outlined key disease pair associations unique to each population as well as categorical enrichments of these pairs. Finally, we identified 51 key ‘hub’ diseases that are the focal points in the race-centric networks and of particular clinical importance. Incorporating race-specific disease comorbidity patterns will produce a more accurate and complete picture of the disease landscape overall and could support more precise understanding of disease relationships and patient management towards improved clinical outcomes. Contacts: rong.chen@mssm.edu or joel.dudley@mssm.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307606

Exploratory Metabolomic Analyses Reveal Compounds Correlated with Lutein Concentration in Frontal Cortex, Hippocampus, and Occipital Cortex of Human Infant Brain.

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Jacqueline C Lieblein-Boff

Full Text Available Lutein is a dietary carotenoid well known for its role as an antioxidant in the macula, and recent reports implicate a role for lutein in cognitive function. Lutein is the dominant carotenoid in both pediatric and geriatric brain tissue. In addition, cognitive function in older adults correlated with macular and postmortem brain lutein concentrations. Furthermore, lutein was found to preferentially accumulate in the infant brain in comparison to other carotenoids that are predominant in diet. While lutein is consistently related to cognitive function, the mechanisms by which lutein may influence cognition are not clear. In an effort to identify potential mechanisms through which lutein might influence neurodevelopment, an exploratory study relating metabolite signatures and lutein was completed. Post-mortem metabolomic analyses were performed on human infant brain tissues in three regions important for learning and memory: the frontal cortex, hippocampus, and occipital cortex. Metabolomic profiles were compared to lutein concentration, and correlations were identified and reported here. A total of 1276 correlations were carried out across all brain regions. Of 427 metabolites analyzed, 257 were metabolites of known identity. Unidentified metabolite correlations (510 were excluded. In addition, moderate correlations with xenobiotic relationships (2 or those driven by single outliers (3 were excluded from further study. Lutein concentrations correlated with lipid pathway metabolites, energy pathway metabolites, brain osmolytes, amino acid neurotransmitters, and the antioxidant homocarnosine. These correlations were often brain region-specific. Revealing relationships between lutein and metabolic pathways may help identify potential candidates on which to complete further analyses and may shed light on important roles of lutein in the human brain during development.

Comparative Genome Analyses of Serratia marcescens FS14 Reveals Its High Antagonistic Potential

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Li, Pengpeng; Kwok, Amy H. Y.; Jiang, Jingwei; Ran, Tingting; Xu, Dongqing; Wang, Weiwu; Leung, Frederick C.

2015-01-01

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens. PMID:25856195

Comparative genome analyses of Serratia marcescens FS14 reveals its high antagonistic potential.

Science.gov (United States)

Li, Pengpeng; Kwok, Amy H Y; Jiang, Jingwei; Ran, Tingting; Xu, Dongqing; Wang, Weiwu; Leung, Frederick C

2015-01-01

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.

Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.

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Banks, Rosamonde E; Craven, Rachel A; Harnden, Patricia A; Selby, Peter J

2003-04-01

Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.

Label-free DNA quantification via a 'pipette, aggregate and blot' (PAB) approach with magnetic silica particles on filter paper.

Science.gov (United States)

Li, Jingyi; Liu, Qian; Alsamarri, Hussein; Lounsbury, Jenny A; Haversitick, Doris M; Landers, James P

2013-03-07

Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

Southern blot analysis of skin biopsies for human papillomavirus DNA: renal allograft recipients in south-eastern Queensland.

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Trenfield, K; Salmond, C A; Pope, J H; Hardie, I R

1993-01-01

The 104 skin biopsies from 34 patients who attended a Renal Transplant Unit in Brisbane over 12 months included 40 squamous cell carcinoma (SCC), 22 solar keratoses, 4 hyperkeratoses, 18 warts and 11 basal cell carcinoma (BCC). Human papillomavirus (HPV) DNA was identified by Southern blot hybridisation using, as individual probes, purified insert DNA from recombinant HPV 1, 2, 3 or 3/10, 4, 5 or 5/8, 7, 11, 16, 18 and 41 under relaxed conditions and characterised by restriction enzyme analysis and Southern blot hybridisation under more stringent conditions. Genomic HPV DNA was characterised in 7 skin biopsies from 4 renal allograft recipients (RARs): HPV 1A in a SCC (20 copies/cell) and a BCC (10 copies/cell) from the one patient, HPV 36 (20 copies/cell) in a SCC, HPV 1A [symbol: see text] 1000 copies/cell) in a wart and HPV 2B (200-800 copies/cell) in 3 warts from the one patient. Only HPV 1A in the SCC exhibited a significant degree of subtype variation. HPV DNA was identified in another 5 skin biopsies from another 4 RARs: HPV 3A in a wart and a hyperkeratosis, HPV 3/10-related DNA in 2 solar keratoses and HPV 5/8-related DNA in another (20-50 copies/cell). The incidence of HPV 5 (or 5-related HPVs) in RAR SCC was very low and that of HPV DNA in RAR warts was lower than that recorded elsewhere but this was not due to insensitivity of the assays. There was no evidence for a role for HPV in the aetiology of skin cancer in RARs in south-eastern Queensland but the possibility remains that as yet unidentified HPV types are involved.

Spatial genetic analyses reveal cryptic population structure and migration patterns in a continuously harvested grey wolf (Canis lupus population in north-eastern Europe.

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Maris Hindrikson

Full Text Available Spatial genetics is a relatively new field in wildlife and conservation biology that is becoming an essential tool for unravelling the complexities of animal population processes, and for designing effective strategies for conservation and management. Conceptual and methodological developments in this field are therefore critical. Here we present two novel methodological approaches that further the analytical possibilities of STRUCTURE and DResD. Using these approaches we analyse structure and migrations in a grey wolf (Canislupus population in north-eastern Europe. We genotyped 16 microsatellite loci in 166 individuals sampled from the wolf population in Estonia and Latvia that has been under strong and continuous hunting pressure for decades. Our analysis demonstrated that this relatively small wolf population is represented by four genetic groups. We also used a novel methodological approach that uses linear interpolation to statistically test the spatial separation of genetic groups. The new method, which is capable of using program STRUCTURE output, can be applied widely in population genetics to reveal both core areas and areas of low significance for genetic groups. We also used a recently developed spatially explicit individual-based method DResD, and applied it for the first time to microsatellite data, revealing a migration corridor and barriers, and several contact zones.

Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays.

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Matee, M I; Lyamuya, E F; Simon, E; Mbena, E C; Kagoma, C; Samaranayake, L P; Scheutz, F

1996-05-01

The present study, based on 158 HIV seropositives and 167 HIV seronegatives, demonstrates that saliva collected with the Omni-SAL device and tested with GACELISA (an IgG antibody capture ELISA) is an effective non-invasive alternative to serum for anti-HIV IgG antibody screening. The study also shows that a conventional serum Western blot kit can be used, with slight modifications, for confirmatory testing of saliva specimens. Collecting saliva with the Omni-SAL device had a very good acceptance rate among Tanzanian subjects, and although this diagnostic method is not yet known by the general public, 65% of the study participants preferred to give saliva instead of blood for HIV testing.

Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis

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Couturier Marie

2012-02-01

Full Text Available Abstract Background Filamentous fungi are potent biomass degraders due to their ability to thrive in ligno(hemicellulose-rich environments. During the last decade, fungal genome sequencing initiatives have yielded abundant information on the genes that are putatively involved in lignocellulose degradation. At present, additional experimental studies are essential to provide insights into the fungal secreted enzymatic pools involved in lignocellulose degradation. Results In this study, we performed a wide analysis of 20 filamentous fungi for which genomic data are available to investigate their biomass-hydrolysis potential. A comparison of fungal genomes and secretomes using enzyme activity profiling revealed discrepancies in carbohydrate active enzymes (CAZymes sets dedicated to plant cell wall. Investigation of the contribution made by each secretome to the saccharification of wheat straw demonstrated that most of them individually supplemented the industrial Trichoderma reesei CL847 enzymatic cocktail. Unexpectedly, the most striking effect was obtained with the phytopathogen Ustilago maydis that improved the release of total sugars by 57% and of glucose by 22%. Proteomic analyses of the best-performing secretomes indicated a specific enzymatic mechanism of U. maydis that is likely to involve oxido-reductases and hemicellulases. Conclusion This study provides insight into the lignocellulose-degradation mechanisms by filamentous fungi and allows for the identification of a number of enzymes that are potentially useful to further improve the industrial lignocellulose bioconversion process.

Analyses in zebrafish embryos reveal that nanotoxicity profiles are dependent on surface-functionalization controlled penetrance of biological membranes.

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Paatero, Ilkka; Casals, Eudald; Niemi, Rasmus; Özliseli, Ezgi; Rosenholm, Jessica M; Sahlgren, Cecilia

2017-08-21

Mesoporous silica nanoparticles (MSNs) are extensively explored as drug delivery systems, but in depth understanding of design-toxicity relationships is still scarce. We used zebrafish (Danio rerio) embryos to study toxicity profiles of differently surface functionalized MSNs. Embryos with the chorion membrane intact, or dechoroniated embryos, were incubated or microinjected with amino (NH 2 -MSNs), polyethyleneimine (PEI-MSNs), succinic acid (SUCC-MSNs) or polyethyleneglycol (PEG-MSNs) functionalized MSNs. Toxicity was assessed by viability and cardiovascular function. NH 2 -MSNs, SUCC-MSNs and PEG-MSNs were well tolerated, 50 µg/ml PEI-MSNs induced 100% lethality 48 hours post fertilization (hpf). Dechoroniated embryos were more sensitive and 10 µg/ml PEI-MSNs reduced viability to 5% at 96hpf. Sensitivity to PEG- and SUCC-, but not NH 2 -MSNs, was also enhanced. Typically cardiovascular toxicity was evident prior to lethality. Confocal microscopy revealed that PEI-MSNs penetrated into the embryos whereas PEG-, NH2- and SUCC-MSNs remained aggregated on the skin surface. Direct exposure of inner organs by microinjecting NH 2 -MSNs and PEI-MSNs demonstrated that the particles displayed similar toxicity indicating that functionalization affects the toxicity profile by influencing penetrance through biological barriers. The data emphasize the need for careful analyses of toxicity mechanisms in relevant models and constitute an important knowledge step towards the development of safer and sustainable nanotherapies.

Transcriptomic analyses reveal novel genes with sexually dimorphic expression in the zebrafish gonad and brain.

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Rajini Sreenivasan

Full Text Available BACKGROUND: Our knowledge on zebrafish reproduction is very limited. We generated a gonad-derived cDNA microarray from zebrafish and used it to analyze large-scale gene expression profiles in adult gonads and other organs. METHODOLOGY/PRINCIPAL FINDINGS: We have identified 116638 gonad-derived zebrafish expressed sequence tags (ESTs, 21% of which were isolated in our lab. Following in silico normalization, we constructed a gonad-derived microarray comprising 6370 unique, full-length cDNAs from differentiating and adult gonads. Labeled targets from adult gonad, brain, kidney and 'rest-of-body' from both sexes were hybridized onto the microarray. Our analyses revealed 1366, 881 and 656 differentially expressed transcripts (34.7% novel that showed highest expression in ovary, testis and both gonads respectively. Hierarchical clustering showed correlation of the two gonadal transcriptomes and their similarities to those of the brains. In addition, we have identified 276 genes showing sexually dimorphic expression both between the brains and between the gonads. By in situ hybridization, we showed that the gonadal transcripts with the strongest array signal intensities were germline-expressed. We found that five members of the GTP-binding septin gene family, from which only one member (septin 4 has previously been implicated in reproduction in mice, were all strongly expressed in the gonads. CONCLUSIONS/SIGNIFICANCE: We have generated a gonad-derived zebrafish cDNA microarray and demonstrated its usefulness in identifying genes with sexually dimorphic co-expression in both the gonads and the brains. We have also provided the first evidence of large-scale differential gene expression between female and male brains of a teleost. Our microarray would be useful for studying gonad development, differentiation and function not only in zebrafish but also in related teleosts via cross-species hybridizations. Since several genes have been shown to play similar

Glycophospholipid Formulation with NADH and CoQ10 Significantly Reduces Intractable Fatigue in Western Blot-Positive ‘Chronic Lyme Disease’ Patients: Preliminary Report

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Garth L. Nicolson

2012-03-01

Full Text Available Background: An open label 8-week preliminary study was conducted in a small number of patients to determine if a combination oral supplement containing a mixture of phosphoglycolipids, coenzyme Q10 and microencapsulated NADH and other nutrients could affect fatigue levels in long-term, Western blot-positive, multi-symptom ‘chronic Lyme disease’ patients (also called ‘post-treatment Lyme disease’ or ‘post Lyme syndrome’ with intractable fatigue. Methods: The subjects in this study were 6 males (mean age = 45.1 ± 12.4 years and 10 females (mean age = 54.6 ± 7.4 years with ‘chronic Lyme disease’ (determined by multiple symptoms and positive Western blot analysis that had been symptomatic with chronic fatigue for an average of 12.7 ± 6.6 years. They had been seen by multiple physicians (13.3 ± 7.6 and had used many other remedies, supplements and drugs (14.4 ± 7.4 without fatigue relief. Fatigue was monitored at 0, 7, 30 and 60 days using a validated instrument, the Piper Fatigue Scale.Results: Patients in this preliminary study responded to the combination test supplement, showing a 26% reduction in overall fatigue by the end of the 8-week trial (p< 0.0003. Analysis of subcategories of fatigue indicated that there were significant improvements in the ability to complete tasks and activities as well as significant improvements in mood and cognitive abilities. Regression analysis of the data indicated that reductions in fatigue were consistent and occurred with a high degree of confidence (R2= 0.998. Functional Foods in Health and Disease 2012, 2(3:35-47 Conclusions: The combination supplement was a safe and effective method to significantly reduce intractable fatigue in long-term patients with Western blot-positive ‘chronic Lyme disease.’

Comparative genome analyses of Serratia marcescens FS14 reveals its high antagonistic potential.

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Pengpeng Li

Full Text Available S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.

Hepatitis B virus X protein (HBx)-induced abnormalities of nucleic acid metabolism revealed by (1)H-NMR-based metabonomics.

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Dan Yue; Zhang, Yuwei; Cheng, Liuliu; Ma, Jinhu; Xi, Yufeng; Yang, Liping; Su, Chao; Shao, Bin; Huang, Anliang; Xiang, Rong; Cheng, Ping

2016-04-14

Hepatitis B virus X protein (HBx) plays an important role in HBV-related hepatocarcinogenesis; however, mechanisms underlying HBx-mediated carcinogenesis remain unclear. In this study, an NMR-based metabolomics approach was applied to systematically investigate the effects of HBx on cell metabolism. EdU incorporation assay was conducted to examine the effects of HBx on DNA synthesis, an important feature of nucleic acid metabolism. The results revealed that HBx disrupted metabolism of glucose, lipids, and amino acids, especially nucleic acids. To understand the potential mechanism of HBx-induced abnormalities of nucleic acid metabolism, gene expression profiles of HepG2 cells expressing HBx were investigated. The results showed that 29 genes involved in DNA damage and DNA repair were differentially expressed in HBx-expressing HepG2 cells. HBx-induced DNA damage was further demonstrated by karyotyping, comet assay, Western blotting, immunofluorescence and immunohistochemistry analyses. Many studies have previously reported that DNA damage can induce abnormalities of nucleic acid metabolism. Thus, our results implied that HBx initially induces DNA damage, and then disrupts nucleic acid metabolism, which in turn blocks DNA repair and induces the occurrence of hepatocellular carcinoma (HCC). These findings further contribute to our understanding of the occurrence of HCC.

Application of FTA sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based Southern blotting.

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Hsiao, K M; Lin, H M; Pan, H; Li, T C; Chen, S S; Jou, S B; Chiu, Y L; Wu, M F; Lin, C C; Li, S Y

1999-01-01

Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.

Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

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MORALES Olga Lucía

2002-01-01

Full Text Available Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB assay was performed using excretory/secretory antigens (E/S of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli.

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Shan Goh

Full Text Available BACKGROUND: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development. METHODOLOGY/PRINCIPAL FINDINGS: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50. When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50 values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required. CONCLUSIONS: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

Comparative analyses of population-scale phenomic data in electronic medical records reveal race-specific disease networks.

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Glicksberg, Benjamin S; Li, Li; Badgeley, Marcus A; Shameer, Khader; Kosoy, Roman; Beckmann, Noam D; Pho, Nam; Hakenberg, Jörg; Ma, Meng; Ayers, Kristin L; Hoffman, Gabriel E; Dan Li, Shuyu; Schadt, Eric E; Patel, Chirag J; Chen, Rong; Dudley, Joel T

2016-06-15

Underrepresentation of racial groups represents an important challenge and major gap in phenomics research. Most of the current human phenomics research is based primarily on European populations; hence it is an important challenge to expand it to consider other population groups. One approach is to utilize data from EMR databases that contain patient data from diverse demographics and ancestries. The implications of this racial underrepresentation of data can be profound regarding effects on the healthcare delivery and actionability. To the best of our knowledge, our work is the first attempt to perform comparative, population-scale analyses of disease networks across three different populations, namely Caucasian (EA), African American (AA) and Hispanic/Latino (HL). We compared susceptibility profiles and temporal connectivity patterns for 1988 diseases and 37 282 disease pairs represented in a clinical population of 1 025 573 patients. Accordingly, we revealed appreciable differences in disease susceptibility, temporal patterns, network structure and underlying disease connections between EA, AA and HL populations. We found 2158 significantly comorbid diseases for the EA cohort, 3265 for AA and 672 for HL. We further outlined key disease pair associations unique to each population as well as categorical enrichments of these pairs. Finally, we identified 51 key 'hub' diseases that are the focal points in the race-centric networks and of particular clinical importance. Incorporating race-specific disease comorbidity patterns will produce a more accurate and complete picture of the disease landscape overall and could support more precise understanding of disease relationships and patient management towards improved clinical outcomes. rong.chen@mssm.edu or joel.dudley@mssm.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Combined Metabonomic and Quantitative RT-PCR Analyses Revealed Metabolic Reprogramming Associated with Fusarium graminearum Resistance in Transgenic Arabidopsis thaliana

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Fangfang Chen

2018-01-01

Full Text Available Fusarium head blight disease resulting from Fusarium graminearum (FG infection causes huge losses in global production of cereals and development of FG-resistant plants is urgently needed. To understand biochemistry mechanisms for FG resistance, here, we have systematically investigated the plant metabolomic phenotypes associated with FG resistance for transgenic Arabidopsis thaliana expressing a class-I chitinase (Chi, a Fusarium-specific recombinant antibody gene (CWP2 and fused Chi-CWP2. Plant disease indices, mycotoxin levels, metabonomic characteristics, and expression levels of several key genes were measured together with their correlations. We found that A. thaliana expressing Chi-CWP2 showed higher FG resistance with much lower disease indices and mycotoxin levels than the wild-type and the plants expressing Chi or CWP2 alone. The combined metabonomic and quantitative RT-PCR analyses revealed that such FG-resistance was closely associated with the promoted biosynthesis of secondary metabolites (phenylpropanoids, alkanoids and organic osmolytes (proline, betaine, glucose, myo-inositol together with enhanced TCA cycle and GABA shunt. These suggest that the concurrently enhanced biosyntheses of the shikimate-mediated secondary metabolites and organic osmolytes be an important strategy for A. thaliana to develop and improve FG resistance. These findings provide essential biochemical information related to FG resistance which is important for developing FG-resistant cereals.

Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens

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Hermes Escalante

2011-09-01

Full Text Available Objetivos. Evaluar la eficacia de la técnica de electroinmunotransferencia (EITB o Western blot utilizando antígenos de excreción-secreción de las formas adultas de Fasciola hepatica (Fh E/S Ag para el diagnóstico de la fasciolosis humana. Materiales y métodos. Los antígenos fueron obtenidos a las 18 horas de incubación en medio Minimum Essential Eagle y preparados a la concentración proteica de 0,15 ug/uL; los cuales, al ser enfrentados con un pool de sueros de pacientes con fasciolosis confirmada por el hallazgo de huevos del parásito en las heces, se detectaron los antígenos de 10, 12, 17, 23, 27, 30, 36, 43, 66 y 136 KDa, con los cuales se desarrolló la técnica de Western blot. La sensibilidad se evaluó empleando sueros de 67 pacientes con fasciolosis, y la especificidad con sueros de 57 pacientes con otras parasitosis y diez sueros de personas no parasitadas. Resultados. De los 67 sueros, 64 reaccionaron con la banda de 23 KDa y 61 con la banda de 17KDa. Estas dos bandas no fueron detectadas por ninguno de los sueros de pacientes con otras parasitosis, ni de personas no parasitadas, siendo por ello consideradas como específicas y diagnósticas. Conclusiones. La sensibilidad de la prueba, utilizando las bandas de 17 y 23 KDa, fue de 95,5 % cuando se presenta reacción positiva en una o en las dos bandas, siendo la especificidad para estos dos antígenos de 100 % con un valor predictivo positivo de 100 % y un valor predictivo negativo de 95,71 %.Objectives. To evaluate the performance of the enzyme-linked immunoelectrotransfer blot assay (EITB, Western blot using excretory/secretory antigens from adult forms of Fasciola hepatica (Fh E/S Ag for the diagnosis of human fasciolosis. Materials and methods. Antigens were obtained after 18 hours of incubation in culture medium Minimum Essential Eagle, prepared at a protein concentration of 0.15 ug/uL and run against a pool of sera of patients with proven fasciolosis (confirmed by the

Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

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Luiz Claudio Pereira Ribeiro

2013-09-01

Full Text Available Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1 antibodies (Abs represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP patients. Western blotting (WB for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I and gag (p24 proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

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Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

2018-02-01

Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

Core microbial functional activities in ocean environments revealed by global metagenomic profiling analyses.

KAUST Repository

Ferreira, Ari J S

2014-06-12

Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world\\'s oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

Core microbial functional activities in ocean environments revealed by global metagenomic profiling analyses.

KAUST Repository

Ferreira, Ari J S; Siam, Rania; Setubal, Joã o C; Moustafa, Ahmed; Sayed, Ahmed; Chambergo, Felipe S; Dawe, Adam S; Ghazy, Mohamed A; Sharaf, Hazem; Ouf, Amged; Alam, Intikhab; Abdel-Haleem, Alyaa M; Lehvä slaiho, Heikki; Ramadan, Eman; Antunes, André ; Stingl, Ulrich; Archer, John A.C.; Jankovic, Boris R; Sogin, Mitchell; Bajic, Vladimir B.; El-Dorry, Hamza

2014-01-01

Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world's oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

Core microbial functional activities in ocean environments revealed by global metagenomic profiling analyses.

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Ari J S Ferreira

Full Text Available Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world's oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

Electrospun nitrocellulose and nylon: Design and fabrication of novel high performance platforms for protein blotting applications

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Bowlin Gary L

2007-10-01

Full Text Available Abstract Background Electrospinning is a non-mechanical processing strategy that can be used to process a variety of native and synthetic polymers into highly porous materials composed of nano-scale to micron-scale diameter fibers. By nature, electrospun materials exhibit an extensive surface area and highly interconnected pore spaces. In this study we adopted a biological engineering approach to ask how the specific unique advantages of the electrospinning process might be exploited to produce a new class of research/diagnostic tools. Methods The electrospinning properties of nitrocellulose, charged nylon and blends of these materials are characterized. Results Nitrocellulose electrospun from a starting concentration of Conclusion The flexibility afforded by electrospinning process makes it possible to tailor blotting membranes to specific applications. Electrospinning has a variety of potential applications in the clinical diagnostic field of use.

Deciphering the Cryptic Genome: Genome-wide Analyses of the Rice Pathogen Fusarium fujikuroi Reveal Complex Regulation of Secondary Metabolism and Novel Metabolites

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Studt, Lena; Niehaus, Eva-Maria; Espino, Jose J.; Huß, Kathleen; Michielse, Caroline B.; Albermann, Sabine; Wagner, Dominik; Bergner, Sonja V.; Connolly, Lanelle R.; Fischer, Andreas; Reuter, Gunter; Kleigrewe, Karin; Bald, Till; Wingfield, Brenda D.; Ophir, Ron; Freeman, Stanley; Hippler, Michael; Smith, Kristina M.; Brown, Daren W.; Proctor, Robert H.; Münsterkötter, Martin; Freitag, Michael; Humpf, Hans-Ulrich; Güldener, Ulrich; Tudzynski, Bettina

2013-01-01

The fungus Fusarium fujikuroi causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F

Diet and stable isotope analyses reveal the feeding ecology of the orangeback squid Sthenoteuthis pteropus (Steenstrup 1855 (Mollusca, Ommastrephidae in the eastern tropical Atlantic.

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Véronique Merten

Full Text Available In the eastern tropical Atlantic, the orangeback flying squid Sthenoteuthis pteropus (Steenstrup 1855 (Cephalopoda, Ommastrephidae is a dominant species of the epipelagic nekton community. This carnivore squid has a short lifespan and is one of the fastest-growing squids. In this study, we characterise the role of S. pteropus in the pelagic food web of the eastern tropical Atlantic by investigating its diet and the dynamics of its feeding habits throughout its ontogeny and migration. During three expeditions in the eastern tropical Atlantic in 2015, 129 specimens were caught by hand jigging. Stomach content analyses (via visual identification and DNA barcoding were combined with stable isotope data (∂15N and ∂13C of muscle tissue to describe diet, feeding habits and trophic ecology of S. pteropus. Additionally, stable isotope analyses of incremental samples along the squid's gladius-the chitinous spiniform structure supporting the muscles and organs-were carried out to explore possible diet shifts through ontogeny and migration. Our results show that S. pteropus preys mainly on myctophid fishes (e.g. Myctophum asperum, Myctophum nitidulum, Vinciguerria spp., but also on other teleost species, cephalopods (e.g. Enoploteuthidae, Bolitinidae, Ommastrephidae, crustaceans and possibly on gelatinous zooplankton as well. The squid shows a highly opportunistic feeding behaviour that includes cannibalism. Our study indicates that the trophic position of S. pteropus may increase by approximately one trophic level from a mantle length of 15 cm to 47 cm. The reconstructed isotope-based feeding chronologies of the gladii revealed high intra- and inter-individual variability in the squid's trophic position and foraging area. These findings are not revealed by diet or muscle tissue stable isotope analysis. This suggests a variable and complex life history involving individual variation and migration. The role of S. pteropus in transferring energy and

Diet and stable isotope analyses reveal the feeding ecology of the orangeback squid Sthenoteuthis pteropus (Steenstrup 1855) (Mollusca, Ommastrephidae) in the eastern tropical Atlantic.

Science.gov (United States)

Merten, Véronique; Christiansen, Bernd; Javidpour, Jamileh; Piatkowski, Uwe; Puebla, Oscar; Gasca, Rebeca; Hoving, Henk-Jan T

2017-01-01

In the eastern tropical Atlantic, the orangeback flying squid Sthenoteuthis pteropus (Steenstrup 1855) (Cephalopoda, Ommastrephidae) is a dominant species of the epipelagic nekton community. This carnivore squid has a short lifespan and is one of the fastest-growing squids. In this study, we characterise the role of S. pteropus in the pelagic food web of the eastern tropical Atlantic by investigating its diet and the dynamics of its feeding habits throughout its ontogeny and migration. During three expeditions in the eastern tropical Atlantic in 2015, 129 specimens were caught by hand jigging. Stomach content analyses (via visual identification and DNA barcoding) were combined with stable isotope data (∂15N and ∂13C) of muscle tissue to describe diet, feeding habits and trophic ecology of S. pteropus. Additionally, stable isotope analyses of incremental samples along the squid's gladius-the chitinous spiniform structure supporting the muscles and organs-were carried out to explore possible diet shifts through ontogeny and migration. Our results show that S. pteropus preys mainly on myctophid fishes (e.g. Myctophum asperum, Myctophum nitidulum, Vinciguerria spp.), but also on other teleost species, cephalopods (e.g. Enoploteuthidae, Bolitinidae, Ommastrephidae), crustaceans and possibly on gelatinous zooplankton as well. The squid shows a highly opportunistic feeding behaviour that includes cannibalism. Our study indicates that the trophic position of S. pteropus may increase by approximately one trophic level from a mantle length of 15 cm to 47 cm. The reconstructed isotope-based feeding chronologies of the gladii revealed high intra- and inter-individual variability in the squid's trophic position and foraging area. These findings are not revealed by diet or muscle tissue stable isotope analysis. This suggests a variable and complex life history involving individual variation and migration. The role of S. pteropus in transferring energy and nutrients from

Imaging and high-sensitivity quantification of chemiluminescent labeled DNA-blots

International Nuclear Information System (INIS)

Dorner, G.

1997-01-01

The present thesis has for objective the development of both, methods of DNA labeling by chemiluminescence (via the catalytic activity of the enzyme alkaline phosphatase - AP) and an appropriate imaging system. Offering a competitive alternative to the detection of classical radio-labels in molecular-biological experiments of the blotting type, this technique should permit the realization of quantitative studies of gene expression at ultra-high sensitivity necessary in particular for differential-screening experiments. To reach our aim. we separated the project into three different parts. In a first step an imager based on a liquid-nitrogen-cooled CCD coupled to a standard optics (50 mm/fl.2) has been installed and characterized. This system offers a sensitive area of up to 625 cm 2 , a spatial resolution of 0.3-1 mm (depending on the field of view) and a sensitivity sufficient to detect 10 fg/mm 2 labeled DNA. In a second part, the chemiluminescent light-generation process in solution has been investigated to optimize the parameters temperature. pH and concentration of the substrate as well as the enzyme. The substrate offering the highest light yield (CDP-Star in addition with the enhancer EMERALD II) allows quantification of AP down to 10 -15 M within a dynamic range of 10 4 in solution. Finally. preparation, immobilization and detection of AP-labeled DNA probes (via a biotin-streptavidin-biotin-AP bridge) on nylon membranes has been optimized. A linear relation between the light intensities and the amount of DNA was observed in a range of 10 fg/mm 2 - 100 pg/mm 2 . Hybridization of the probes to bacterial cloned target-DNA has been addressed after examination of the best hybridization conditions. Our protocol includes the treatment of a proteinase, which resulted in a significantly lower background on the filter. The results of our investigations suggest that the main conditions for a reliable differential-screening experiment are fulfilled when using

Transcriptomics and proteomics analyses of the PACAP38 influenced ischemic brain in permanent middle cerebral artery occlusion model mice

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Hori Motohide

2012-11-01

Full Text Available Abstract Introduction The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP is considered to be a potential therapeutic agent for prevention of cerebral ischemia. Ischemia is a most common cause of death after heart attack and cancer causing major negative social and economic consequences. This study was designed to investigate the effect of PACAP38 injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO along with corresponding SHAM control that used 0.9% saline injection. Methods Ischemic and non-ischemic brain tissues were sampled at 6 and 24 hours post-treatment. Following behavioral analyses to confirm whether the ischemia has occurred, we investigated the genome-wide changes in gene and protein expression using DNA microarray chip (4x44K, Agilent and two-dimensional gel electrophoresis (2-DGE coupled with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS, respectively. Western blotting and immunofluorescent staining were also used to further examine the identified protein factor. Results Our results revealed numerous changes in the transcriptome of ischemic hemisphere (ipsilateral treated with PACAP38 compared to the saline-injected SHAM control hemisphere (contralateral. Previously known (such as the interleukin family and novel (Gabra6, Crtam genes were identified under PACAP influence. In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic hemisphere that was identified as dihydropyrimidinase-related protein 2 (DPYL2. The DPYL2, also known as Crmp2, is a marker for the axonal growth and nerve development. Interestingly, PACAP treatment slightly increased its abundance (by 2-DGE and immunostaining at 6 h but not at 24 h in the ischemic hemisphere, suggesting PACAP activates neuronal defense mechanism early on. Conclusions This study provides a detailed inventory of PACAP influenced gene expressions

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

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Wang, Hye-young; Kim, Hyunjung; Kim, Yeun; Bang, Hyeeun; Kim, Jong-Pill; Hwang, Joo Hwan; Cho, Sang-Nae; Kim, Tae Ue; Lee, Hyeyoung

2015-10-01

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

Detection of Rickettsia in Rhipicephalus sanguineus Ticks and Ctenocephalides felis Fleas from Southeastern Tunisia by Reverse Line Blot Assay

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Khrouf, Fatma; M'Ghirbi, Youmna; Znazen, Abir; Ben Jemaa, Mounir; Hammami, Adnene

2014-01-01

Ticks (n = 663) and fleas (n = 470) collected from domestic animals from southeastern Tunisia were screened for Rickettsia infection using reverse line blot assay. Evidence of spotted fever group Rickettsia was obtained. We detected Rickettsia felis in fleas, Rickettsia massiliae Bar 29 and the Rickettsia conorii Israeli spotted fever strain in ticks, and Rickettsia conorii subsp. conorii and Rickettsia spp. in both arthropods. The sensitivity of the adopted technique allowed the identification of a new association between fleas and R. conorii subsp. conorii species. The presence of these vector-borne Rickettsia infections should be considered when diagnosing this disease in humans in Tunisia. PMID:24226919

Genome size analyses of Pucciniales reveal the largest fungal genomes.

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Tavares, Sílvia; Ramos, Ana Paula; Pires, Ana Sofia; Azinheira, Helena G; Caldeirinha, Patrícia; Link, Tobias; Abranches, Rita; Silva, Maria do Céu; Voegele, Ralf T; Loureiro, João; Talhinhas, Pedro

2014-01-01

Rust fungi (Basidiomycota, Pucciniales) are biotrophic plant pathogens which exhibit diverse complexities in their life cycles and host ranges. The completion of genome sequencing of a few rust fungi has revealed the occurrence of large genomes. Sequencing efforts for other rust fungi have been hampered by uncertainty concerning their genome sizes. Flow cytometry was recently applied to estimate the genome size of a few rust fungi, and confirmed the occurrence of large genomes in this order (averaging 225.3 Mbp, while the average for Basidiomycota was 49.9 Mbp and was 37.7 Mbp for all fungi). In this work, we have used an innovative and simple approach to simultaneously isolate nuclei from the rust and its host plant in order to estimate the genome size of 30 rust species by flow cytometry. Genome sizes varied over 10-fold, from 70 to 893 Mbp, with an average genome size value of 380.2 Mbp. Compared to the genome sizes of over 1800 fungi, Gymnosporangium confusum possesses the largest fungal genome ever reported (893.2 Mbp). Moreover, even the smallest rust genome determined in this study is larger than the vast majority of fungal genomes (94%). The average genome size of the Pucciniales is now of 305.5 Mbp, while the average Basidiomycota genome size has shifted to 70.4 Mbp and the average for all fungi reached 44.2 Mbp. Despite the fact that no correlation could be drawn between the genome sizes, the phylogenomics or the life cycle of rust fungi, it is interesting to note that rusts with Fabaceae hosts present genomes clearly larger than those with Poaceae hosts. Although this study comprises only a small fraction of the more than 7000 rust species described, it seems already evident that the Pucciniales represent a group where genome size expansion could be a common characteristic. This is in sharp contrast to sister taxa, placing this order in a relevant position in fungal genomics research.

Use of the water-soluble fluor sodium salicylate for fluorographic detection of tritium in thin-layer chromatograms and nitrocellulose blots

International Nuclear Information System (INIS)

Lucher, L.A.; Lego, T.

1989-01-01

We have determined that sodium salicylate, a water-soluble fluor which we use routinely for fluorography with polyacrylamide gels, is also useful for fluorography with thin-layer media. Detection of 3 H-labeled material applied to thin-layer chromatography plates, or nitrocellulose membranes, can be enhanced up to 150-fold after treatment with an aqueous solution of 2 M sodium salicylate, while detection of 35 S-labeled material is enhanced only about 2-fold. We demonstrate the utility of sodium salicylate fluorography in detecting 3H-labeled palmitic acid following thin-layer chromatography and 3 H-labeled proteins following blotting to nitrocellulose

Use of nitrocellulose blotting for the study of hepatitis B surface antigen electrophoresed in agarose gels

Energy Technology Data Exchange (ETDEWEB)

McMichael, J C; Greisiger, L M; Millman, I [Institute for Cancer Research, Philadelphia, PA (USA). Fox Chase Cancer Center

1981-08-28

Nitrocellulose-protein blotting of serum electrophoresed in agarose gels has been adapted for the study of hepatitis B surface antigen (HBsAg). /sup 125/I-labeled anti-HBs was used as the antigen probe, and the electrophoretic migration was monitored by autoradiography. The method required 3 ..mu..l or less of serum and could detect as little as 1 pg of purified HBsAg. Typically, the authors observed two bands of HBsAg; a moving band which migrated about one-third the distance moved by human serum albumin and a non-migratory band which remained at the loading site. Some examples of the use of the method include: (1) empirical methods for correlating HBsAg concentration in serum to film darkness; (2) observations of mobility changes in serial sera from dialysis patients with chronic HBsAg antigenemia; and (3) detection of related antigens such as antigen from the PLC/PRF/5 hepatoma tissue culture line and the cross-reacting woodchuck hepatitis virus surface antigen (WHsAg).

Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

DEFF Research Database (Denmark)

Sahm, K.; MacGregor, BJ; Jørgensen, BB

1999-01-01

In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell. Here, we report evidence that quantification of SRB rRNA by slot-blot hybridization......, directly above the sulphate reduction maximum. Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment. Cellular sulphate reduction rates calculated on the basis...

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii

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Johnson David R

2011-11-01

opposite effect and decreased after perturbation with PEG8000. Conclusions A combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

Ancient ancestry of KFDV and AHFV revealed by complete genome analyses of viruses isolated from ticks and mammalian hosts.

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Kimberly A Dodd

2011-10-01

Full Text Available BACKGROUND: Alkhurma hemorrhagic fever virus (AHFV and Kyasanur forest disease virus (KFDV cause significant human disease and mortality in Saudi Arabia and India, respectively. Despite their distinct geographic ranges, AHFV and KFDV share a remarkably high sequence identity. Given its emergence decades after KFDV, AHFV has since been considered a variant of KFDV and thought to have arisen from an introduction of KFDV to Saudi Arabia from India. To gain a better understanding of the evolutionary history of AHFV and KFDV, we analyzed the full length genomes of 16 AHFV and 3 KFDV isolates. METHODOLOGY/PRINCIPAL FINDINGS: Viral genomes were sequenced and compared to two AHFV sequences available in GenBank. Sequence analyses revealed higher genetic diversity within AHFVs isolated from ticks than human AHFV isolates. A Bayesian coalescent phylogenetic analysis demonstrated an ancient divergence of AHFV and KFDV of approximately 700 years ago. CONCLUSIONS/SIGNIFICANCE: The high sequence diversity within tick populations and the presence of competent tick vectors in the surrounding regions, coupled with the recent identification of AHFV in Egypt, indicate possible viral range expansion or a larger geographic range than previously thought. The divergence of AHFV from KFDV nearly 700 years ago suggests other AHFV/KFDV-like viruses might exist in the regions between Saudi Arabia and India. Given the human morbidity and mortality associated with these viruses, these results emphasize the importance of more focused study of these significant public health threats.

Integrative analyses reveal novel strategies in HPV11,-16 and-45 early infection

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Kaczkowski, Bogumil; Rossing, Maria; Andersen, Ditte

2012-01-01

of genes not previously implicated in HPV biology, such as the PSG family and ANKRD1, and of genes implicated in the biology of other viruses, e. g. MX1, IFI44 and DDX60. Carcinogenesis-related genes, e. g. ABL2, MGLL and CYR61, were upregulated by high-risk HPV16 and -45. The integrative analysis revealed...... the suppression of DNA repair by HPV11 and -16, and downregulation of cytoskeleton genes by all HPV types. Various signalling pathways were affected by the HPVs: IL-2 by HPV11; JAK-STAT by HPV16; and TGF-beta, NOTCH and tyrosine kinase signalling by HPV45. This study uncovered novel strategies employed by HPV...... to establish infection and promote uncontrolled growth....

Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts.

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Morinaga, Osamu; Uto, Takuhiro; Yuan, Chun-Su; Tanaka, Hiroyuki; Shoyama, Yukihiro

2010-06-01

A new eastern blotting technique has been established for ginsenoside Re (G-Re) contained in American ginseng berry pulp extracts. G-Re in American ginseng berry pulp was extracted using 100% methanol, 100% ethanol, 50% aqueous methanol, and 50% aqueous ethanol. The combined crude extracts were applied onto a polyethersulfone membrane and developed using the methanol-water-acetic acid solvent system (45:55:1 v/v). Separated components were immunostained using anti-G-Re monoclonal antibody. G-Re was first specifically detected and then quantitatively analyzed using NIH Imaging software. We also confirmed that the most suitable solvent was 50% aqueous methanol for extracting G-Re from American ginseng berry pulp. (c) 2009 Elsevier B.V. All rights reserved.

Northern blot analysis to investigate the abundance of microorganisms

International Nuclear Information System (INIS)

Krause, D.O.

2005-01-01

areas known as hyper-variable regions which have a high degree of sequence variation. As a result of this structure, it is possible to design signature oligonucleotide probes varying in length from about 15 to 30 nucleotides that are diagnostic of microorganisms at the kingdom, domain, genus and even species level. These signature sequences can be used in a variety of applications such as PCR analysis, construction of clone libraries or direct probing of bulk rRNA. In this chapter, I provide detailed protocols for the analysis of extracted rRNA and give detailed procedures that must be followed to do northern blot analysis of bulk RNA extracted from the rumen

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

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Lončar-Brzak, Božana; Klobučar, Marko; Veliki-Dalić, Irena; Sabol, Ivan; Kraljević Pavelić, Sandra; Krušlin, Božo; Mravak-Stipetić, Marinka

2018-03-01

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p < 0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p < 0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.

Diagnostic potential of Western blot analysis of sera from dogs with leishmaniasis in endemic areas and significance of the pattern.

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Aisa, M J; Castillejo, S; Gallego, M; Fisa, R; Riera, M C; de Colmenares, M; Torras, S; Roura, X; Sentis, J; Portus, M

1998-02-01

Serum samples collected from 237 dogs in Catalonia (northeastern Spain) were screened by Western blot analysis to detect the presence of antibodies specific to different Leishmania infantum polypeptide fractions. Leishmaniasis was confirmed in 72 of these dogs by direct examination and/or culture. Another 165 animals from the Priorat region were studied periodically for 2-8 years between 1987 and 1995, giving a total of 565 determinations. A control group of 93 dogs from nonendemic areas was also studied. Sera from dogs with leishmaniasis recognized antigens with molecular weights ranging from 12 to 85 kD. The most sensitive antigens were those of 70, 65, 46, 30, 28, 14, and 12 kD, which were recognized by 75%, 75%, 78%, 75%, 81%, 79%, and 75%, respectively, of the sera from dogs with positive parasitologic examination results. Antigens of 70 and 65 kD were also recognized by two dogs from nonendemic areas. Antigens of 14 and 12 kD were the first to be recognized by sera of asymptomatic dogs with titers less than the cut-off value of the dot-ELISA that increased during the longitudinal study, and the presence of antibodies specific for these fractions was observed for up to six years before seroconversion observed by dot-ELISA. These antibodies were also the first to disappear in dogs in which the disease was self-limited. The study corroborates the high sensitivity and specificity of Western blots in the diagnosis of canine leishmaniasis when the bands of low molecular weight (less than 46 kD) are considered, and indicates that fractions of 14 and 12 kD are useful in detecting early forms of the disease.

Blot hybridization analysis of TCR genes of T cells for five people exposed in a radiation accident

International Nuclear Information System (INIS)

Min Rui; Liu Benti; Cheng Tianmin; Yang Rujun; Meng Xiangshun; Xiao Jinsong

1996-01-01

Human lymphocyte total DNA was prepared in agarose plug by mixing cells with low melting agarose, and two restriction endonucleases were used for digestion of the total DNA with human α and β TCR cDNA probes. The total digested DNA from five people who were whole body exposed to 2.0-2.5 Gy ionizing radiation in an accident 4.5 years ago was hybridized by Southern blot method. The results showed that no obvious difference in hybridization bands was found between controls and the five victims when hybridizations were fulfilled in the total DNA which was digested by Hind III restriction endonuclease with both α and β probes. However, when the total DNA was digested with restriction endonuclease EcoR I and was hybridized with TCR α probe, four of the five exposed people showed a different hybridizing band pattern compared with the controls. The results are also discussed

Development of a dot blot assay with antibodies to recombinant “core” 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt–Jakob disease

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Sarada Subramanian

2016-01-01

Full Text Available Background and Purpose: Definitive diagnosis of Creutzfeldt–Jakob disease (CJD requires demonstration of infective prion protein (PrPSc in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy.This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD. Materials and Methods: A minigene expressing the “core” 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF. Results: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001 in the CSF levels of 14-3-3 protein between the CJD cases (N= 50 and disease controls (N= 70. The receiver operating characteristic (ROC analysis of the results suggested an optimal cutoff value of 2 ng/mL. Conclusions: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF.

Analyses of Catharanthus roseus and Arabidopsis thaliana WRKY transcription factors reveal involvement in jasmonate signaling.

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Schluttenhofer, Craig; Pattanaik, Sitakanta; Patra, Barunava; Yuan, Ling

2014-06-20

To combat infection to biotic stress plants elicit the biosynthesis of numerous natural products, many of which are valuable pharmaceutical compounds. Jasmonate is a central regulator of defense response to pathogens and accumulation of specialized metabolites. Catharanthus roseus produces a large number of terpenoid indole alkaloids (TIAs) and is an excellent model for understanding the regulation of this class of valuable compounds. Recent work illustrates a possible role for the Catharanthus WRKY transcription factors (TFs) in regulating TIA biosynthesis. In Arabidopsis and other plants, the WRKY TF family is also shown to play important role in controlling tolerance to biotic and abiotic stresses, as well as secondary metabolism. Here, we describe the WRKY TF families in response to jasmonate in Arabidopsis and Catharanthus. Publically available Arabidopsis microarrays revealed at least 30% (22 of 72) of WRKY TFs respond to jasmonate treatments. Microarray analysis identified at least six jasmonate responsive Arabidopsis WRKY genes (AtWRKY7, AtWRKY20, AtWRKY26, AtWRKY45, AtWRKY48, and AtWRKY72) that have not been previously reported. The Catharanthus WRKY TF family is comprised of at least 48 members. Phylogenetic clustering reveals 11 group I, 32 group II, and 5 group III WRKY TFs. Furthermore, we found that at least 25% (12 of 48) were jasmonate responsive, and 75% (9 of 12) of the jasmonate responsive CrWRKYs are orthologs of AtWRKYs known to be regulated by jasmonate. Overall, the CrWRKY family, ascertained from transcriptome sequences, contains approximately 75% of the number of WRKYs found in other sequenced asterid species (pepper, tomato, potato, and bladderwort). Microarray and transcriptomic data indicate that expression of WRKY TFs in Arabidopsis and Catharanthus are under tight spatio-temporal and developmental control, and potentially have a significant role in jasmonate signaling. Profiling of CrWRKY expression in response to jasmonate treatment

Transcriptome, expression, and activity analyses reveal a vital heat shock protein 70 in the stress response of stony coral Pocillopora damicornis.

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Zhang, Yidan; Zhou, Zhi; Wang, Lingui; Huang, Bo

2018-02-12

Coral bleaching occurs worldwide with increasing frequencies and intensities, which is caused by the stress response of stony coral to environmental change, especially increased sea surface temperature. In the present study, transcriptome, expression, and activity analyses were employed to illustrate the underlying molecular mechanisms of heat shock protein 70 (HSP70) in the stress response of coral to environmental changes. The domain analyses of assembled transcripts revealed 30 HSP70 gene contigs in stony coral Pocillopora damicornis. One crucial HSP70 (PdHSP70) was observed, whose expressions were induced by both elevated temperature and ammonium after expression difference analysis. The complete complementary DNA (cDNA) sequence of PdHSP70 was identified, which encoded a polypeptide of 650 amino acids with a molecular weight of 71.93 kDa. The deduced amino acid sequence of PdHSP70 contained a HSP70 domain (from Pro8 to Gly616), and it shared the highest similarity (95%) with HSP70 from Stylophora pistillata. The expression level of PdHSP70 gene increased significantly at 12 h, and returned to the initial level at 24 h after the stress of high temperature (32 °C). The cDNA fragment encoding the mature peptide of PdHSP70 was recombined and expressed in the prokaryotic expression system. The ATPase activity of recombinant PdHSP70 protein was determined, and it did not change significantly in a wide range of temperature from 25 to 40 °C. These results collectively suggested that PdHSP70 was a vital heat shock protein 70 in the stony coral P. damicornis, whose mRNA expression could be induced by diverse environmental stress and whose activity could remain stable under heat stress. PdHSP70 might be involved in the regulation of the bleaching owing to heat stress in the stony coral P. damicornis.

Possible Factors Promoting Car Evacuation in the 2011 Tohoku Tsunami Revealed by Analysing a Large-Scale Questionnaire Survey in Kesennuma City

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Fumiyasu Makinoshima

2017-11-01

Full Text Available Excessive car evacuation can cause severe traffic jams that can lead to large numbers of casualties during tsunami disasters. Investigating the possible factors that lead to unnecessary car evacuation can ensure smoother tsunami evacuations and mitigate casualty damages in future tsunami events. In this study, we quantitatively investigated the possible factors that promote car evacuation, including both necessary and unnecessary usages, by statistically analysing a large amount of data on actual tsunami evacuation behaviours surveyed in Kesennuma, where devastating damage occurred during the 2011 Tohoku Tsunami. A straightforward statistical analysis revealed a high percentage of car evacuations (approx. 50%; however, this fraction includes a high number of unnecessary usage events that were distinguished based on mode choice reasons. In addition, a binary logistic regression was conducted to quantitatively evaluate the effects of several factors and to identify the dominant factor that affected evacuation mode choice. The regression results suggested that the evacuation distance was the dominant factor for choosing car evacuation relative to other factors, such as age and sex. The cross-validation test of the regression model demonstrated that the considered factors were useful for decision making and the prediction of evacuation mode choice in the target area.

Comparative genome analyses reveal distinct structure in the saltwater crocodile MHC.

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Weerachai Jaratlerdsiri

Full Text Available The major histocompatibility complex (MHC is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2-6 times longer than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs.

Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

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Lee, Hong-Won; Kyung, Taeyoon; Yoo, Janghyun; Kim, Tackhoon; Chung, Chaeuk; Ryu, Ji Young; Lee, Hanki; Park, Kihyun; Lee, Sangkyu; Jones, Walton D.; Lim, Dae-Sik; Hyeon, Changbong; Do Heo, Won; Yoon, Tae-Young

2013-01-01

Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level. PMID:23422673

Complex analyses of inverted repeats in mitochondrial genomes revealed their importance and variability.

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Cechová, Jana; Lýsek, Jirí; Bartas, Martin; Brázda, Václav

2018-04-01

The NCBI database contains mitochondrial DNA (mtDNA) genomes from numerous species. We investigated the presence and locations of inverted repeat sequences (IRs) in these mtDNA sequences, which are known to be important for regulating nuclear genomes. IRs were identified in mtDNA in all species. IR lengths and frequencies correlate with evolutionary age and the greatest variability was detected in subgroups of plants and fungi and the lowest variability in mammals. IR presence is non-random and evolutionary favoured. The frequency of IRs generally decreased with IR length, but not for IRs 24 or 30 bp long, which are 1.5 times more abundant. IRs are enriched in sequences from the replication origin, followed by D-loop, stem-loop and miscellaneous sequences, pointing to the importance of IRs in regulatory regions of mitochondrial DNA. Data were produced using Palindrome analyser, freely available on the web at http://bioinformatics.ibp.cz. vaclav@ibp.cz. Supplementary data are available at Bioinformatics online.

Physiological and Proteomics Analyses Reveal Low-Phosphorus Stress Affected the Regulation of Photosynthesis in Soybean.

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Chu, Shanshan; Li, Hongyan; Zhang, Xiangqian; Yu, Kaiye; Chao, Maoni; Han, Suoyi; Zhang, Dan

2018-06-06

Previous studies have revealed a significant genetic relationship between phosphorus (P)-efficiency and photosynthesis-related traits in soybean. In this study, we used proteome profiling in combination with expression analysis, biochemical investigations, and leaf ultrastructural analysis to identify the underlying physiological and molecular responses. The expression analysis and ultrastructural analysis showed that the photosynthesis key genes were decreased at transcript levels and the leaf mesophyll and chloroplast were severely damaged after low-P stress. Approximately 55 protein spots showed changes under low-P condition by mass spectrometry, of which 17 were involved in various photosynthetic processes. Further analysis revealed the depression of photosynthesis caused by low-P stress mainly involves the regulation of leaf structure, adenosine triphosphate (ATP) synthesis, absorption and transportation of CO₂, photosynthetic electron transport, production of assimilatory power, and levels of enzymes related to the Calvin cycle. In summary, our findings indicated that the existence of a stringent relationship between P supply and the genomic control of photosynthesis in soybean. As an important strategy to protect soybean photosynthesis, P could maintain the stability of cell structure, up-regulate the enzymes’ activities, recover the process of photosystem II (PSII), and induce the expression of low-P responsive genes and proteins.

Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

DEFF Research Database (Denmark)

Hansen, M.C.; Nielsen, A.K.; Molin, Søren

2001-01-01

obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting...... the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell...... decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes...

Reverse line blot probe design and polymerase chain reaction optimization for bloodmeal analysis of ticks from the eastern United States.

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Scott, M C; Harmon, J R; Tsao, J I; Jones, C J; Hickling, G J

2012-05-01

Determining the host preference of vector ticks is vital to elucidating the eco-epidemiology of the diseases they spread. Detachment of ticks from captured hosts can provide evidence of feeding on those host species, but only for those species that are feasible to capture. Recently developed, highly sensitive molecular assays show great promise in allowing host selection to be determined from minute traces of host DNA that persist in recently molted ticks. Using methods developed in Europe as a starting-point, we designed 12S rDNA mitochondrial gene probes suitable for use in a reverse line blot (RLB) assay of ticks feeding on common host species in the eastern United States. This is the first study to use the 12S mitochondrial gene in a RLB bloodmeal assay in North America. The assay combines conventional PCR with a biotin-labeled primer and reverse line blots that can be stripped and rehybridized up to 20 times, making the method less expensive and more straightforward to interpret than previous methods of tick bloodmeal identification. Probes were designed that target the species, genus, genus group, family, order, or class of eight reptile, 13 birds, and 32 mammal hosts. After optimization, the RLB assay correctly identified the current hostspecies for 99% of ticks [Amblyomma americanum (L.) and eight other ixodid tick species] collected directly from known hosts. The method identified previous-host DNA for approximately half of all questing ticks assayed. Multiple bloodmeal determinations were obtained in some instances from feeding and questing ticks; this pattern is consistent with previous RLB studies but requires further investigation. Development of this probe library, suitable for eastern U.S. ecosystems, opens new avenues for eco-epidemiological investigations of this region's tick-host systems.

Retrospective study of hemoparasites in cattle in southern Italy by reverse line blot hybridization.

Science.gov (United States)

Ceci, Luigi; Iarussi, Fabrizio; Greco, Beatrice; Lacinio, Rosanna; Fornelli, Stefania; Carelli, Grazia

2014-06-01

Tick-borne diseases are widespread in tropical and temperate regions and are responsible for important economic losses in those areas. In order to assess the presence and prevalence of various pathogens in southern Italy, we retrospectively analyzed cattle blood samples collected for a previous study in 2000 using reverse line blot (RLB) hybridization. The study had been carried out in three regions of southern Italy on 1,500 randomly selected and apparently healthy adult cattle. RLB showed that 43.7% of the cattle were positive for nine different species of hemoparasites with either a single infection or a mixed infection. Theileria buffeli was the most common species found, being present in 27.3% of the animals, followed by Anaplasma marginale in 18.1%, Anaplasma centrale in 13.8%, Babesia bigemina and Anaplasma bovis in 4.2%, Anaplasma phagocytophilum in 1.7%, Babesia bovis in 1.6%, Babesia major in 0.2% and Babesia divergens in 0.1%. Complete blood counts showed different degrees of anemia in 363 animals (24.2%) and of these, 169 were RLB-positive for at least one pathogen. Among the ticks that were collected from the cattle, the following species were identified: Rhipicephalus bursa, Ixodes ricinus, Hyalomma marginatum, Boophilus annulatus, Dermacentor marginatus and Haemaphysalis (sulcata, parva, inermis and punctata). The results obtained confirmed the spread of endemic tick-borne pathogens in the regions studied.

Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

Science.gov (United States)

Ogden, Nicholas H; Arsenault, Julie; Hatchette, Todd F; Mechai, Samir; Lindsay, L Robbin

2017-01-01

Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6)-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

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Nicholas H Ogden

Full Text Available Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]. Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

Science.gov (United States)

Lamer, S; Jungblut, P R

2001-03-10

In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

Reticulate evolution: frequent introgressive hybridization among chinese hares (genus lepus revealed by analyses of multiple mitochondrial and nuclear DNA loci

Directory of Open Access Journals (Sweden)

Wu Shi-Fang

2011-07-01

Full Text Available Abstract Background Interspecific hybridization may lead to the introgression of genes and genomes across species barriers and contribute to a reticulate evolutionary pattern and thus taxonomic uncertainties. Since several previous studies have demonstrated that introgressive hybridization has occurred among some species within Lepus, therefore it is possible that introgressive hybridization events also occur among Chinese Lepus species and contribute to the current taxonomic confusion. Results Data from four mtDNA genes, from 116 individuals, and one nuclear gene, from 119 individuals, provides the first evidence of frequent introgression events via historical and recent interspecific hybridizations among six Chinese Lepus species. Remarkably, the mtDNA of L. mandshuricus was completely replaced by mtDNA from L. timidus and L. sinensis. Analysis of the nuclear DNA sequence revealed a high proportion of heterozygous genotypes containing alleles from two divergent clades and that several haplotypes were shared among species, suggesting repeated and recent introgression. Furthermore, results from the present analyses suggest that Chinese hares belong to eight species. Conclusion This study provides a framework for understanding the patterns of speciation and the taxonomy of this clade. The existence of morphological intermediates and atypical mitochondrial gene genealogies resulting from frequent hybridization events likely contribute to the current taxonomic confusion of Chinese hares. The present study also demonstrated that nuclear gene sequence could offer a powerful complementary data set with mtDNA in tracing a complete evolutionary history of recently diverged species.

Infra-Red Gas Analysers of Liquid Crystal Type for Environmental Monitoring

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V. I. Nazarov

2004-01-01

Full Text Available The paper reveals an opportunity to use infra-red gas analysers on the basis of the developed dichroic liquid crystal cells for investigation of absorption bands of various gases in the near infrared spectral region.

Complete mitochondrial genome sequences of three bats species and whole genome mitochondrial analyses reveal patterns of codon bias and lend support to a basal split in Chiroptera.

Science.gov (United States)

Meganathan, P R; Pagan, Heidi J T; McCulloch, Eve S; Stevens, Richard D; Ray, David A

2012-01-15

Order Chiroptera is a unique group of mammals whose members have attained self-powered flight as their main mode of locomotion. Much speculation persists regarding bat evolution; however, lack of sufficient molecular data hampers evolutionary and conservation studies. Of ~1200 species, complete mitochondrial genome sequences are available for only eleven. Additional sequences should be generated if we are to resolve many questions concerning these fascinating mammals. Herein, we describe the complete mitochondrial genomes of three bats: Corynorhinus rafinesquii, Lasiurus borealis and Artibeus lituratus. We also compare the currently available mitochondrial genomes and analyze codon usage in Chiroptera. C. rafinesquii, L. borealis and A. lituratus mitochondrial genomes are 16438 bp, 17048 bp and 16709 bp, respectively. Genome organization and gene arrangements are similar to other bats. Phylogenetic analyses using complete mitochondrial genome sequences support previously established phylogenetic relationships and suggest utility in future studies focusing on the evolutionary aspects of these species. Comprehensive analyses of available bat mitochondrial genomes reveal distinct nucleotide patterns and synonymous codon preferences corresponding to different chiropteran families. These patterns suggest that mutational and selection forces are acting to different extents within Chiroptera and shape their mitochondrial genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

Proteomic profiling of human keratinocytes undergoing UVB-induced alternative differentiation reveals TRIpartite Motif Protein 29 as a survival factor.

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Véronique Bertrand-Vallery

Full Text Available BACKGROUND: Repeated exposures to UVB of human keratinocytes lacking functional p16(INK-4a and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence. METHODOLOGY/PRINCIPAL FINDINGS: A 2D-DIGE proteomic profiling of this alternative state of differentiation was performed herein at various times after the exposures to UVB. Sixty-nine differentially abundant protein species were identified by mass spectrometry, many of which are involved in keratinocyte differentiation and survival. Among these protein species was TRIpartite Motif Protein 29 (TRIM29. Increased abundance of TRIM29 following UVB exposures was validated by Western blot using specific antibody and was also further analysed by immunochemistry and by RT-PCR. TRIM29 was found very abundant in keratinocytes and reconstructed epidermis. Knocking down the expression of TRIM29 by short-hairpin RNA interference decreased the viability of keratinocytes after UVB exposure. The abundance of involucrin mRNA, a marker of late differentiation, increased concomitantly. In TRIM29-knocked down reconstructed epidermis, the presence of picnotic cells revealed cell injury. Increased abundance of TRIM29 was also observed upon exposure to DNA damaging agents and PKC activation. The UVB-induced increase of TRIM29 abundance was dependent on a PKC signaling pathway, likely PKCdelta. CONCLUSIONS/SIGNIFICANCE: These findings suggest that TRIM29 allows keratinocytes to enter a protective alternative differentiation process rather than die massively after stress.

Dot Blot para determinar la identidad antigénica en vacunas conjugadas contra Streptococcus pneumoniae serotipo 19F

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Osmir Cabrera-Blanco

2017-04-01

Full Text Available Las autoridades regulatorias recomiendan el uso de técnicas de Resonancia Magnética Nuclear o técnicas serológicas para la determinación de la identidad de los antígenos presentes en las vacunas conjugadas. Con la aparición de las vacunas conjugadas multivalentes, se ha hecho necesario recurrir a técnicas inmunoquímicas con la utilización de anticuerpos monoclonales para aumentar la sensibilidad en la determinación de la identidad de los antígenos en dichas vacunas conjugadas. El objetivo del presente trabajo fue establecer las condiciones óptimas de trabajo que permitieran utilizar la técnica del Dot Blot para determinar la identidad de los antígenos en vacunas conjugadas de Streptococcus pneumoniae serotipo 19F. Para ello se estudiaron los tiempos de incubación, la influencia del reactivo en la solución de bloqueo; también las concentraciones óptimas del anticuerpo monoclonal y de los ingredientes farmacéuticos activos, así como los volúmenes de aplicación óptimos para estos y vacunas. Se utilizó un anticuerpo monoclonal contra el polisacárido capsular del serotipo 19F de neumococo. Las muestras empleadas en este trabajo fueron lotes de ingredientes farmacéuticos activos de conjugados de polisacárido capsular 19F y lotes de un candidato vacunal cubano conjugado heptavalente contra neumococos. Los resultados mostraron que para la determinación de la identidad antigénica fueron suficientes 10 µL de muestras de los principios activos a una concentración de 125 µg/mL e igual volumen para las vacunas heptavalentes. Quedó demostrado que una concentración de 1 µg/mL para el anticuerpo monoclonal y tiempos de incubación de 30 min a 37 °C fueron suficientes para la determinación. Estos resultados permiten concluir que quedaron establecidas las condiciones óptimas de trabajo para determinar la identidad antigénica por Dot Blot del polisacárido capsular de S. pneumoniae serotipo 19F presente en las vacunas

Comparative metagenomic and metatranscriptomic analyses of microbial communities in acid mine drainage.

Science.gov (United States)

Chen, Lin-xing; Hu, Min; Huang, Li-nan; Hua, Zheng-shuang; Kuang, Jia-liang; Li, Sheng-jin; Shu, Wen-sheng

2015-07-01

The microbial communities in acid mine drainage have been extensively studied to reveal their roles in acid generation and adaption to this environment. Lacking, however, are integrated community- and organism-wide comparative gene transcriptional analyses that could reveal the response and adaptation mechanisms of these extraordinary microorganisms to different environmental conditions. In this study, comparative metagenomics and metatranscriptomics were performed on microbial assemblages collected from four geochemically distinct acid mine drainage (AMD) sites. Taxonomic analysis uncovered unexpectedly high microbial biodiversity of these extremely acidophilic communities, and the abundant taxa of Acidithiobacillus, Leptospirillum and Acidiphilium exhibited high transcriptional activities. Community-wide comparative analyses clearly showed that the AMD microorganisms adapted to the different environmental conditions via regulating the expression of genes involved in multiple in situ functional activities, including low-pH adaptation, carbon, nitrogen and phosphate assimilation, energy generation, environmental stress resistance, and other functions. Organism-wide comparative analyses of the active taxa revealed environment-dependent gene transcriptional profiles, especially the distinct strategies used by Acidithiobacillus ferrivorans and Leptospirillum ferrodiazotrophum in nutrients assimilation and energy generation for survival under different conditions. Overall, these findings demonstrate that the gene transcriptional profiles of AMD microorganisms are closely related to the site physiochemical characteristics, providing clues into the microbial response and adaptation mechanisms in the oligotrophic, extremely acidic environments.

High overlap of CNVs and selection signatures revealed by varLD analyses of taurine and zebu cattle

Science.gov (United States)

Selection Signatures (SS) assessed through analysis of genomic data are being widely studied to discover population specific regions selected via artificial or natural selection. Different methodologies have been proposed for these analyses, each having specific limitations as to the age of the sele...

Family-based Association Analyses of Imputed Genotypes Reveal Genome-Wide Significant Association of Alzheimer’s disease with OSBPL6, PTPRG and PDCL3

Science.gov (United States)

Herold, Christine; Hooli, Basavaraj V.; Mullin, Kristina; Liu, Tian; Roehr, Johannes T; Mattheisen, Manuel; Parrado, Antonio R.; Bertram, Lars; Lange, Christoph; Tanzi, Rudolph E.

2015-01-01

The genetic basis of Alzheimer's disease (AD) is complex and heterogeneous. Over 200 highly penetrant pathogenic variants in the genes APP, PSEN1 and PSEN2 cause a subset of early-onset familial Alzheimer's disease (EOFAD). On the other hand, susceptibility to late-onset forms of AD (LOAD) is indisputably associated to the ε4 allele in the gene APOE, and more recently to variants in more than two-dozen additional genes identified in the large-scale genome-wide association studies (GWAS) and meta-analyses reports. Taken together however, although the heritability in AD is estimated to be as high as 80%, a large proportion of the underlying genetic factors still remain to be elucidated. In this study we performed a systematic family-based genome-wide association and meta-analysis on close to 15 million imputed variants from three large collections of AD families (~3,500 subjects from 1,070 families). Using a multivariate phenotype combining affection status and onset age, meta-analysis of the association results revealed three single nucleotide polymorphisms (SNPs) that achieved genome-wide significance for association with AD risk: rs7609954 in the gene PTPRG (P-value = 3.98·10−08), rs1347297 in the gene OSBPL6 (P-value = 4.53·10−08), and rs1513625 near PDCL3 (P-value = 4.28·10−08). In addition, rs72953347 in OSBPL6 (P-value = 6.36·10−07) and two SNPs in the gene CDKAL1 showed marginally significant association with LOAD (rs10456232, P-value: 4.76·10−07; rs62400067, P-value: 3.54·10−07). In summary, family-based GWAS meta-analysis of imputed SNPs revealed novel genomic variants in (or near) PTPRG, OSBPL6, and PDCL3 that influence risk for AD with genome-wide significance. PMID:26830138

Effect of propofol in the immature rat brain on short- and long-term neurodevelopmental outcome.

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Tanja Karen

Full Text Available BACKGROUND: Propofol is commonly used as sedative in newborns and children. Recent experimental studies led to contradictory results, revealing neurodegenerative or neuroprotective properties of propofol on the developing brain. We investigated neurodevelopmental short- and long-term effects of neonatal propofol treatment. METHODS: 6-day-old Wistar rats (P6, randomised in two groups, received repeated intraperitoneal injections (0, 90, 180 min of 30 mg/kg propofol or normal saline and sacrificed 6, 12 and 24 hrs following the first injection. Cortical and thalamic areas were analysed by Western blot and quantitative real-time PCR (qRT-PCR for expression of apoptotic and neurotrophin-dependent signalling pathways. Long-term effects were assessed by Open-field and Novel-Object-Recognition at P30 and P120. RESULTS: Western blot analyses revealed a transient increase of activated caspase-3 in cortical, and a reduction of active mitogen-activated protein kinases (ERK1/2, AKT in cortical and thalamic areas. qRT-PCR analyses showed a down-regulation of neurotrophic factors (BDNF, NGF, NT-3 in cortical and thalamic regions. Minor impairment in locomotive activity was observed in propofol treated adolescent animals at P30. Memory or anxiety were not impaired at any time point. CONCLUSION: Exposing the neonatal rat brain to propofol induces acute neurotrophic imbalance and neuroapoptosis in a region- and time-specific manner and minor behavioural changes in adolescent animals.

Combined metabolomic and correlation networks analyses reveal fumarase insufficiency altered amino acid metabolism.

Science.gov (United States)

Hou, Entai; Li, Xian; Liu, Zerong; Zhang, Fuchang; Tian, Zhongmin

2018-04-01

Fumarase catalyzes the interconversion of fumarate and l-malate in the tricarboxylic acid cycle. Fumarase insufficiencies were associated with increased levels of fumarate, decreased levels of malate and exacerbated salt-induced hypertension. To gain insights into the metabolism profiles induced by fumarase insufficiency and identify key regulatory metabolites, we applied a GC-MS based metabolomics platform coupled with a network approach to analyze fumarase insufficient human umbilical vein endothelial cells (HUVEC) and negative controls. A total of 24 altered metabolites involved in seven metabolic pathways were identified as significantly altered, and enriched for the biological module of amino acids metabolism. In addition, Pearson correlation network analysis revealed that fumaric acid, l-malic acid, l-aspartic acid, glycine and l-glutamic acid were hub metabolites according to Pagerank based on their three centrality indices. Alanine aminotransferase and glutamate dehydrogenase activities increased significantly in fumarase deficiency HUVEC. These results confirmed that fumarase insufficiency altered amino acid metabolism. The combination of metabolomics and network methods would provide another perspective on expounding the molecular mechanism at metabolomics level. Copyright © 2017 John Wiley & Sons, Ltd.

Expression and phylogenetic analyses reveal paralogous lineages of putatively classical and non-classical MHC-I genes in three sparrow species (Passer).

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Drews, Anna; Strandh, Maria; Råberg, Lars; Westerdahl, Helena

2017-06-26

The Major Histocompatibility Complex (MHC) plays a central role in immunity and has been given considerable attention by evolutionary ecologists due to its associations with fitness-related traits. Songbirds have unusually high numbers of MHC class I (MHC-I) genes, but it is not known whether all are expressed and equally important for immune function. Classical MHC-I genes are highly expressed, polymorphic and present peptides to T-cells whereas non-classical MHC-I genes have lower expression, are more monomorphic and do not present peptides to T-cells. To get a better understanding of the highly duplicated MHC genes in songbirds, we studied gene expression in a phylogenetic framework in three species of sparrows (house sparrow, tree sparrow and Spanish sparrow), using high-throughput sequencing. We hypothesize that sparrows could have classical and non-classical genes, as previously indicated though never tested using gene expression. The phylogenetic analyses reveal two distinct types of MHC-I alleles among the three sparrow species, one with high and one with low level of polymorphism, thus resembling classical and non-classical genes, respectively. All individuals had both types of alleles, but there was copy number variation both within and among the sparrow species. However, the number of highly polymorphic alleles that were expressed did not vary between species, suggesting that the structural genomic variation is counterbalanced by conserved gene expression. Overall, 50% of the MHC-I alleles were expressed in sparrows. Expression of the highly polymorphic alleles was very variable, whereas the alleles with low polymorphism had uniformly low expression. Interestingly, within an individual only one or two alleles from the polymorphic genes were highly expressed, indicating that only a single copy of these is highly expressed. Taken together, the phylogenetic reconstruction and the analyses of expression suggest that sparrows have both classical and non

Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

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Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

2011-04-01

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

Simultaneous Detection of Bovine Theileria and Babesia Species by Reverse Line Blot Hybridization

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Gubbels, J. M.; de Vos, A. P.; van der Weide, M.; Viseras, J.; Schouls, L. M.; de Vries, E.; Jongejan, F.

1999-01-01

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species. PMID:10325324

Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.

Science.gov (United States)

Frantzen, Cyril A; Kleppen, Hans Petter; Holo, Helge

2018-02-01

Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp . lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR , a core gene, and epsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk. IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of

Post-facta Analyses of Fukushima Accident and Lessons Learned

Energy Technology Data Exchange (ETDEWEB)

Tanabe, Fumiya [Sociotechnical Systems Safety Research Institute, Ichige (Japan)

2014-08-15

Independent analyses have been performed of the core melt behavior of the Unit 1, Unit 2 and Unit 3 reactors of Fukushima Daiichi Nuclear Power Station on 11-15 March 2011. The analyses are based on a phenomenological methodology with measured data investigation and a simple physical model calculation. Estimated are time variation of core water level, core material temperature and hydrogen generation rate. The analyses have revealed characteristics of accident process of each reactor. In the case of Unit 2 reactor, the calculated result suggests little hydrogen generation because of no steam generation in the core for zirconium-steam reaction during fuel damage process. It could be the reason of no hydrogen explosion in the Unit 2 reactor building. Analyses have been performed also on the core material behavior in another chaotic period of 19-31 March 2011, and it resulted in a re-melt hypothesis that core material in each reactor should have melted again due to shortage of cooling water. The hypothesis is consistent with many observed features of radioactive materials dispersion into the environment.

New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization▿

Science.gov (United States)

Traversa, Donato; Iorio, Raffaella; Klei, Thomas R.; Kharchenko, Vitaliy A.; Gawor, Jakub; Otranto, Domenico; Sparagano, Olivier A. E.

2007-01-01

The ability of a reverse line blot (RLB) assay to identify 13 common species of equine small strongyles (cyathostomins) and to discriminate them from three Strongylus spp. (large strongyles) was demonstrated. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound species-specific probes. All cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles (Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris). This assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. Also, it could represent a basic step toward the development of a rapid and simple molecular test for the early detection of drug-resistant genotypes of horse strongyle species. PMID:17626168

Chemical analyses of wasp-associated streptomyces bacteria reveal a prolific potential for natural products discovery

DEFF Research Database (Denmark)

Poulsen, Michael; Oh, Dong-Chan; Clardy, Jon

2011-01-01

and solitary Hymenoptera. Here we test this possibility by examining two species of solitary mud dauber wasps, Sceliphron caementarium and Chalybion californicum. We performed enrichment isolations from 33 wasps and obtained more than 200 isolates of Streptomyces Actinobacteria. Chemical analyses of 15...... and antibacterial activity. The prevalence and anti-microbial properties of Actinobacteria associated with these two solitary wasp species suggest the potential role of these Streptomyces as antibiotic-producing symbionts, potentially helping defend their wasp hosts from pathogenic microbes. Finding...... phylogenetically diverse and chemically prolific Actinobacteria from solitary wasps suggests that insect-associated Actinobacteria can provide a valuable source of novel natural products of pharmaceutical interest....

ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE Análise de antígenos recombinantes de Treponema pallidum no diagnóstico da sífilis utilizando a técnica de Western Blotting

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Neuza Satomi SATO

1999-03-01

Full Text Available Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.Os antígenos recombinantes de Treponema pallidum GST-rTp47, GST-rTp17 e GST-rTp15, produzidos em fusão com glutationa S-transferase (GST em E. coli, foram analisados quanto ao potencial diagnóstico da sífilis pela técnica de Western blotting. Foram testadas 53 amostras, sendo 25 de pacientes em diferentes estágios clínicos da sífilis, com resultados positivos no teste treponêmico clássico; 25 amostras procedentes de doadores de banco de sangue, com sorologia negativa e 3 de pacientes com doença sexualmente transmissível não relacionado à sífilis. Todas as amostras de pacientes com sífilis apresentaram alta reatividade com o antígeno GST-rTp17. Quanto aos antígenos GST-rTp47 e GST-Tp15 verificou-se uma variação na presença ou na intensidade da reação em diferentes amostras de pacientes com sífilis, sem mostrar correlação com o estágio da doença. Nenhuma reatividade contra quaisquer desses antígenos foi observada com as amostras do grupo controle. Nenhuma das amostras testadas apresentaram reatividade com a GST purificada. A

Combined genomic and structural analyses of a cultured magnetotactic bacterium reveals its niche adaptation to a dynamic environment

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Ana Carolina Vieira Araujo

2016-10-01

Full Text Available Abstract Background Magnetotactic bacteria (MTB are a unique group of prokaryotes that have a potentially high impact on global geochemical cycling of significant primary elements because of their metabolic plasticity and the ability to biomineralize iron-rich magnetic particles called magnetosomes. Understanding the genetic composition of the few cultivated MTB along with the unique morphological features of this group of bacteria may provide an important framework for discerning their potential biogeochemical roles in natural environments. Results Genomic and ultrastructural analyses were combined to characterize the cultivated magnetotactic coccus Magnetofaba australis strain IT-1. Cells of this species synthesize a single chain of elongated, cuboctahedral magnetite (Fe3O4 magnetosomes that cause them to align along magnetic field lines while they swim being propelled by two bundles of flagella at velocities up to 300 μm s−1. High-speed microscopy imaging showed the cells move in a straight line rather than in the helical trajectory described for other magnetotactic cocci. Specific genes within the genome of Mf. australis strain IT-1 suggest the strain is capable of nitrogen fixation, sulfur reduction and oxidation, synthesis of intracellular polyphosphate granules and transporting iron with low and high affinity. Mf. australis strain IT-1 and Magnetococcus marinus strain MC-1 are closely related phylogenetically although similarity values between their homologous proteins are not very high. Conclusion Mf. australis strain IT-1 inhabits a constantly changing environment and its complete genome sequence reveals a great metabolic plasticity to deal with these changes. Aside from its chemoautotrophic and chemoheterotrophic metabolism, genomic data indicate the cells are capable of nitrogen fixation, possess high and low affinity iron transporters, and might be capable of reducing and oxidizing a number of sulfur compounds. The relatively

Temporal Fluctuation in North East Baltic Sea Region Cattle Population Revealed by Mitochondrial and Y-Chromosomal DNA Analyses

Science.gov (United States)

Niemi, Marianna; Bläuer, Auli; Iso-Touru, Terhi; Harjula, Janne; Nyström Edmark, Veronica; Rannamäe, Eve; Lõugas, Lembi; Sajantila, Antti; Lidén, Kerstin; Taavitsainen, Jussi-Pekka

2015-01-01

Background Ancient DNA analysis offers a way to detect changes in populations over time. To date, most studies of ancient cattle have focused on their domestication in prehistory, while only a limited number of studies have analysed later periods. Conversely, the genetic structure of modern cattle populations is well known given the undertaking of several molecular and population genetic studies. Results Bones and teeth from ancient cattle populations from the North-East Baltic Sea region dated to the Prehistoric (Late Bronze and Iron Age, 5 samples), Medieval (14), and Post-Medieval (26) periods were investigated by sequencing 667 base pairs (bp) from the mitochondrial DNA (mtDNA) and 155 bp of intron 19 in the Y-chromosomal UTY gene. Comparison of maternal (mtDNA haplotypes) genetic diversity in ancient cattle (45 samples) with modern cattle populations in Europe and Asia (2094 samples) revealed 30 ancient mtDNA haplotypes, 24 of which were shared with modern breeds, while 6 were unique to the ancient samples. Of seven Y-chromosomal sequences determined from ancient samples, six were Y2 and one Y1 haplotype. Combined data including Swedish samples from the same periods (64 samples) was compared with the occurrence of Y-chromosomal haplotypes in modern cattle (1614 samples). Conclusions The diversity of haplogroups was highest in the Prehistoric samples, where many haplotypes were unique. The Medieval and Post-Medieval samples also show a high diversity with new haplotypes. Some of these haplotypes have become frequent in modern breeds in the Nordic Countries and North-Western Russia while other haplotypes have remained in only a few local breeds or seem to have been lost. A temporal shift in Y-chromosomal haplotypes from Y2 to Y1 was detected that corresponds with the appearance of new mtDNA haplotypes in the Medieval and Post-Medieval period. This suggests a replacement of the Prehistoric mtDNA and Y chromosomal haplotypes by new types of cattle. PMID:25992976

Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells

DEFF Research Database (Denmark)

Ding, Li; Paszkowski-Rogacz, Maciej; Winzi, Maria

2015-01-01

We combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization, and "protein-level dependency" studies-a systematic technique that uncovers post-transcriptional regulation-to delineate the network of factors that control the expression...... of Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell...... identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide...

The genetic diversity of genus Bacillus and the related genera revealed by 16S rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey

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Arzu Coleri Cihan

2012-03-01

Full Text Available Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%, the facultative thermophiles (14% and the mesophiles (12%. These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16, Brevibacillus (13, Paenibacillus (1 and Thermoactinomycetes (2 were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4-100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6, B. lichenformis (3, B. subtilis (3, B. agri (3, B. smithii (2, T. vulgaris (2 and finally P. barengoltzii (1. In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ≤ 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.

Comparative Biochemical and Proteomic Analyses of Soybean Seed Cultivars Differing in Protein and Oil Content.

Science.gov (United States)

Min, Chul Woo; Gupta, Ravi; Kim, So Wun; Lee, So Eui; Kim, Yong Chul; Bae, Dong Won; Han, Won Young; Lee, Byong Won; Ko, Jong Min; Agrawal, Ganesh Kumar; Rakwal, Randeep; Kim, Sun Tae

2015-08-19

This study develops differential protein profiles of soybean (Glycine max) seeds (cv. Saedanbaek and Daewon) varying in protein (47.9 and 39.2%) and oil (16.3 and 19.7%) content using protamine sulfate (PS) precipitation method coupled with a 2D gel electrophoresis (2DGE) approach. Of 71 detected differential spots between Daewon and Saedanbaek, 48 were successfully identified by MALDI-TOF/TOF. Gene ontology analysis revealed that up-regulated proteins in Saedanbaek were largely associated with nutrient reservoir activity (42.6%), which included mainly seed-storage proteins (SSPs; subunits of glycinin and β-conglycinin). Similar results were also obtained in two cultivars of wild soybean (G. soja cv. WS22 and WS15) differing in protein content. Western blots confirmed higher accumulation of SSPs in protein-rich Saedanbaek. Findings presented and discussed in this study highlight a possible involvement of the urea cycle for increased accumulation of SSPs and hence the higher protein content in soybean seeds.

Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis

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William H Roldán

2009-05-01

Full Text Available To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis. Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3%, whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100% concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3% and 53.7%, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

Phylogenomic analyses data of the avian phylogenomics project

DEFF Research Database (Denmark)

Jarvis, Erich D; Mirarab, Siavash; Aberer, Andre J

2015-01-01

BACKGROUND: Determining the evolutionary relationships among the major lineages of extant birds has been one of the biggest challenges in systematic biology. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae...... and two of the five Palaeognathae orders. We used these genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomic analyses. FINDINGS: Here we present the datasets associated with the phylogenomic analyses, which include sequence alignment files consisting of nucleotides......ML algorithm or when using statistical binning with the coalescence-based MP-EST algorithm (which we refer to as MP-EST*). Other data sets, such as the coding sequence of some exons, revealed other properties of genome evolution, namely convergence. CONCLUSIONS: The Avian Phylogenomics Project is the largest...

Study on sensitivity of southern blotting hybridization using a 32P-labeled probe of PCR products in detecting human cytomegalovirus

International Nuclear Information System (INIS)

Bu Hengfu; Chen Juan; Shen Rongsen; Ma Liren; Xu Yongqiang

1996-01-01

Southern blotting hybridization (SBH) using a 32 P-labeled probe is one of the most practical methods for genetic diagnosis of pathogen. On the basis of establishing PCR and nested PCR for detecting human cytomegalovirus (HCMV), a 32 P-labeled probe was prepared with the amplified products of 613 bp PCR outer primers and hybridized with 300 bp inner primer amplified product, resulting in increase in detecting sensitivity from 17 ng (in 1.2% agarose electrophoresis) before SBH to 500 pg (autoradiographed), in other words, increasing the sensitivity of detecting HCMV by 10 2 dilutions after using SBH. The method of PCR and SBH using a 32 P-labeled probe could detect less than 1 gene copy of HCMV, therefore, it is a rapid and reliable diagnosis method for detecting HCMV latent infection

Comparative live-cell imaging analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa reveal novel features of the filamentous fungal polarisome.

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Alexander Lichius

Full Text Available A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk, whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.

Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

Science.gov (United States)

Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

2009-12-01

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay.

Science.gov (United States)

Tsaltas, G; Ford, C H; Gallant, M

1992-01-01

One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).

A review of bioinformatic methods for forensic DNA analyses.

Science.gov (United States)

Liu, Yao-Yuan; Harbison, SallyAnn

2018-03-01

Short tandem repeats, single nucleotide polymorphisms, and whole mitochondrial analyses are three classes of markers which will play an important role in the future of forensic DNA typing. The arrival of massively parallel sequencing platforms in forensic science reveals new information such as insights into the complexity and variability of the markers that were previously unseen, along with amounts of data too immense for analyses by manual means. Along with the sequencing chemistries employed, bioinformatic methods are required to process and interpret this new and extensive data. As more is learnt about the use of these new technologies for forensic applications, development and standardization of efficient, favourable tools for each stage of data processing is being carried out, and faster, more accurate methods that improve on the original approaches have been developed. As forensic laboratories search for the optimal pipeline of tools, sequencer manufacturers have incorporated pipelines into sequencer software to make analyses convenient. This review explores the current state of bioinformatic methods and tools used for the analyses of forensic markers sequenced on the massively parallel sequencing (MPS) platforms currently most widely used. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysing Access Control Specifications

DEFF Research Database (Denmark)

Probst, Christian W.; Hansen, René Rydhof

2009-01-01

When prosecuting crimes, the main question to answer is often who had a motive and the possibility to commit the crime. When investigating cyber crimes, the question of possibility is often hard to answer, as in a networked system almost any location can be accessed from almost anywhere. The most...... common tool to answer this question, analysis of log files, faces the problem that the amount of logged data may be overwhelming. This problems gets even worse in the case of insider attacks, where the attacker’s actions usually will be logged as permissible, standard actions—if they are logged at all....... Recent events have revealed intimate knowledge of surveillance and control systems on the side of the attacker, making it often impossible to deduce the identity of an inside attacker from logged data. In this work we present an approach that analyses the access control configuration to identify the set...

Een kwantitatieve analyse van Nederlandse bijdragen aan internationaal arbeids- en organisatiepsychologisch onderzoek

NARCIS (Netherlands)

de Dreu, C.K.W.

2010-01-01

The author reports a quantitative analysis of the absolute and relative numbers of publications in six high-ranking journals on work and organisational psychology, (co-)authored by scientists with a Dutch, German, or British affiliation. In contrast to Germany and Great Britain, analyses reveal a

Comprehensive genetic analyses reveal evolutionary distinction of a mouse (Zapus hudsonius preblei) proposed for delisting from the US Endangered Species Act.

Science.gov (United States)

King, Tim L; Switzer, John F; Morrison, Cheryl L; Eackles, Michael S; Young, Colleen C; Lubinski, Barbara A; Cryan, Paul

2006-12-01

Zapus hudsonius preblei, listed as threatened under the US Endangered Species Act (ESA), is one of 12 recognized subspecies of meadow jumping mice found in North America. Recent morphometric and phylogenetic comparisons among Z. h. preblei and neighbouring conspecifics questioned the taxonomic status of selected subspecies, resulting in a proposal to delist the Z. h. preblei from the ESA. We present additional analyses of the phylogeographic structure within Z. hudsonius that calls into question previously published data (and conclusions) and confirms the original taxonomic designations. A survey of 21 microsatellite DNA loci and 1380 base pairs from two mitochondrial DNA (mtDNA) regions (control region and cytochrome b) revealed that each Z. hudsonius subspecies is genetically distinct. These data do not support the null hypothesis of a homogeneous gene pool among the five subspecies found within the southwestern portion of the species' range. The magnitude of the observed differentiation was considerable and supported by significant findings for nearly every statistical comparison made, regardless of the genome or the taxa under consideration. Structuring of nuclear multilocus genotypes and subspecies-specific mtDNA haplotypes corresponded directly with the disjunct distributions of the subspecies investigated. Given the level of correspondence between the observed genetic population structure and previously proposed taxonomic classification of subspecies (based on the geographic separation and surveys of morphological variation), we conclude that the nominal subspecies surveyed in this study do not warrant synonymy, as has been proposed for Z. h. preblei, Z. h. campestris, and Z. h. intermedius.

Porcine Cysticercosis: Possible Cross-Reactivity of Taenia hydatigena to GP50 Antigen in the Enzyme-Linked Immunoelectrotransfer Blot Assay.

Science.gov (United States)

Muro, Claudio; Gomez-Puerta, Luis A; Flecker, Robert H; Gamboa, Ricardo; Barreto, Percy Vilchez; Dorny, Pierre; Tsang, Victor C W; Gilman, Robert H; Gonzalez, Armando E; Garcia, Hector H; O'Neal, Seth E; For The Cysticercosis Working Group In Peru

2017-12-01

The lentil lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB, reported sensitivity 99% and specificity 100%) is used as a serologic marker of exposure to Taenia solium in pigs. However, only a limited number of parasites have been evaluated for cross reactivity. Pigs may host other related cestode infections, including Taenia hydatigena, which have not been formally evaluated for cross-reactions. We investigated a corral in Tumbes, Peru, a region where a cysticercosis elimination demonstration project was completed in 2012. In this corral, 14/19 (73.7%) 6-8-week-old piglets were reactive to GP50 on LLGP EITB, and all had circulating Taenia sp. antigens. From eight necropsied piglets; four were infected with T. hydatigena metacestodes whereas none had evidence of T. solium infection. Two resident dogs were subsequently confirmed to have T. hydatigena taeniasis. These results suggest GP50 cross-reactivity in T. hydatigena- infected pigs, although controlled experimental infection is needed to confirm this hypothesis.

Data-Independent Acquisition-Based Quantitative Proteomic Analysis Reveals Potential Biomarkers of Kidney Cancer.

Science.gov (United States)

Song, Yimeng; Zhong, Lijun; Zhou, Juntuo; Lu, Min; Xing, Tianying; Ma, Lulin; Shen, Jing

2017-12-01

Renal cell carcinoma (RCC) is a malignant and metastatic cancer with 95% mortality, and clear cell RCC (ccRCC) is the most observed among the five major subtypes of RCC. Specific biomarkers that can distinguish cancer tissues from adjacent normal tissues should be developed to diagnose this disease in early stages and conduct a reliable prognostic evaluation. Data-independent acquisition (DIA) strategy has been widely employed in proteomic analysis because of various advantages, including enhanced protein coverage and reliable data acquisition. In this study, a DIA workflow is constructed on a quadrupole-Orbitrap LC-MS platform to reveal dysregulated proteins between ccRCC and adjacent normal tissues. More than 4000 proteins are identified, 436 of these proteins are dysregulated in ccRCC tissues. Bioinformatic analysis reveals that multiple pathways and Gene Ontology items are strongly associated with ccRCC. The expression levels of L-lactate dehydrogenase A chain, annexin A4, nicotinamide N-methyltransferase, and perilipin-2 examined through RT-qPCR, Western blot, and immunohistochemistry confirm the validity of the proteomic analysis results. The proposed DIA workflow yields optimum time efficiency and data reliability and provides a good choice for proteomic analysis in biological and clinical studies, and these dysregulated proteins might be potential biomarkers for ccRCC diagnosis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rendimiento diagnóstico del Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana

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Kelly Davelois

Full Text Available Objetivo. Determinar el rendimiento diagnóstico de la técnica de Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana. Materiales y métodos. Estudio transversal de evaluación de prueba diagnóstica. Se obtuvieron los antígenos de excreción-secreción de las larvas de Taenia solium, quistes de Echinococcus granulosus; y la forma adulta de Fasciola hepática; que luego fueron separados electroforéticamente en geles de poliacrilamida individuales, transferidos y fijados a una membrana de nitrocelulosa para ser enfrentados con sueros de pacientes con las tres parasitosis. La sensibilidad de la técnica se evaluó empleando 300 sueros individuales, 60 pools de dos parasitosis y 20 pools de tres parasitosis y la especificidad con 75 sueros de pacientes con otras parasitosis, 10 de pacientes con otras enfermedades y 15 sueros de personas no parasitadas. Resultados. La técnica reconoció trece glicoproteínas (GP: GP 35, 31, 24, 23, 18, 17, 14 y 13 kDa para cisticercosis, GP 8,16 y 21 kDa para hidatidosis y GP: 17 y 23 kDa para fascioliasis. La prueba detectó la presencia de anticuerpos alcanzando una sensibilidad de 96% (IC95%: 94,62-98,54% en la detección de una o las trece bandas, una especificidad de 100% (IC95%: 99,50 - 100,00%; individualmente, se tuvo una sensibilidad para cisticercosis de 97% (IC95%: 93,16-100%, para hidatidosis de 94% (IC95%: 88,85-99,15% y para fascioliasis de 96% (IC95%: 91,66-100%. Conclusiones. La prueba de Western blot es eficaz en la detección, simultanea de anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana, y puede ser utilizada como prueba de descarte o confirmatoria en zonas endémicas.

Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors

International Nuclear Information System (INIS)

Klajic, Jovana; Tost, Jörg; Kristensen, Vessela N; Fleischer, Thomas; Dejeux, Emelyne; Edvardsen, Hege; Warnberg, Fredrik; Bukholm, Ida; Lønning, Per Eystein; Solvang, Hiroko; Børresen-Dale, Anne-Lise

2013-01-01

Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above

Changes in HSP gene and protein expression in natural scrapie with brain damage

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2011-01-01

Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease. PMID:21314976

Changes in HSP gene and protein expression in natural scrapie with brain damage

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Serrano Carmen

2011-01-01

Full Text Available Abstract Heat shock proteins (Hsp perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

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Stankowski, L.F. Jr.; Tindall, K.R.; Hsie, A.W.

1983-01-01

Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10 6 clonable cells. As with HGPRT - mutants, XGPRT - mutants can be selected in 6-thioguanine. AS52 (XGPRT + ) and wild type CHO (HGPRT + ) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT - to HGPRT - mutants induced per unit dose (J/m 2 for UV light and μg/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT - AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment

Genome-based comparative analyses of Antarctic and temperate species of Paenibacillus.

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Melissa Dsouza

Full Text Available Antarctic soils represent a unique environment characterised by extremes of temperature, salinity, elevated UV radiation, low nutrient and low water content. Despite the harshness of this environment, members of 15 bacterial phyla have been identified in soils of the Ross Sea Region (RSR. However, the survival mechanisms and ecological roles of these phyla are largely unknown. The aim of this study was to investigate whether strains of Paenibacillus darwinianus owe their resilience to substantial genomic changes. For this, genome-based comparative analyses were performed on three P. darwinianus strains, isolated from gamma-irradiated RSR soils, together with nine temperate, soil-dwelling Paenibacillus spp. The genome of each strain was sequenced to over 1,000-fold coverage, then assembled into contigs totalling approximately 3 Mbp per genome. Based on the occurrence of essential, single-copy genes, genome completeness was estimated at approximately 88%. Genome analysis revealed between 3,043-3,091 protein-coding sequences (CDSs, primarily associated with two-component systems, sigma factors, transporters, sporulation and genes induced by cold-shock, oxidative and osmotic stresses. These comparative analyses provide an insight into the metabolic potential of P. darwinianus, revealing potential adaptive mechanisms for survival in Antarctic soils. However, a large proportion of these mechanisms were also identified in temperate Paenibacillus spp., suggesting that these mechanisms are beneficial for growth and survival in a range of soil environments. These analyses have also revealed that the P. darwinianus genomes contain significantly fewer CDSs and have a lower paralogous content. Notwithstanding the incompleteness of the assemblies, the large differences in genome sizes, determined by the number of genes in paralogous clusters and the CDS content, are indicative of genome content scaling. Finally, these sequences are a resource for further

Molecular analyses reveal two geographic and genetic lineages for tapeworms, Taenia solium and Taenia saginata, from Ecuador using mitochondrial DNA.

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Solano, Danilo; Navarro, Juan Carlos; León-Reyes, Antonio; Benítez-Ortiz, Washington; Rodríguez-Hidalgo, Richar

2016-12-01

Tapeworms Taenia solium and Taenia saginata are the causative agents of taeniasis/cysticercosis. These are diseases with high medical and veterinary importance due to their impact on public health and rural economy in tropical countries. The re-emergence of T. solium as a result of human migration, the economic burden affecting livestock industry, and the large variability of symptoms in several human cysticercosis, encourage studies on genetic diversity, and the identification of these parasites with molecular phylogenetic tools. Samples collected from the Ecuadorian provinces: Loja, Guayas, Manabí, Tungurahua (South), and Imbabura, Pichincha (North) from 2000 to 2012 were performed under Maximum Parsimony analyses and haplotype networks using partial sequences of mitochondrial DNA, cytochrome oxidase subunit I (COI) and NADH subunit I (NDI), from Genbank and own sequences of Taenia solium and Taenia saginata from Ecuador. Both species have shown reciprocal monophyly, which confirms its molecular taxonomic identity. The COI and NDI genes results suggest phylogenetic structure for both parasite species from south and north of Ecuador. In T. solium, both genes gene revealed greater geographic structure, whereas in T. saginata, the variability for both genes was low. In conclusion, COI haplotype networks of T. solium suggest two geographical events in the introduction of this species in Ecuador (African and Asian lineages) and occurring sympatric, probably through the most common routes of maritime trade between the XV-XIX centuries. Moreover, the evidence of two NDI geographical lineages in T. solium from the north (province of Imbabura) and the south (province of Loja) of Ecuador derivate from a common Indian ancestor open new approaches for studies on genetic populations and eco-epidemiology. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

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Shin, Kwang-Soo, E-mail: shinks@dju.kr [Division of Life Science, Daejeon University, Daejeon, 300-716 (Korea, Republic of); Kim, Young Hwan [Biomedical Omics Team, Korea Basic Science Institute (KBSI), Ohcang, 368-883 (Korea, Republic of); Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Department of Bio-Analytical Science, University of Science and Technology, Daejeon, 305-333 (Korea, Republic of); Yu, Jae-Hyuk, E-mail: jyu1@wisc.edu [Departments of Bacteriology and Genetics, The University of Wisconsin–Madison, Madison, WI, 53706 (United States)

2015-07-31

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus.

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

International Nuclear Information System (INIS)

Shin, Kwang-Soo; Kim, Young Hwan; Yu, Jae-Hyuk

2015-01-01

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus

The early asthmatic response is associated with glycolysis, calcium binding and mitochondria activity as revealed by proteomic analysis in rats

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Xu Yu-Dong

2010-08-01

Full Text Available Abstract Background The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR, which is characterized by acute airway obstruction beginning within a few minutes. The EAR is the earliest indicator of the pathological progression of allergic asthma. Because the molecular mechanism underlying the EAR is not fully defined, this study will contribute to a better understanding of asthma. Methods In order to gain insight into the molecular basis of the EAR, we examined changes in protein expression patterns in the lung tissue of asthmatic rats during the EAR using 2-DE/MS-based proteomic techniques. Bioinformatic analysis of the proteomic data was then performed using PPI Spider and KEGG Spider to investigate the underlying molecular mechanism. Results In total, 44 differentially expressed protein spots were detected in the 2-DE gels. Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry. During subsequent bioinformatic analysis, the gene ontology classification, the protein-protein interaction networking and the biological pathway exploration demonstrated that the identified proteins were mainly involved in glycolysis, calcium binding and mitochondrial activity. Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses. Conclusions Our results reveal that the allergen-induced EAR in asthmatic rats is associated with glycolysis, calcium binding and mitochondrial activity, which could establish a functional network in which calcium binding may play a central role in promoting the progression of asthma.

Whole Genome Analyses of a Well-Differentiated Liposarcoma Reveals Novel SYT1 and DDR2 Rearrangements

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Egan, Jan B.; Barrett, Michael T.; Champion, Mia D.; Middha, Sumit; Lenkiewicz, Elizabeth; Evers, Lisa; Francis, Princy; Schmidt, Jessica; Shi, Chang-Xin; Van Wier, Scott; Badar, Sandra; Ahmann, Gregory; Kortuem, K. Martin; Boczek, Nicole J.; Fonseca, Rafael; Craig, David W.; Carpten, John D.; Borad, Mitesh J.; Stewart, A. Keith

2014-01-01

Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2. PMID:24505276

Whole genome analyses of a well-differentiated liposarcoma reveals novel SYT1 and DDR2 rearrangements.

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Jan B Egan

Full Text Available Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.

Structural Analyses of Avocado sunblotch viroid Reveal Differences in the Folding of Plus and Minus RNA Strands

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Clémentine Delan-Forino

2014-01-01

Full Text Available Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd, a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle.

Self-disclosure on Facebook: How much do we really reveal?

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Stephanie Day

Full Text Available This paper investigates the use of the social networking site Facebook to self-disclose and analyses the responses of a small group of Facebook users surveyed about their own willingness to self-disclose. An online survey was used to ask Facebook users about their level of Facebook use, what types of personal information they are willing to reveal and the frequency of these personal revelations. The survey also asked the participants to take a look at their publicly viewable profile and the types of information revealed there. Results indicated that overall, most people tended to be cautious about the types of information they revealed, posted mainly positive statements about themselves and were aware of personal privacy issues.

Comparison of RT-PCR-Dot blot hybridization based on radioisotope 32P with conventional RT-PCR and commercial ELISA Assays for blood screening of HIV-1

International Nuclear Information System (INIS)

Maria Lina R; Andi Yasmon

2011-01-01

There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32 P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked lmmunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-l RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RTPCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) were negative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has high potential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA. (author)

Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

DEFF Research Database (Denmark)

Sahm, K.; MacGregor, BJ; Jørgensen, BB

1999-01-01

In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell. Here, we report evidence that quantification of SRB rRNA by slot-blot hybridization...... between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulpho-bacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates......, directly above the sulphate reduction maximum. Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment. Cellular sulphate reduction rates calculated on the basis...

Variability of protein level and phosphorylation status caused by biopsy protocol design in human skeletal muscle analyses

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Caron Marc-André

2011-11-01

Full Text Available Abstract Background Bergström needle biopsy is widely used to sample skeletal muscle in order to study cell signaling directly in human tissue. Consequences of the biopsy protocol design on muscle protein quantity and quality remain unclear. The aim of the present study was to assess the impact of different events surrounding biopsy protocol on the stability of the Western blot signal of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, Akt, glycogen synthase kinase-3β (GSK-3β, muscle RING finger protein 1 (MuRF1 and p70 S6 kinase (p70 S6K. Six healthy subjects underwent four biopsies of the vastus lateralis, distributed into two distinct visits spaced by 48 hrs. At visit 1, a basal biopsy in the right leg was performed in the morning (R1 followed by a second in the left leg in the afternoon (AF. At visit 2, a second basal biopsy (R2 was collected from the right leg. Low intensity mobilization (3 × 20 right leg extensions was performed and a final biopsy (Mob was collected using the same incision site as R2. Results Akt and p70 S6K phosphorylation levels were increased by 83% when AF biopsy was compared to R1. Mob condition induced important phosphorylation of p70 S6K when compared to R2. Comparison of R1 and R2 biopsies revealed a relative stability of the signal for both total and phosphorylated proteins. Conclusions This study highlights the importance to standardize muscle biopsy protocols in order to minimize the method-induced variation when analyzing Western blot signals.

Categorizing words using 'frequent frames': what cross-linguistic analyses reveal about distributional acquisition strategies.

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Chemla, Emmanuel; Mintz, Toben H; Bernal, Savita; Christophe, Anne

2009-04-01

Mintz (2003) described a distributional environment called a frame, defined as the co-occurrence of two context words with one intervening target word. Analyses of English child-directed speech showed that words that fell within any frequently occurring frame consistently belonged to the same grammatical category (e.g. noun, verb, adjective, etc.). In this paper, we first generalize this result to French, a language in which the function word system allows patterns that are potentially detrimental to a frame-based analysis procedure. Second, we show that the discontinuity of the chosen environments (i.e. the fact that target words are framed by the context words) is crucial for the mechanism to be efficient. This property might be relevant for any computational approach to grammatical categorization. Finally, we investigate a recursive application of the procedure and observe that the categorization is paradoxically worse when context elements are categories rather than actual lexical items. Item-specificity is thus also a core computational principle for this type of algorithm. Our analysis, along with results from behavioural studies (Gómez, 2002; Gómez and Maye, 2005; Mintz, 2006), provides strong support for frames as a basis for the acquisition of grammatical categories by infants. Discontinuity and item-specificity appear to be crucial features.

Transcriptome-wide analyses indicate mitochondrial responses to particulate air pollution exposure

DEFF Research Database (Denmark)

Winckelmans, Ellen; Nawrot, Tim S.; Tsamou, Maria

2017-01-01

validation cohort (n = 169, 55.6% women). Results: Overrepresentation analyses revealed significant pathways (p-value transport chain (ETC) for medium-term exposure in women. For men, medium-term PM10....... Conclusions: In this exploratory study, we identified mitochondrial genes and pathways associated with particulate air pollution indicating upregulation of energy producing pathways as a potential mechanism to compensate for PM-induced mitochondrial damage....

Identification of Bombyx mori bidensovirus VD1-ORF4 reveals a novel protein associated with viral structural component.

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Li, Guohui; Hu, Zhaoyang; Guo, Xuli; Li, Guangtian; Tang, Qi; Wang, Peng; Chen, Keping; Yao, Qin

2013-06-01

Bombyx mori bidensovirus (BmBDV) VD1-ORF4 (open reading frame 4, ORF4) consists of 3,318 nucleotides, which codes for a predicted 1,105-amino acid protein containing a conserved DNA polymerase motif. However, its functions in viral propagation remain unknown. In the current study, the transcription of VD1-ORF4 was examined from 6 to 96 h postinfection (p.i.) by RT-PCR, 5'-RACE revealed the transcription initiation site of BmBDV ORF4 to be -16 nucleotides upstream from the start codon, and 3'-RACE revealed the transcription termination site of VD1-ORF4 to be +7 nucleotides downstream from termination codon. Three different proteins were examined in the extracts of BmBDV-infected silkworms midguts by Western blot using raised antibodies against VD1-ORF4 deduced amino acid, and a specific protein band about 53 kDa was further detected in purified virions using the same antibodies. Taken together, BmBDV VD1-ORF4 codes for three or more proteins during the viral life cycle, one of which is a 53 kDa protein and confirmed to be a component of BmBDV virion.

Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

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Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

2009-01-01

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

Pancreatic Adenocarcinoma Therapeutic Targets Revealed by Tumor-Stroma Cross-Talk Analyses in Patient-Derived Xenografts

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Rémy Nicolle

2017-11-01

Full Text Available Preclinical models based on patient-derived xenografts have remarkable specificity in distinguishing transformed human tumor cells from non-transformed murine stromal cells computationally. We obtained 29 pancreatic ductal adenocarcinoma (PDAC xenografts from either resectable or non-resectable patients (surgery and endoscopic ultrasound-guided fine-needle aspirate, respectively. Extensive multiomic profiling revealed two subtypes with distinct clinical outcomes. These subtypes uncovered specific alterations in DNA methylation and transcription as well as in signaling pathways involved in tumor-stromal cross-talk. The analysis of these pathways indicates therapeutic opportunities for targeting both compartments and their interactions. In particular, we show that inhibiting NPC1L1 with Ezetimibe, a clinically available drug, might be an efficient approach for treating pancreatic cancers. These findings uncover the complex and diverse interplay between PDAC tumors and the stroma and demonstrate the pivotal role of xenografts for drug discovery and relevance to PDAC.

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

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Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

2007-12-01

A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

Genomic and transcriptomic analyses reveal differential regulation of diverse terpenoid and polyketides secondary metabolites in Hericium erinaceus.

Science.gov (United States)

Chen, Juan; Zeng, Xu; Yang, Yan Long; Xing, Yong Mei; Zhang, Qi; Li, Jia Mei; Ma, Ke; Liu, Hong Wei; Guo, Shun Xing

2017-08-31

The lion's mane mushroom Hericium erinaceus is a famous traditional medicinal fungus credited with anti-dementia activity and a producer of cyathane diterpenoid natural products (erinacines) useful against nervous system diseases. To date, few studies have explored the biosynthesis of these compounds, although their chemical synthesis is known. Here, we report the first genome and tanscriptome sequence of the medicinal fungus H. erinaceus. The size of the genome is 39.35 Mb, containing 9895 gene models. The genome of H. erinaceus reveals diverse enzymes and a large family of cytochrome P450 (CYP) proteins involved in the biosynthesis of terpenoid backbones, diterpenoids, sesquiterpenes and polyketides. Three gene clusters related to terpene biosynthesis and one gene cluster for polyketides biosynthesis (PKS) were predicted. Genes involved in terpenoid biosynthesis were generally upregulated in mycelia, while the PKS gene was upregulated in the fruiting body. Comparative genome analysis of 42 fungal species of Basidiomycota revealed that most edible and medicinal mushroom show many more gene clusters involved in terpenoid and polyketide biosynthesis compared to the pathogenic fungi. None of the gene clusters for terpenoid or polyketide biosynthesis were predicted in the poisonous mushroom Amanita muscaria. Our findings may facilitate future discovery and biosynthesis of bioactive secondary metabolites from H. erinaceus and provide fundamental information for exploring the secondary metabolites in other Basidiomycetes.

Differential co-expression and regulation analyses reveal different mechanisms underlying major depressive disorder and subsyndromal symptomatic depression.

Science.gov (United States)

Xu, Fan; Yang, Jing; Chen, Jin; Wu, Qingyuan; Gong, Wei; Zhang, Jianguo; Shao, Weihua; Mu, Jun; Yang, Deyu; Yang, Yongtao; Li, Zhiwei; Xie, Peng

2015-04-03

Recent depression research has revealed a growing awareness of how to best classify depression into depressive subtypes. Appropriately subtyping depression can lead to identification of subtypes that are more responsive to current pharmacological treatment and aid in separating out depressed patients in which current antidepressants are not particularly effective. Differential co-expression analysis (DCEA) and differential regulation analysis (DRA) were applied to compare the transcriptomic profiles of peripheral blood lymphocytes from patients with two depressive subtypes: major depressive disorder (MDD) and subsyndromal symptomatic depression (SSD). Six differentially regulated genes (DRGs) (FOSL1, SRF, JUN, TFAP4, SOX9, and HLF) and 16 transcription factor-to-target differentially co-expressed gene links or pairs (TF2target DCLs) appear to be the key differential factors in MDD; in contrast, one DRG (PATZ1) and eight TF2target DCLs appear to be the key differential factors in SSD. There was no overlap between the MDD target genes and SSD target genes. Venlafaxine (Efexor™, Effexor™) appears to have a significant effect on the gene expression profile of MDD patients but no significant effect on the gene expression profile of SSD patients. DCEA and DRA revealed no apparent similarities between the differential regulatory processes underlying MDD and SSD. This bioinformatic analysis may provide novel insights that can support future antidepressant R&D efforts.

Presence and seeding activity of pathological prion protein (PrP(TSE in skeletal muscles of white-tailed deer infected with chronic wasting disease.

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Martin L Daus

Full Text Available Chronic wasting disease (CWD is a contagious, rapidly spreading transmissible spongiform encephalopathy (TSE, or prion disease, occurring in cervids such as white tailed-deer (WTD, mule deer or elk in North America. Despite efficient horizontal transmission of CWD among cervids natural transmission of the disease to other species has not yet been observed. Here, we report for the first time a direct biochemical demonstration of pathological prion protein PrP(TSE and of PrP(TSE-associated seeding activity, the static and dynamic biochemical markers for biological prion infectivity, respectively, in skeletal muscles of CWD-infected cervids, i. e. WTD for which no clinical signs of CWD had been recognized. The presence of PrP(TSE was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrP(TSE was revealed by protein misfolding cyclic amplification (PMCA. Semi-quantitative Western blotting indicated that the concentration of PrP(TSE in skeletal muscles of CWD-infected WTD was approximately 2000-10,000-fold lower than in brain tissue. Tissue-blot-analyses revealed that PrP(TSE was located in muscle-associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrP(TSE in skeletal muscle from CWD-infected cervids suggests prevention of such tissue in the human diet as a precautionary measure for food safety, pending on further clarification of whether CWD may be transmissible to humans.

Divergent and convergent modes of interaction between wheat and Puccinia graminis f. sp. tritici isolates revealed by the comparative gene co-expression network and genome analyses.

Science.gov (United States)

Rutter, William B; Salcedo, Andres; Akhunova, Alina; He, Fei; Wang, Shichen; Liang, Hanquan; Bowden, Robert L; Akhunov, Eduard

2017-04-12

Two opposing evolutionary constraints exert pressure on plant pathogens: one to diversify virulence factors in order to evade plant defenses, and the other to retain virulence factors critical for maintaining a compatible interaction with the plant host. To better understand how the diversified arsenals of fungal genes promote interaction with the same compatible wheat line, we performed a comparative genomic analysis of two North American isolates of Puccinia graminis f. sp. tritici (Pgt). The patterns of inter-isolate divergence in the secreted candidate effector genes were compared with the levels of conservation and divergence of plant-pathogen gene co-expression networks (GCN) developed for each isolate. Comprative genomic analyses revealed substantial level of interisolate divergence in effector gene complement and sequence divergence. Gene Ontology (GO) analyses of the conserved and unique parts of the isolate-specific GCNs identified a number of conserved host pathways targeted by both isolates. Interestingly, the degree of inter-isolate sub-network conservation varied widely for the different host pathways and was positively associated with the proportion of conserved effector candidates associated with each sub-network. While different Pgt isolates tended to exploit similar wheat pathways for infection, the mode of plant-pathogen interaction varied for different pathways with some pathways being associated with the conserved set of effectors and others being linked with the diverged or isolate-specific effectors. Our data suggest that at the intra-species level pathogen populations likely maintain divergent sets of effectors capable of targeting the same plant host pathways. This functional redundancy may play an important role in the dynamic of the "arms-race" between host and pathogen serving as the basis for diverse virulence strategies and creating conditions where mutations in certain effector groups will not have a major effect on the pathogen

Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.

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Maite Sabalza

Full Text Available In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP and reverse dot-blot for detection (RDB and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.

Nuclear localization of the CK2α-subunit correlates with poor prognosis in Clear Cell Renal Cell Carcinoma

DEFF Research Database (Denmark)

Rabjerg, Maj; Guerra, Barbara; Oliván-Viguera, Aida

2017-01-01

Protein kinase CK2a, one of the two catalytic isoforms of the protein kinase CK2 has been shown to contribute to tumor development, tumor proliferation and suppression of apoptosis in various malignancies. We conducted this study to investigate CK2 expression in different subtypes of Renal Cell...... Carcinoma (RCC) and in the benign oncocytoma. qRT-PCR, immunohistochemistry and Western blot analyses revealed that CK2a expression was significantly increased at the mRNA and protein levels in clear cell RCC (ccRCC). Also the kinase activity of CK2 was significantly increased in ccRCC compared to normal...... renal cortex. Nuclear protein expression of CK2a correlated in univariate analysis with poor Progression Free Survival (HR = 8.11, p = 0.016). Functional analyses (cell proliferation assay) revealed an inhibitory effect of Caki-2 cell growth following CK2 inhibition with CX-4945. Our results suggest...

Analysing public relations education through international standards: The Portuguese case

OpenAIRE

Gonçalves, Gisela Marques Pereira; Spínola, Susana de Carvalho; Padamo, Celma

2013-01-01

By using international reports on PR education as a benchmark we analyse the status of PR higher education in Portugal. Despite differences among the study programs, the findings reveal that the standard five courses recommendation by the Commission on Public Relations Education (CPRE) are a part of Portuguese undergraduate curriculum. This includes 12 of the 14 content field guidelines needed to achieve the ideal master's program. Data shows, however, the difficulty of positioning public rel...

Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene

Science.gov (United States)

Macovei, Anca; Faè, Matteo; Biggiogera, Marco; de Sousa Araújo, Susana; Carbonera, Daniela; Balestrazzi, Alma

2018-01-01

The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively) and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL), the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL) and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells. PMID:29868059

Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene

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Anca Macovei

2018-05-01

Full Text Available The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2 involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively and a control (CTRL line carrying the empty vector were investigated. Transmission electron microscopy (TEM revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL, the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells.

Evolutionary trajectories of snake genes and genomes revealed by comparative analyses of five-pacer viper

Science.gov (United States)

Yin, Wei; Wang, Zong-ji; Li, Qi-ye; Lian, Jin-ming; Zhou, Yang; Lu, Bing-zheng; Jin, Li-jun; Qiu, Peng-xin; Zhang, Pei; Zhu, Wen-bo; Wen, Bo; Huang, Yi-jun; Lin, Zhi-long; Qiu, Bi-tao; Su, Xing-wen; Yang, Huan-ming; Zhang, Guo-jie; Yan, Guang-mei; Zhou, Qi

2016-01-01

Snakes have numerous features distinctive from other tetrapods and a rich history of genome evolution that is still obscure. Here, we report the high-quality genome of the five-pacer viper, Deinagkistrodon acutus, and comparative analyses with other representative snake and lizard genomes. We map the evolutionary trajectories of transposable elements (TEs), developmental genes and sex chromosomes onto the snake phylogeny. TEs exhibit dynamic lineage-specific expansion, and many viper TEs show brain-specific gene expression along with their nearby genes. We detect signatures of adaptive evolution in olfactory, venom and thermal-sensing genes and also functional degeneration of genes associated with vision and hearing. Lineage-specific relaxation of functional constraints on respective Hox and Tbx limb-patterning genes supports fossil evidence for a successive loss of forelimbs then hindlimbs during snake evolution. Finally, we infer that the ZW sex chromosome pair had undergone at least three recombination suppression events in the ancestor of advanced snakes. These results altogether forge a framework for our deep understanding into snakes' history of molecular evolution. PMID:27708285

Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

Science.gov (United States)

Richards, Mark J; Daniel, Susan

2017-02-07

The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

Science.gov (United States)

Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2017-02-22

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

Divergent Significance of Bone Mineral Density Changes in Aging Depending on Sites and Sex Revealed through Separate Analyses of Bone Mineral Content and Area

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Yasumoto Matsui

2012-01-01

Full Text Available Bone mineral density (aBMD is equivalent to bone mineral content (BMC divided by area. We rechecked the significance of aBMD changes in aging by examining BMC and area separately. Subjects were 1167 community-dwelling Japanese men and women, aged 40–79 years. ABMDs of femoral neck and lumbar spine were assessed by DXA twice, at 6-year intervals. The change rates of BMC and area, as well as aBMD, were calculated and described separately by the age stratum and by sex. In the femoral neck region, aBMDs were significantly decreased in all age strata by an increase in area as well as BMC loss in the same pattern in both sexes. In the lumbar spine region, aBMDs decreased until the age of 60 in women, caused by the significant BMC decrease accompanying the small area change. Very differently in men, aBMDs increased after their 50s due to BMC increase, accompanied by an area increase. Separate analyses of BMC and area change revealed that the significance of aBMD changes in aging was very divergent among sites and between sexes. This may explain in part the dissociation of aBMD change and bone strength, suggesting that we should be more cautious when interpreting the meaning of aBMD change.

Ubiquity of Polynucleobacter necessarius subspecies asymbioticus results from ecological diversification

Science.gov (United States)

Jezbera, Jan; Jezberová, Jitka; Brandt, Ulrike; Hahn, Martin W

2011-01-01

The subspecies Polynucleobacter necessarius asymbioticus (> 99% 16S rRNA similarity) has a cosmopolitan distribution and a ubiquitous occurrence in lentic freshwater habitats. We tested if the observed ubiquity of these free-living planktonic freshwater bacteria results from a euryoecious (generalist) adaptation of P. n. asymbioticus strains, or from ecological diversification within the subspecies. We developed a reverse line blot hybridization assay enabling the cultivation-independent detection of 13 groups within the subspecies in environmental samples. A set of 121 lentic freshwater habitats, spanning a broad variety of habitat types (e.g. pH levels ranging from 3.8 to 8.5) was investigated for the presence of these 13 P. n. asymbioticus groups. Statistical analyses of the reverse line blot hybridization detections revealed pronounced differences in habitat preferences of several of the groups. Their preferences differed regarding pH, conductivity, dissolved organic carbon and oxygen concentration of habitats. For some groups, differences in environmental preferences resulted even in complete niche separation between them. The revealed differences in habitat preferences suggest that the previously reported ubiquity of P. n. asymbioticus results from ecological diversification within the taxon and not from generalist adaptation of strains. PMID:21208356

Analysing the Improper Pronunciation of Diphthongs by Iraqi EFL learners

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Mukhalad Malik Almutalabi

2018-04-01

Full Text Available The current study aims at analysing the improper pronunciation of Iraqi EFL learners concerning the pronunciation of diphthongs in words of various syllables. It describes and identifies thoroughly the mispronunciations of such important sounds in English language. The study attempts at analysing such mispronunciations by clarifying and assigning the phonetic deviations of Iraqi EFL learners when they pronounce diphthongs. So the main objective of the study is to analyse the errors committed by Iraqi learners in the pronunciation of diphthongs grouping each error into its specific category. To verify the objective of the paper, 25 Iraqi EFL learners from the department of English at Cihan University/ Slemani are chosen to be the main participants of the study. The test which was conducted in the laboratory of the Department of English contained 10 words comprising various diphthongs. The results clearly revealed that mispronouncing English diphthongs by Iraqi EFL were mostly observed by replacing the required diphthong with another improper one and they also tended to use simple vowels instead of the correct required diphthongs.

Financial relationships in economic analyses of targeted therapies in oncology.

Science.gov (United States)

Valachis, Antonis; Polyzos, Nikolaos P; Nearchou, Andreas; Lind, Pehr; Mauri, Davide

2012-04-20

A potential financial relationship between investigators and pharmaceutical manufacturers has been associated with an increased likelihood of reporting favorable conclusions about a sponsor's proprietary agent in pharmacoeconomic studies. The purpose of this study is to investigate whether there is an association between financial relationships and outcome in economic analyses of new targeted therapies in oncology. We searched PubMed (last update June 2011) for economic analyses of targeted therapies (including monoclonal antibodies, tyrosine-kinase inhibitors, and mammalian target of rapamycin inhibitors) in oncology. The trials were qualitatively rated regarding the cost assessment as favorable, neutral, or unfavorable on the basis of prespecified criteria. Overall, 81 eligible studies were identified. Economic analyses that were funded by pharmaceutical companies were more likely to report favorable qualitative cost estimates (28 [82%] of 34 v 21 [45%] of 47; P = .003). The presence of an author affiliated with manufacturer was not associated with study outcome. Furthermore, if only studies including a conflict of interest statement were included (66 of 81), studies that reported any financial relationship with manufacturers (author affiliation and/or funding and/or other financial relationship) were more likely to report favorable results of targeted therapies compared with studies without financial relationship (32 [71%] of 45 v nine [43%] of 21; P = .025). Our study reveals a potential threat for industry-related bias in economic analyses of targeted therapies in oncology in favor of analyses with financial relationships between authors and manufacturers. A more balanced funding of economic analyses from other sources may allow greater confidence in the interpretation of their results.

Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

Science.gov (United States)

Morgon, Adriano M; Belisario-Ferrari, Matheus R; Trevisan-Silva, Dilza; Meissner, Gabriel O; Vuitika, Larissa; Marin, Brenda; Tashima, Alexandre K; Gremski, Luiza H; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Veiga, Silvio S; Chaim, Olga M

2016-01-01

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

Science.gov (United States)

Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

2016-08-01

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Floral reversion mechanism in longan (Dimocarpus longan Lour.) revealed by proteomic and anatomic analyses.

Science.gov (United States)

You, Xiangrong; Wang, Lingxia; Liang, Wenyu; Gai, Yonghong; Wang, Xiaoyan; Chen, Wei

2012-02-02

Two-dimensional gel electrophoresis (2-DE) was used to analyze the proteins related to floral reversion in Dimocarpus longan Lour. Proteins were extracted from buds undergoing the normal process of flowering and from those undergoing floral reversion in three developing stages in D. longan. Differentially expressed proteins were identified from the gels after 2-DE analysis, which were confirmed using matrix-assisted laser desorption/ionization-time of flying-mass spectroscopy and protein database search. A total of 39 proteins, including 18 up-regulated and 21 down-regulated proteins, were classified into different categories, such as energy and substance metabolism, protein translation, secondary metabolism, phytohormone, cytoskeleton structure, regulation, and stress tolerance. Among these, the largest functional class was associated with primary metabolism. Down-regulated proteins were involved in photosynthesis, transcription, and translation, whereas up-regulated proteins were involved in respiration. Decreased flavonoid synthesis and up-regulated GA20ox might be involved in the floral reversion process. Up-regulated 14-3-3 proteins played a role in the regulation of floral reversion in D. longan by responding to abiotic stress. Observations via transmission electron microscopy revealed the ultrastructure changes in shedding buds undergoing floral reversion. Overall, the results provided insights into the molecular basis for the floral reversion mechanism in D. longan. Copyright © 2011 Elsevier B.V. All rights reserved.

Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2

DEFF Research Database (Denmark)

Witting, Nanna; Duno, M; Vissing, J

2013-01-01

Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic......, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest...

Extensive expansion of A1 family aspartic proteinases in fungi revealed by evolutionary analyses of 107 complete eukaryotic proteomes

NARCIS (Netherlands)

Revuelta, M.V.; Kan, van J.A.L.; Kay, J.; Have, ten A.

2014-01-01

The A1 family of eukaryotic aspartic proteinases (APs) forms one of the 16 AP families. Although one of the best characterized families, the recent increase in genome sequence data has revealed many fungal AP homologs with novel sequence characteristics. This study was performed to explore the

Advanced exergy-based analyses applied to a system including LNG regasification and electricity generation

Energy Technology Data Exchange (ETDEWEB)

Morosuk, Tatiana; Tsatsaronis, George; Boyano, Alicia; Gantiva, Camilo [Technische Univ. Berlin (Germany)

2012-07-01

Liquefied natural gas (LNG) will contribute more in the future than in the past to the overall energy supply in the world. The paper discusses the application of advanced exergy-based analyses to a recently developed LNG-based cogeneration system. These analyses include advanced exergetic, advanced exergoeconomic, and advanced exergoenvironmental analyses in which thermodynamic inefficiencies (exergy destruction), costs, and environmental impacts have been split into avoidable and unavoidable parts. With the aid of these analyses, the potentials for improving the thermodynamic efficiency and for reducing the overall cost and the overall environmental impact are revealed. The objectives of this paper are to demonstrate (a) the potential for generating electricity while regasifying LNG and (b) some of the capabilities associated with advanced exergy-based methods. The most important subsystems and components are identified, and suggestions for improving them are made. (orig.)

Synthesis, Hirshfeld surface analyses and magnetism of a 1D Mn(II ...

African Journals Online (AJOL)

A new Mn-based complex of {[Mn(L)2(mi)]·H2O}n (1) (HL = p-hydroxy phenylacetic acid; mi = 1,1'-(1,4-butanediyl)bis(imidazole)), has been synthesized and structurally characterized. Single-crystal X-ray analyses reveal that compound 1 has a dinuclear Mn(II) unit linking by four carboxylate groups. The bridging N-donor ...

Chemical analyses of wasp-associated streptomyces bacteria reveal a prolific potential for natural products discovery.

Directory of Open Access Journals (Sweden)

Michael Poulsen

2011-02-01

Full Text Available Identifying new sources for small molecule discovery is necessary to help mitigate the continuous emergence of antibiotic-resistance in pathogenic microbes. Recent studies indicate that one potentially rich source of novel natural products is Actinobacterial symbionts associated with social and solitary Hymenoptera. Here we test this possibility by examining two species of solitary mud dauber wasps, Sceliphron caementarium and Chalybion californicum. We performed enrichment isolations from 33 wasps and obtained more than 200 isolates of Streptomyces Actinobacteria. Chemical analyses of 15 of these isolates identified 11 distinct and structurally diverse secondary metabolites, including a novel polyunsaturated and polyoxygenated macrocyclic lactam, which we name sceliphrolactam. By pairing the 15 Streptomyces strains against a collection of fungi and bacteria, we document their antifungal and antibacterial activity. The prevalence and anti-microbial properties of Actinobacteria associated with these two solitary wasp species suggest the potential role of these Streptomyces as antibiotic-producing symbionts, potentially helping defend their wasp hosts from pathogenic microbes. Finding phylogenetically diverse and chemically prolific Actinobacteria from solitary wasps suggests that insect-associated Actinobacteria can provide a valuable source of novel natural products of pharmaceutical interest.

Indian Point 2 steam generator tube rupture analyses

International Nuclear Information System (INIS)

Dayan, A.

1985-01-01

Analyses were conducted with RETRAN-02 to study consequences of steam generator tube rupture (SGTR) events. The Indian Point, Unit 2, power plant (IP2, PWR) was modeled as a two asymmetric loops, consisting of 27 volumes and 37 junctions. The break section was modeled once, conservatively, as a 150% flow area opening at the wall of the steam generator cold leg plenum, and once as a 200% double-ended tube break. Results revealed 60% overprediction of breakflow rates by the traditional conservative model. Two SGTR transients were studied, one with low-pressure reactor trip and one with an earlier reactor trip via over temperature ΔT. The former is more typical to a plant with low reactor average temperature such as IP2. Transient analyses for a single tube break event over 500 seconds indicated continued primary subcooling and no need for steam line pressure relief. In addition, SGTR transients with reactor trip while the pressurizer still contains water were found to favorably reduce depressurization rates. Comparison of the conservative results with independent LOFTRAN predictions showed good agreement

Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

Science.gov (United States)

Ravera, Ivan; Ferreira, Diana; Gallego, Laia Solano; Bardagí, Mar; Ferrer, Lluís

2015-08-01

The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

Science.gov (United States)

Paoletta, Martina Soledad; López Arias, Ludmila; de la Fournière, Sofía; Guillemi, Eliana Carolina; Luciani, Carlos; Sarmiento, Néstor Fabián; Mosqueda, Juan; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

2018-02-01

Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens. Copyright © 2017 Elsevier GmbH. All rights reserved.

Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

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Amrendra Kumar

2016-10-01

Full Text Available Objective: To identify white spot syndrome virus (WSSV entry into the host-cells of the cultured shrimp Penaeus monodon, we have attempted to localize PmRab7 (Ras-related in brain which is playing a vital role in the WSSV internalization. Methods: In this study, we have cloned PmRab7 and expressed in Escherichia coli, further purified rPmRab7 was used for antibody production, isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein. Moreover, high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR. Results: 651 bp amplicon size gene was successfully amplified, ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel. Subcloned (pRSET-B 651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page, furthermore soluble fraction of rPmRab7 (26 kDa was purified by ni-NTA column. AntiPmRab7 antibody was received by Merk Pvt. Ltd., and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction. Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct = 1.0 to Ct = 8.5. Conclusions: Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation, other hand WSSV may follow the same route of entry. WSSV internalization has directly linked with regulation of PmRab7.

Non-invasive PGAA, PIXE and ToF-ND analyses on Hungarian Bronze Age defensive armour

International Nuclear Information System (INIS)

Marianne Moedlinger; Imre Kovacs; Zoltan Szoekefalvi-Nagy; Ziad El Morr

2014-01-01

Non-invasive, archaeometric analyses on selected Hungarian Bronze Age defensive armour is presented here: three greaves, three helmets two shields as well as one vessel fragment were analysed with PIXE, PGAA and TOF-ND. The detected alloy elemental and phase composition as well as its intergranular or spatial concentration distribution reveals important insights into the alloys used and the manufacturing techniques applied c. 1200-950 BC, and allows to reconstruct the production techniques used during the Late Bronze Age. (author)

Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

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Kaspar-Schoenefeld, Stephanie; Merx, Kathleen; Jozefowicz, Anna Maria; Hartmann, Anja; Seiffert, Udo; Weschke, Winfriede; Matros, Andrea; Mock, Hans-Peter

2016-06-30

Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage

Genetic Structuration, Demography and Evolutionary History of Mycobacterium tuberculosis LAM9 Sublineage in the Americas as Two Distinct Subpopulations Revealed by Bayesian Analyses

Science.gov (United States)

Reynaud, Yann; Millet, Julie; Rastogi, Nalin

2015-01-01

Tuberculosis (TB) remains broadly present in the Americas despite intense global efforts for its control and elimination. Starting from a large dataset comprising spoligotyping (n = 21183 isolates) and 12-loci MIRU-VNTRs data (n = 4022 isolates) from a total of 31 countries of the Americas (data extracted from the SITVIT2 database), this study aimed to get an overview of lineages circulating in the Americas. A total of 17119 (80.8%) strains belonged to the Euro-American lineage 4, among which the most predominant genotypic family belonged to the Latin American and Mediterranean (LAM) lineage (n = 6386, 30.1% of strains). By combining classical phylogenetic analyses and Bayesian approaches, this study revealed for the first time a clear genetic structuration of LAM9 sublineage into two subpopulations named LAM9C1 and LAM9C2, with distinct genetic characteristics. LAM9C1 was predominant in Chile, Colombia and USA, while LAM9C2 was predominant in Brazil, Dominican Republic, Guadeloupe and French Guiana. Globally, LAM9C2 was characterized by higher allelic richness as compared to LAM9C1 isolates. Moreover, LAM9C2 sublineage appeared to expand close to twenty times more than LAM9C1 and showed older traces of expansion. Interestingly, a significant proportion of LAM9C2 isolates presented typical signature of ancestral LAM-RDRio MIRU-VNTR type (224226153321). Further studies based on Whole Genome Sequencing of LAM strains will provide the needed resolution to decipher the biogeographical structure and evolutionary history of this successful family. PMID:26517715

The role of laboratory diagnostic of boreliose in the environment of forest workers

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Patryk Matuszek

2013-12-01

Full Text Available Background: The laboratory diagnostics of boreliose is based on detection of specific IgG and IgM antibodies by ELISA test followed by confirmation test of Line-blot. Material and methods: Testing of the presence of specific anti-Borrelia antibodies was performed in the group of 568 forest workers. The ELISA tests were performed in serum, saliva, and dry spot of blood. The confirmation tests Line blot were performed in serum then the prevalence of particular antibodies against Borrelia was calculated. The ELISA test consists complete extract from Borrelia whereas the Line-blot test consists antigens from B. afzelii, B. burdorferi, and B. garinii. Results: The samples from 201 (35% forest workers have revealed the positive results in ELISA in IgG class and 171 (30 results from this group were confirmed by Line-blot. Only in 40 (7 samples have revealed the positive results in Line-blot but were negative in ELISA tests. The main antigen was VIsE (77% in IgG class. It has been shown that 119 (21% samples have revealed positive results in the IgM class in ELISA tests, and from this group 59 (10% cases were confirmed by Line-blot. Antibody against antigen OspC revealed the highest prevalence (71% in IgM class. The correlation between samples from sera and mucosal transudate have shown r40.736 in the case of IgG and r40.162 in the case of IgM. Conclusion: The frequency, of anti-Borrelia antibodies occurrence was very high and appeared in 42% of forest workers. It has revealed that the correlation of results between serum and mucosal transudate was not satisfactory in the case of ELISA test. In contrary, the correlation of results between serum and dry spot of blood was very high and should be consider as alternative material for boreliose diagnosis.

Indeterminate human immunodeficiency virus western blot results in Iranian patients with discordant screening assay results

International Nuclear Information System (INIS)

Ravanshad, M.; Sabahi, F.; Mahboudi, F.; Sabahi, F.

2006-01-01

The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection and confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) and 2 (HIV-2). However, indeterminate WB reactivity to HIV-1 and HIV-2 proteins may occur in individuals who do not appear to be infected with HIV. In this study, we describe the results of indeterminate WB reactivity in Iranian patients with discordant screening assays. The samples were obtained from Iranian Blood Transfusion Center, Tehran, Iran and evaluated in the Biotechnology Process Development Center, Pasteur Institute of Iran, Tehran, Iran between 2003 and 2004. A total of 4707 were tested for the presence of HIV-1 antibodies. Six hundred and four (12.8%) patients tested for HIV were positive for HIV-1 antibody. Nine (1.49%) have discordant results among screening assays and indeterminate WB results as interpreted by Centers for Disease Control and Prevention (CDC) criteria. Most (66.7%) of these indeterminate WB results were due to p24 reactivity. However, 2(22.2%) display reactivity to both gp41 and gp120 proteins [Positive by World Health Organization (WHO) criteria]. Of 9 WB assays initially indeterminate by the CDC criteria and with follow-up samples 8(88.8%) became negative when retested subsequently while one (11.1%) remained indeterminate for more than a year and were thus considered negative. In addition all the indeterminate samples were negative when assessed by polymerase chain reaction assay. In general, there were was an 88.8% concordance between the CDC and WHO criteria for an indeterminate WB result. The CDC II criteria for an indeterminate WB result. The CDC II criteria best met the specified objectives for diagnosis in our setting. (author)

Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

Energy Technology Data Exchange (ETDEWEB)

Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

2011-12-16

Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

Using team cognitive work analysis to reveal healthcare team interactions in a birthing unit.

Science.gov (United States)

Ashoori, Maryam; Burns, Catherine M; d'Entremont, Barbara; Momtahan, Kathryn

2014-01-01

Cognitive work analysis (CWA) as an analytical approach for examining complex sociotechnical systems has shown success in modelling the work of single operators. The CWA approach incorporates social and team interactions, but a more explicit analysis of team aspects can reveal more information for systems design. In this paper, Team CWA is explored to understand teamwork within a birthing unit at a hospital. Team CWA models are derived from theories and models of teamwork and leverage the existing CWA approaches to analyse team interactions. Team CWA is explained and contrasted with prior approaches to CWA. Team CWA does not replace CWA, but supplements traditional CWA to more easily reveal team information. As a result, Team CWA may be a useful approach to enhance CWA in complex environments where effective teamwork is required. This paper looks at ways of analysing cognitive work in healthcare teams. Team Cognitive Work Analysis, when used to supplement traditional Cognitive Work Analysis, revealed more team information than traditional Cognitive Work Analysis. Team Cognitive Work Analysis should be considered when studying teams.

Using team cognitive work analysis to reveal healthcare team interactions in a birthing unit

Science.gov (United States)

Ashoori, Maryam; Burns, Catherine M.; d'Entremont, Barbara; Momtahan, Kathryn

2014-01-01

Cognitive work analysis (CWA) as an analytical approach for examining complex sociotechnical systems has shown success in modelling the work of single operators. The CWA approach incorporates social and team interactions, but a more explicit analysis of team aspects can reveal more information for systems design. In this paper, Team CWA is explored to understand teamwork within a birthing unit at a hospital. Team CWA models are derived from theories and models of teamworkand leverage the existing CWA approaches to analyse team interactions. Team CWA is explained and contrasted with prior approaches to CWA. Team CWA does not replace CWA, but supplements traditional CWA to more easily reveal team information. As a result, Team CWA may be a useful approach to enhance CWA in complex environments where effective teamwork is required. Practitioner Summary: This paper looks at ways of analysing cognitive work in healthcare teams. Team Cognitive Work Analysis, when used to supplement traditional Cognitive Work Analysis, revealed more team information than traditional Cognitive Work Analysis. Team Cognitive Work Analysis should be considered when studying teams PMID:24837514

Comparative analyses of genetic risk prediction methods reveal ...

Indian Academy of Sciences (India)

2015-03-12

Mar 12, 2015 ... 1National Institute of Biomedical Genomics, Netaji Subhas Sanatorium (T. B. ... 2Institute of Post Graduate Medical Education and Research, J. C. Bose Road, ...... Torres D. M. and Harrison S. A 2008 Diagnosis and therapy of.

Comparative analyses of genetic risk prediction methods reveal ...

Indian Academy of Sciences (India)

0.85. 0.8. 0.95. 0.93. 0.81 rs1227756. 0.5. 0.56. 0.75. 0.55. 0.7. 0.71. 0.58. 0.81. 0.25. 0.5. 0.53. 0.48. 0.64. 0.58. 0.58. 0.8. 0.68. 0.78. 0.48. 0.65. NA. NA. NA. NA rs1501299. 0.64. 0.78. 0.75. 0.83. 0.83. 0.89. 0.83. 0.72. 0.63. 0.73. 0.78. 0.75. 0.84. 0.79. 0.61. 0.78. 0.78. 0.7. 0.73. 0.73. NA. NA. NA. NA rs16944. 0.64. 0.72. 0.68.

The postglacial recolonization of Northern Europe by Rana arvalis as revealed by microsatellite and mitochondrial DNA analyses.

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Knopp, T; Merilä, J

2009-02-01

The postglacial history of the moor frog (Rana arvalis) in Northern Europe was investigated with the aid of eight variable microsatellite loci and a 661 bp sequence of the mitochondrial cytochrome b gene. A division between eastern and western mitochondrial lineages was discovered, supporting two recolonization routes to Fennoscandia since the last glacial maximum. This result was corroborated by the microsatellite data, which revealed a contact zone between the two lineages in Northern Sweden. These findings add to the increasing evidence that an intraspecific genetic biodiversity founded on the existence of eastern and western clades is a common element in Fennoscandian fauna and flora.

Distinct signaling roles of ceramide species in yeast revealed through systematic perturbation and systems biology analyses.

Science.gov (United States)

Montefusco, David J; Chen, Lujia; Matmati, Nabil; Lu, Songjian; Newcomb, Benjamin; Cooper, Gregory F; Hannun, Yusuf A; Lu, Xinghua

2013-10-29

Ceramide, the central molecule of sphingolipid metabolism, is an important bioactive molecule that participates in various cellular regulatory events and that has been implicated in disease. Deciphering ceramide signaling is challenging because multiple ceramide species exist, and many of them may have distinct functions. We applied systems biology and molecular approaches to perturb ceramide metabolism in the yeast Saccharomyces cerevisiae and inferred causal relationships between ceramide species and their potential targets by combining lipidomic, genomic, and transcriptomic analyses. We found that during heat stress, distinct metabolic mechanisms controlled the abundance of different groups of ceramide species and provided experimental support for the importance of the dihydroceramidase Ydc1 in mediating the decrease in dihydroceramides during heat stress. Additionally, distinct groups of ceramide species, with different N-acyl chains and hydroxylations, regulated different sets of functionally related genes, indicating that the structural complexity of these lipids produces functional diversity. The transcriptional modules that we identified provide a resource to begin to dissect the specific functions of ceramides.

Less is less: a systematic review of graph use in meta-analyses.

Science.gov (United States)

Schild, Anne H E; Voracek, Martin

2013-09-01

Graphs are an essential part of scientific communication. Complex datasets, of which meta-analyses are textbook examples, benefit the most from visualization. Although a number of graph options for meta-analyses exist, the extent to which these are used was hitherto unclear. A systematic review on graph use in meta-analyses in three disciplines (medicine, psychology, and business) and nine journals was conducted. Interdisciplinary differences, which are mirrored in the respective journals, were revealed, that is, graph use correlates with external factors rather than methodological considerations. There was only limited variation in graph types (with forest plots as the most important representatives), and diagnostic plots were very rare. Although an increase in graph use over time could be observed, it is unlikely that this phenomenon is specific to meta-analyses. There is a gaping discrepancy between available graphic methods and their application in meta-analyses. This may be rooted in a number of factors, namely, (i) insufficient dissemination of new developments, (ii) unsatisfactory implementation in software packages, and (iii) minor attention on graphics in meta-analysis reporting guidelines. Using visualization methods to their full capacity is a further step in using meta-analysis to its full potential. Copyright © 2013 John Wiley & Sons, Ltd.

Comparative Genomics Analyses Reveal Extensive Chromosome Colinearity and Novel Quantitative Trait Loci in Eucalyptus

Science.gov (United States)

Weng, Qijie; Li, Mei; Yu, Xiaoli; Guo, Yong; Wang, Yu; Zhang, Xiaohong; Gan, Siming

2015-01-01

Dense genetic maps, along with quantitative trait loci (QTLs) detected on such maps, are powerful tools for genomics and molecular breeding studies. In the important woody genus Eucalyptus, the recent release of E. grandis genome sequence allows for sequence-based genomic comparison and searching for positional candidate genes within QTL regions. Here, dense genetic maps were constructed for E. urophylla and E. tereticornis using genomic simple sequence repeats (SSR), expressed sequence tag (EST) derived SSR, EST-derived cleaved amplified polymorphic sequence (EST-CAPS), and diversity arrays technology (DArT) markers. The E. urophylla and E. tereticornis maps comprised 700 and 585 markers across 11 linkage groups, totaling at 1,208.2 and 1,241.4 cM in length, respectively. Extensive synteny and colinearity were observed as compared to three earlier DArT-based eucalypt maps (two maps with E. grandis × E. urophylla and one map of E. globulus) and with the E. grandis genome sequence. Fifty-three QTLs for growth (10–56 months of age) and wood density (56 months) were identified in 22 discrete regions on both maps, in which only one colocalizaiton was found between growth and wood density. Novel QTLs were revealed as compared with those previously detected on DArT-based maps for similar ages in Eucalyptus. Eleven to 585 positional candidate genes were obained for a 56-month-old QTL through aligning QTL confidence interval with the E. grandis genome. These results will assist in comparative genomics studies, targeted gene characterization, and marker-assisted selection in Eucalyptus and the related taxa. PMID:26695430

Comparative Genomics Analyses Reveal Extensive Chromosome Colinearity and Novel Quantitative Trait Loci in Eucalyptus.

Directory of Open Access Journals (Sweden)

Fagen Li

Full Text Available Dense genetic maps, along with quantitative trait loci (QTLs detected on such maps, are powerful tools for genomics and molecular breeding studies. In the important woody genus Eucalyptus, the recent release of E. grandis genome sequence allows for sequence-based genomic comparison and searching for positional candidate genes within QTL regions. Here, dense genetic maps were constructed for E. urophylla and E. tereticornis using genomic simple sequence repeats (SSR, expressed sequence tag (EST derived SSR, EST-derived cleaved amplified polymorphic sequence (EST-CAPS, and diversity arrays technology (DArT markers. The E. urophylla and E. tereticornis maps comprised 700 and 585 markers across 11 linkage groups, totaling at 1,208.2 and 1,241.4 cM in length, respectively. Extensive synteny and colinearity were observed as compared to three earlier DArT-based eucalypt maps (two maps with E. grandis × E. urophylla and one map of E. globulus and with the E. grandis genome sequence. Fifty-three QTLs for growth (10-56 months of age and wood density (56 months were identified in 22 discrete regions on both maps, in which only one colocalizaiton was found between growth and wood density. Novel QTLs were revealed as compared with those previously detected on DArT-based maps for similar ages in Eucalyptus. Eleven to 585 positional candidate genes were obained for a 56-month-old QTL through aligning QTL confidence interval with the E. grandis genome. These results will assist in comparative genomics studies, targeted gene characterization, and marker-assisted selection in Eucalyptus and the related taxa.

MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias

Science.gov (United States)

Calin, George Adrian; Liu, Chang-Gong; Sevignani, Cinzia; Ferracin, Manuela; Felli, Nadia; Dumitru, Calin Dan; Shimizu, Masayoshi; Cimmino, Amelia; Zupo, Simona; Dono, Mariella; Dell'Aquila, Marie L.; Alder, Hansjuerg; Rassenti, Laura; Kipps, Thomas J.; Bullrich, Florencia; Negrini, Massimo; Croce, Carlo M.

2004-01-01

Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673–676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253–258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia. PMID:15284443

Medulloblastoma in China: clinicopathologic analyses of SHH, WNT, and non-SHH/WNT molecular subgroups reveal different therapeutic responses to adjuvant chemotherapy.

Directory of Open Access Journals (Sweden)

Zhen-Yu Zhang

Full Text Available Medulloblastoma (MB is one of the most common primary central nervous system tumors in children. Data is lacking of a large cohort of medulloblastoma patients in China. Also, our knowledge on the sensitivity of different molecular subgroups of MB to adjuvant radiation therapy (RT or chemotherapy (CHT is still limited. The authors performed a retrospective study of 173 medulloblastoma patients treated at two institutions from 2002 to 2011. Formalin-fixed paraffin embedded (FFPE tissues were available in all the cases and sections were stained to classify histological and molecular subgroups. Univariate and multivariate analyses were used to investigate prognostic factors. Of 173 patients, there were 118 children and 55 adults, 112 males and 61 females. Estimated 5-year overall survival (OS rates for all patients, children and adults were 52%, 48% and 63%, respectively. After multivariate analysis, postoperative primary radiation therapy (RT and chemotherapy (CHT were revealed as favorable prognostic factors influencing OS and EFS. Postoperative primary chemotherapy (CHT was found significantly improving the survival of children (p<0.001 while it was not a significant prognostic factor for adult patients. Moreover, patients in WNT subtype had better OS (p = 0.028 than others (SHH and Non-SHH/WNT subtypes given postoperative adjuvant therapies. Postoperative primary RT was found to be a strong prognostic factor influencing the survival in all histological and molecular subgroups (p<0.001. Postoperative primary CHT was found significantly to influence the survival of classic medulloblastoma (CMB (OS p<0.001, EFS p<0.001, SHH subgroup (OS p = 0.020, EFS p = 0.049 and WNT subgroup (OS p = 0.003, EFS p = 0.016 but not in desmoplastic/nodular medulloblastoma (DMB (OS p = 0.361, EFS p = 0.834 and Non-SHH/WNT subgroup (OS p = 0.127, EFS p = 0.055. Our study showed postoperative primary CHT significantly influence the

Analyses of Mitogenome Sequences Revealed that Asian Citrus Psyllids (Diaphorina citri) from California Were Related to Those from Florida.

Science.gov (United States)

Wu, Fengnian; Kumagai, Luci; Cen, Yijing; Chen, Jianchi; Wallis, Christopher M; Polek, MaryLou; Jiang, Hongyan; Zheng, Zheng; Liang, Guangwen; Deng, Xiaoling

2017-08-31

Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) transmits "Candidatus Liberibacter asiaticus" (CLas), an unculturable alpha-proteobacterium associated with citrus Huanglongbing (HLB). CLas has recently been found in California. Understanding ACP population diversity is necessary for HLB regulatory practices aimed at reducing CLas spread. In this study, two circular ACP mitogenome sequences from California (mt-CApsy, ~15,027 bp) and Florida (mt-FLpsy, ~15,012 bp), USA, were acquired. Each mitogenome contained 13 protein coding genes, 2 ribosomal RNA and 22 transfer RNA genes, and a control region varying in sizes. The Californian mt-CApsy was identical to the Floridian mt-FLpsy, but different from the mitogenome (mt-GDpsy) of Guangdong, China, in 50 single nucleotide polymorphisms (SNPs). Further analyses were performed on sequences in cox1 and trnAsn regions with 100 ACPs, SNPs in nad1-nad4-nad5 locus through PCR with 252 ACP samples. All results showed the presence of a Chinese ACP cluster (CAC) and an American ACP cluster (AAC). We proposed that ACP in California was likely not introduced from China based on our current ACP collection but somewhere in America. However, more studies with ACP samples from around the world are needed. ACP mitogenome sequence analyses will facilitate ACP population research.

Development of a dot blot assay using gene probes for the detection of enteroviruses in water

International Nuclear Information System (INIS)

Margolin, A.B.

1986-01-01

Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with 32 P dCTP and 32 P dATP to a specific activity greater then 1.0 x 10 9 cpm/ug DNA. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tap water. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses

Whole-exome sequencing in a single proband reveals a mutation in the CHST8 gene in autosomal recessive peeling skin syndrome.

Science.gov (United States)

Cabral, Rita M; Kurban, Mazen; Wajid, Muhammad; Shimomura, Yutaka; Petukhova, Lynn; Christiano, Angela M

2012-04-01

Generalized peeling skin syndrome (PSS) is an autosomal recessive genodermatosis characterized by lifelong, continuous shedding of the upper epidermis. Using whole-genome homozygozity mapping and whole-exome sequencing, we identified a novel homozygous missense mutation (c.229C>T, R77W) within the CHST8 gene, in a large consanguineous family with non-inflammatory PSS type A. CHST8 encodes a Golgi transmembrane N-acetylgalactosamine-4-O-sulfotransferase (GalNAc4-ST1), which we show by immunofluorescence staining to be expressed throughout normal epidermis. A colorimetric assay for total sulfated glycosaminoglycan (GAG) quantification, comparing human keratinocytes (CCD1106 KERTr) expressing wild type and mutant recombinant GalNAc4-ST1, revealed decreased levels of total sulfated GAGs in cells expressing mutant GalNAc4-ST1, suggesting loss of function. Western blotting revealed lower expression levels of mutant recombinant GalNAc4-ST1 compared to wild type, suggesting that accelerated degradation may result in loss of function, leading to PSS type A. This is the first report describing a mutation as the cause of PSS type A. Copyright © 2012 Elsevier Inc. All rights reserved.

Combined Analyses of Chloroplast DNA Haplotypes and Microsatellite Markers Reveal New Insights Into the Origin and Dissemination Route of Cultivated Pears Native to East Asia

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Xiaoyan Yue

2018-05-01

Full Text Available Asian pear plays an important role in the world pear industry, accounting for over 70% of world total production volume. Commercial Asian pear production relies on four major pear cultivar groups, Japanese pear (JP, Chinese white pear (CWP, Chinese sand pear (CSP, and Ussurian pear (UP, but their origins remain controversial. We estimated the genetic diversity levels and structures in a large sample of existing local cultivars to investigate the origins of Asian pears using twenty-five genome-covering nuclear microsatellite (simple sequence repeats, nSSR markers and two non-coding chloroplast DNA (cpDNA regions (trnL-trnF and accD-psaI. High levels of genetic diversity were detected for both nSSRs (HE = 0.744 and cpDNAs (Hd = 0.792. The major variation was found within geographic populations of cultivated pear groups, demonstrating a close relationship among cultivar groups. CSPs showed a greater genetic diversity than CWPs and JPs, and lowest levels of genetic differentiation were detected among them. Phylogeographical analyses indicated that the CSP, CWP, and JP were derived from the same progenitor of Pyrus pyrifolia in China. A dissemination route of cultivated P. pyrifolia estimated by approximate Bayesian computation suggested that cultivated P. pyrifolia from the Middle Yangtze River Valley area contributed the major genetic resources to the cultivars, excluding those of southwestern China. Three major genetic groups of cultivated Pyrus pyrifolia were revealed using nSSRs and a Bayesian statistical inference: (a JPs; (b cultivars from South-Central China northward to northeastern China, covering the main pear production area in China; (c cultivars from southwestern China to southeastern China, including Yunnan, Guizhou, Guangdong, Guangxi, and Fujian Provinces. This reflected the synergistic effects of ecogeographical factors and human selection during cultivar spread and improvement. The analyses indicated that UP cultivars might be

Mitochondrial DNA analyses and ecological niche modeling reveal post-LGM expansion of the Assam macaque (Macaca assamensis) in the foothills of Nepal Himalaya.

Science.gov (United States)

Khanal, Laxman; Chalise, Mukesh K; He, Kai; Acharya, Bipin K; Kawamoto, Yoshi; Jiang, Xuelong

2018-03-01

Genetic diversity of a species is influenced by multiple factors, including the Quaternary glacial-interglacial cycles and geophysical barriers. Such factors are not yet well documented for fauna from the southern border of the Himalayan region. This study used mitochondrial DNA (mtDNA) sequences and ecological niche modeling (ENM) to explore how the late Pleistocene climatic fluctuations and complex geography of the Himalayan region have shaped genetic diversity, population genetic structure, and demographic history of the Nepalese population of Assam macaques (Macaca assamensis) in the Himalayan foothills. A total of 277 fecal samples were collected from 39 wild troops over almost the entire distribution of the species in Nepal. The mtDNA fragment encompassing the complete control region (1121 bp) was recovered from 208 samples, thus defining 54 haplotypes. Results showed low nucleotide diversity (0.0075 ± SD 0.0001) but high haplotype diversity (0.965 ± SD 0.004). The mtDNA sequences revealed a shallow population genetic structure with a moderate but statistically significant effect of isolation by distance. Demographic history analyses using mtDNA sequences suggested a post-pleistocene population expansion. Paleodistribution reconstruction projected that the potential habitat of the Assam macaque was confined to the lower elevations of central Nepal during the Last Glacial Maximum. With the onset of the Holocene climatic optimum, the glacial refugia population experienced eastward range expansion to higher elevations. We conclude that the low genetic diversity and shallow population genetic structure of the Assam macaque population in the Nepal Himalaya region are the consequence of recent demographic and spatial expansion. © 2018 Wiley Periodicals, Inc.

Temporal analyses of Salmonellae in a headwater spring ecosystem reveals the effects of precipitation and runoff events.

Science.gov (United States)

Gaertner, James P; Garres, Tiffany; Becker, Jesse C; Jimenez, Maria L; Forstner, Michael R J; Hahn, Dittmar

2009-03-01

Sediments and water from the spring and slough arm of Spring Lake, the pristine headwaters of the San Marcos River, Texas, were analyzed for Salmonellae by culture and molecular techniques before and after three major precipitation events, each with intermediate dry periods. Polymerase chain reaction (PCR)-assisted analyses of enrichment cultures detected Salmonellae in samples after all three precipitation events, but failed to detect them immediately prior to the rainfall events. Detection among individual locations differed with respect to the precipitation event analyzed, and strains isolated were highly variable with respect to serovars. These results demonstrate that rainwater associated effects, most likely surface runoff, provide an avenue for short-term pollution of aquatic systems with Salmonellae that do not, however, appear to establish for the long-term in water nor sediments.

Integrative analyses of leprosy susceptibility genes indicate a common autoimmune profile.

Science.gov (United States)

Zhang, Deng-Feng; Wang, Dong; Li, Yu-Ye; Yao, Yong-Gang

2016-04-01

Leprosy is an ancient chronic infection in the skin and peripheral nerves caused by Mycobacterium leprae. The development of leprosy depends on genetic background and the immune status of the host. However, there is no systematic view focusing on the biological pathways, interaction networks and overall expression pattern of leprosy-related immune and genetic factors. To identify the hub genes in the center of leprosy genetic network and to provide an insight into immune and genetic factors contributing to leprosy. We retrieved all reported leprosy-related genes and performed integrative analyses covering gene expression profiling, pathway analysis, protein-protein interaction network, and evolutionary analyses. A list of 123 differentially expressed leprosy related genes, which were enriched in activation and regulation of immune response, was obtained in our analyses. Cross-disorder analysis showed that the list of leprosy susceptibility genes was largely shared by typical autoimmune diseases such as lupus erythematosus and arthritis, suggesting that similar pathways might be affected in leprosy and autoimmune diseases. Protein-protein interaction (PPI) and positive selection analyses revealed a co-evolution network of leprosy risk genes. Our analyses showed that leprosy associated genes constituted a co-evolution network and might undergo positive selection driven by M. leprae. We suggested that leprosy may be a kind of autoimmune disease and the development of leprosy is a matter of defect or over-activation of body immunity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

International Nuclear Information System (INIS)

Armstrong, S.K.

1986-01-01

Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and 125 I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulated strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25 0 C instead of 100 0 C

Antigenic Cross-reactivity among Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis of Goat

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Ruma JAS

2016-12-01

Full Text Available Background: Cross antigenicity is the major problem in developing a reliable tool for immunodiagnosis and immunoprophylaxis of parasitic diseases. Mixed infection due to different types of gastrointestinal parasites is more common than single species infection under field condition.Methods: The present study was undertaken to detect antigenic cross-reactivity among Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis of goats by SDS-PAGE and western blot analysis using hyperimmune sera (HIS rose in rabbit separately against the antigens of the three nematode species.Results: Thirteen, 16 and 14 polypeptides in crude somatic antigen (CSAg of H. contortus (CSAg-Hc, O. columbianum (CSAg-Oc and T. ovis (CSAg-To, respectively, were resolved in SDS PAGE analyses. It was revealed that 54 kDa peptide was shared by H.contortus and O. columbianum, whereas 47 kDa peptide was shared by O. columbianum and T. ovis. Western blot analyses revealed that three immunogenic polypeptides (MW 54, 49 and 42 kDa in CSAg-Hc, five in CSAg-Oc (54, 47, 44, 38 and 35.5 kDa and CSAg-To and five polypeptides (90, 51, 47, 39.5 and 31 kDa in CSAg-To cross-reacted with the heterologous HIS. Four species-specific immunoreactive polypeptides (92, 85, 65 and 39 kDa of H. contortus and two (72 & 26 kDa in O. columbianum were also identified in the study. Conclusion: The shared polypeptides and species-specific polypeptides might be evaluated as protective antigen and subsequently exploitation for developing immunodiagnostic and for immunoprophylactic tools of for these common nematode species. 

Antigenic Cross-reactivity among Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis of Goat.

Science.gov (United States)

Jas, Ruma; Ghosh, Joydeb; DAS, Kinsuk

2016-01-01

Cross antigenicity is the major problem in developing a reliable tool for immunodiagnosis and immunoprophylaxis of parasitic diseases. Mixed infection due to different types of gastrointestinal parasites is more common than single species infection under field condition. The present study was undertaken to detect antigenic cross-reactivity among Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis of goats by SDS-PAGE and western blot analysis using hyperimmune sera (HIS) rose in rabbit separately against the antigens of the three nematode species. Thirteen, 16 and 14 polypeptides in crude somatic antigen (CSAg) of H. contortus (CSAg-Hc), O. columbianum (CSAg-Oc) and T. ovis (CSAg-To), respectively, were resolved in SDS PAGE analyses. It was revealed that 54 kDa peptide was shared by H.contortus and O. columbianum , whereas 47 kDa peptide was shared by O. columbianum and T. ovis . Western blot analyses revealed that three immunogenic polypeptides (MW 54, 49 and 42 kDa) in CSAg-Hc, five in CSAg-Oc (54, 47, 44, 38 and 35.5 kDa) and CSAg-To and five polypeptides (90, 51, 47, 39.5 and 31 kDa) in CSAg-To cross-reacted with the heterologous HIS. Four species-specific immunoreactive polypeptides (92, 85, 65 and 39 kDa) of H. contortus and two (72 & 26 kDa) in O. columbianum were also identified in the study. The shared polypeptides and species-specific polypeptides might be evaluated as protective antigen and subsequently exploitation for developing immunodiagnostic and for immunoprophylactic tools of for these common nematode species.

Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic.

Science.gov (United States)

Cernanská, Dana; Paoletti, Barbara; Králová-Hromadová, Ivica; Iorio, Raffaella; Cudeková, Patrícia; Milillo, Piermarino; Traversa, Donato

2009-03-09

Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These data demonstrate that resistance to benzimidazoles is well established in cyathostomin populations from horse farms in the Slovak Republic and that the molecular assay was able to determine the species-specific distribution of resistant cyathostomins under field conditions.

Differential metabolism of Mycoplasma species as revealed by their genomes

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Fabricio B.M. Arraes

2007-01-01

Full Text Available The annotation and comparative analyses of the genomes of Mycoplasma synoviae and Mycoplasma hyopneumonie, as well as of other Mollicutes (a group of bacteria devoid of a rigid cell wall, has set the grounds for a global understanding of their metabolism and infection mechanisms. According to the annotation data, M. synoviae and M. hyopneumoniae are able to perform glycolytic metabolism, but do not possess the enzymatic machinery for citrate and glyoxylate cycles, gluconeogenesis and the pentose phosphate pathway. Both can synthesize ATP by lactic fermentation, but only M. synoviae can convert acetaldehyde to acetate. Also, our genome analysis revealed that M. synoviae and M. hyopneumoniae are not expected to synthesize polysaccharides, but they can take up a variety of carbohydrates via the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS. Our data showed that these two organisms are unable to synthesize purine and pyrimidine de novo, since they only possess the sequences which encode salvage pathway enzymes. Comparative analyses of M. synoviae and M. hyopneumoniae with other Mollicutes have revealed differential genes in the former two genomes coding for enzymes that participate in carbohydrate, amino acid and nucleotide metabolism and host-pathogen interaction. The identification of these metabolic pathways will provide a better understanding of the biology and pathogenicity of these organisms.

Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli.

Directory of Open Access Journals (Sweden)

Yanlong Wang

Full Text Available We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli, Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology and KEGG (Kyoto encyclopedia of genes and genomes enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species scavenging, membrane proteins and ABC (ATP binding cassette transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli.

Mere end blot pirringer

DEFF Research Database (Denmark)

Jantzen, Christian; Østergaard, Per

2006-01-01

Med udgangspunkt i en række cases på vellykkede oplevelsesøkonomiske forretningsmodeller argumenterer artiklen for, at oplevelsesprodukter skal bygge på et klart tema, som forbrugeren kan koble sig sanse- og følelsesmæssigt op på. Forbrugeren skal kunne omsætte produktets pirringer til egne erfar...

Ej blot til lyst

DEFF Research Database (Denmark)

Leroyer, Patrick

2010-01-01

The purpose of this article is to reassert the crucial importance of access to data in lexicographic information tools and, expanding on this, to establish the existence of two distinct lexicographic access modes - consultation and navigation. It is explained how the tools can be decalibrated whe...... balance between user, access, and data is disturbed, and how access to data then is jeopardized. Taking online wine guides as a case in point, it is shown how such multifunctional information tools do benefit from a lexicographic design featuring both access modes....

Patterns of Limnohabitans microdiversity across a large set of freshwater habitats as revealed by reverse line blot hybridization

Czech Academy of Sciences Publication Activity Database

Jezbera, Jan; Jezberová, Jitka; Kasalický, Vojtěch; Šimek, Karel; Hahn, M.W.

2013-01-01

Roč. 8, č. 3 (2013), e58527 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GAP504/10/0566; GA ČR(CZ) GEEEF/10/E011 Institutional support: RVO:60077344 Keywords : Limnohabitans * microdiversity * habitats * hybridization Subject RIV: DA - Hydrology ; Limnology Impact factor: 3.534, year: 2013

Prey-capture success revealed by echolocation signals in pipistrelle bats (Pipistrellus pygmaeus)

DEFF Research Database (Denmark)

Surlykke, Annemarie; Futtrup, Vibeke; Tougaard, Jakob

2003-01-01

Three Pipistrellus pygmaeus bats were trained to capture prey on the wing while flying in the laboratory. The bats' capture behaviour and capture success were determined and correlated with acoustic analyses of post-buzz echolocation signals. Three acoustic parameters revealed capture success: in...

USE OF BOUNDING ANALYSES TO ESTIMATE THE PREFORMANCE OF A SEISMICALLY ISOLATED STRUCTURE

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Gökhan ÖZDEMİR

2017-03-01

Full Text Available Current design approach for seismic isolated structures is to perform bounding analyses. These analyses provide an envelope for the response of the seismic isolated structure rather than focusing on the actual performance. In this study, the success of bounding analyses to estimate performance of a seismic isolated structure, in which the isolation is provided by means of lead rubber bearings (LRBs, is evaluated in a comparative manner. For this purpose, nonlinear response history analyses were performed under the effect of bidirectional ground motion excitations. In bounding analyses, non-deteriorating hysteretic representations were used to model the hysteretic behavior of LRBs. On the other hand, to estimate the actual performance of both the superstructure and isolator units, deteriorating hysteretic idealizations were employed. The deterioration in strength of LRBs was defined as a function of temperature rise in the lead core. The analyzed structure is an existing seismically isolated hospital building and analytically modeled in accordance with its reported design properties for both isolation units and superstructure. Results obtained from analyses where LRBs are idealized by both deteriorating and non-deteriorating hysteretic representations are used in the comparisons. The response quantities used in the comparisons are maximum isolator displacement, maximum isolator force, maximum absolute floor acceleration, and maximum relative story displacements. In an average sense, bounding analyses is found to provide conservative estimates for the selected response quantities and fulfills its intended purpose. However, it is revealed that there may be individual cases where bounding analyses fails to provide a safe envelope.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

Science.gov (United States)

Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

2015-05-01

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method.

Science.gov (United States)

Silva, M G; Marques, P X; Oliva, A

2010-12-15

Babesiosis and Theileriosis are tick-borne diseases widespread in tropical and sub-tropical regions with high economic impact worldwide. In Portugal there are at least 4 tick vectors known to be competent for the transmission of Babesia and Theileria sp. identified: Rhipicephalus bursa, Rhipicephalus (Boophilus) annulatus, Ixodes ricinus and Haemaphysalis punctata. All these potential Babesia and Theileria tick vectors are widely distributed in Portugal, although they are predominant in the Southern region. In this study, 1104 cattle blood samples were randomly collected from Central and Southern regions of Portugal and analyzed by PCR-reverse line blotting (RLB) for the detection of Babesia and Theileria sp. Testing indicated that 74.7% of the bovines tested were positive for either Babesia and/or Theileria sp. In addition, five different apicomplexan species, namely, Theileria buffeli, Theileria annulata, Babesia divergens, Babesia bovis, and Babesia bigemina were detected by RLB among the bovines tested. T. buffeli was the most frequently found species, being present in 69.9% of the positive samples either as single infections (52.4%), or as mixed infections (17.5%). The Babesia specie most frequently found was B. divergens, detected in 4.2% of the infected bovines. Overall, infected bovines were found in all regions tested; however the highest number of infected bovines was observed in Évora district (96.2%) and in cattle from Limousin breeds (81.7%). The results indicate widespread Babesia and Theileria infections in Portuguese bovines, suggesting the need for improved control of ticks and tick-borne diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

Insect-resistance and high-yield transgenic tobacco obtained by ...

African Journals Online (AJOL)

The modified synthesized VHb gene and insectidal gene (GFMcryIA) were transferred to tobacco plants by Agrobacterium-mediated transformation. The bivalent genes were inserted successfully into the tobacco genome and detected by PCR amplification. Southern blot and Western blot analyses showed that VHb gene ...

Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

Science.gov (United States)

Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

2005-04-01

This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

Analyses of soil microbial community compositions and functional genes reveal potential consequences of natural forest succession.

Science.gov (United States)

Cong, Jing; Yang, Yunfeng; Liu, Xueduan; Lu, Hui; Liu, Xiao; Zhou, Jizhong; Li, Diqiang; Yin, Huaqun; Ding, Junjun; Zhang, Yuguang

2015-05-06

The succession of microbial community structure and function is a central ecological topic, as microbes drive the Earth's biogeochemical cycles. To elucidate the response and mechanistic underpinnings of soil microbial community structure and metabolic potential relevant to natural forest succession, we compared soil microbial communities from three adjacent natural forests: a coniferous forest (CF), a mixed broadleaf forest (MBF) and a deciduous broadleaf forest (DBF) on Shennongjia Mountain in central China. In contrary to plant communities, the microbial taxonomic diversity of the DBF was significantly (P the DBF. Furthermore, a network analysis of microbial carbon and nitrogen cycling genes showed the network for the DBF samples was relatively large and tight, revealing strong couplings between microbes. Soil temperature, reflective of climate regimes, was important in shaping microbial communities at both taxonomic and functional gene levels. As a first glimpse of both the taxonomic and functional compositions of soil microbial communities, our results suggest that microbial community structure and function potentials will be altered by future environmental changes, which have implications for forest succession.

Genetic and molecular analyses of UV radiation-induced mutations in the fem-3 gene of Caenorhabditis elegans

Energy Technology Data Exchange (ETDEWEB)

Hartman, P S; De Wilde, D; Dwarakanath, V N [Texas Christian Univ., Fort Worth, TX (United States). Dept. of Biology

1995-06-01

The utility of a new target gene (fem-3) is described for investigating the molecular nature of mutagenesis in the nematode Caenorhabditis elegans. As a principal attribute, this system allows for the selection, maintenance and molecular analysis of any type of mutation that disrupts the gene, including deletions. In this study, 86 mutant strains were isolated, of which 79 proved to have mutations in fem-3. Twenty of these originally tested as homozygous inviable. Homozygous inviability was expected, as Stewart and coworkers had previously observed that, unlike in other organisms, most UV radiation-induced mutations in C. elegans are chromosomal rearrangements of deficiencies (Mutat. Res 249, 37-54, 1991). However, additional data, including Southern blot analyses on 49 of the strains, indicated that most of the UV radiation-induced fem-3 mutations were not deficiencies, as originally inferred from their homozygous inviability. Instead, the lethals were most likely ``coincident mutations`` in linked, essential genes that were concomitantly induced. As such, they were lost owing to genetic recombination during stock maintenance. As in mammalian cells, yeast and bacteria, the frequency of coincident mutations was much higher than would be predicted by chance. (Author).

Sequence and expression analyses of porcine ISG15 and ISG43 genes.

Science.gov (United States)

Huang, Jiangnan; Zhao, Shuhong; Zhu, Mengjin; Wu, Zhenfang; Yu, Mei

2009-08-01

The coding sequences of porcine interferon-stimulated gene 15 (ISG15) and the interferon-stimulated gene (ISG43) were cloned from swine spleen mRNA. The amino acid sequences deduced from porcine ISG15 and ISG43 genes coding sequence shared 24-75% and 29-83% similarity with ISG15s and ISG43s from other vertebrates, respectively. Structural analyses revealed that porcine ISG15 comprises two ubiquitin homologues motifs (UBQ) domain and a conserved C-terminal LRLRGG conjugating motif. Porcine ISG43 contains an ubiquitin-processing proteases-like domain. Phylogenetic analyses showed that porcine ISG15 and ISG43 were mostly related to rat ISG15 and cattle ISG43, respectively. Using quantitative real-time PCR assay, significant increased expression levels of porcine ISG15 and ISG43 genes were detected in porcine kidney endothelial cells (PK15) cells treated with poly I:C. We also observed the enhanced mRNA expression of three members of dsRNA pattern-recognition receptors (PRR), TLR3, DDX58 and IFIH1, which have been reported to act as critical receptors in inducing the mRNA expression of ISG15 and ISG43 genes. However, we did not detect any induced mRNA expression of IFNalpha and IFNbeta, suggesting that transcriptional activations of ISG15 and ISG43 were mediated through IFN-independent signaling pathway in the poly I:C treated PK15 cells. Association analyses in a Landrace pig population revealed that ISG15 c.347T>C (BstUI) polymorphism and the ISG43 c.953T>G (BccI) polymorphism were significantly associated with hematological parameters and immune-related traits.

Quantitative Proteomic Analysis Reveals that Antioxidation Mechanisms Contribute to Cold Tolerance in Plantain (Musa paradisiaca L.; ABB Group) Seedlings*

Science.gov (United States)

Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A.; Chen, Wei; Yang, Yong; Rose, Jocelyn K. C.; Zhang, Sheng; Yi, Gan-Jun

2012-01-01

Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by

Quantitative proteomic analysis reveals that antioxidation mechanisms contribute to cold tolerance in plantain (Musa paradisiaca L.; ABB Group) seedlings.

Science.gov (United States)

Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A; Chen, Wei; Yang, Yong; Rose, Jocelyn K C; Zhang, Sheng; Yi, Gan-Jun

2012-12-01

Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by

Phylogeographic and population genetic analyses reveal multiple species of Boa and independent origins of insular dwarfism.

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Card, Daren C; Schield, Drew R; Adams, Richard H; Corbin, Andrew B; Perry, Blair W; Andrew, Audra L; Pasquesi, Giulia I M; Smith, Eric N; Jezkova, Tereza; Boback, Scott M; Booth, Warren; Castoe, Todd A

2016-09-01

Boa is a Neotropical genus of snakes historically recognized as monotypic despite its expansive distribution. The distinct morphological traits and color patterns exhibited by these snakes, together with the wide diversity of ecosystems they inhabit, collectively suggest that the genus may represent multiple species. Morphological variation within Boa also includes instances of dwarfism observed in multiple offshore island populations. Despite this substantial diversity, the systematics of the genus Boa has received little attention until very recently. In this study we examined the genetic structure and phylogenetic relationships of Boa populations using mitochondrial sequences and genome-wide SNP data obtained from RADseq. We analyzed these data at multiple geographic scales using a combination of phylogenetic inference (including coalescent-based species delimitation) and population genetic analyses. We identified extensive population structure across the range of the genus Boa and multiple lines of evidence for three widely-distributed clades roughly corresponding with the three primary land masses of the Western Hemisphere. We also find both mitochondrial and nuclear support for independent origins and parallel evolution of dwarfism on offshore island clusters in Belize and Cayos Cochinos Menor, Honduras. Copyright © 2016 Elsevier Inc. All rights reserved.

Biochemical and full genome sequence analyses of clinical Vibrio cholerae isolates in Mexico reveals the presence of novel V. cholerae strains.

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Díaz-Quiñonez, José Alberto; Hernández-Monroy, Irma; Montes-Colima, Norma Angélica; Moreno-Pérez, María Asunción; Galicia-Nicolás, Adriana Guadalupe; López-Martínez, Irma; Ruiz-Matus, Cuitláhuac; Kuri-Morales, Pablo; Ortíz-Alcántara, Joanna María; Garcés-Ayala, Fabiola; Ramírez-González, José Ernesto

2016-05-01

The first week of September 2013, the National Epidemiological Surveillance System identified two cases of cholera in Mexico City. The cultures of both samples were confirmed as Vibrio cholerae serogroup O1, serotype Ogawa, biotype El Tor. Initial analyses by PFGE and by PCR-amplification of the virulence genes, suggested that both strains were similar, but different from those previously reported in Mexico. The following week, four more cases were identified in a community in the state of Hidalgo, located 121 km northeast of Mexico City. Thereafter a cholera outbreak started in the region of La Huasteca. Genomic analyses of the four strains obtained in this study confirmed the presence of Pathogenicity Islands VPI-1 and -2, VSP-1 and -2, and of the integrative element SXT. The genomic structure of the 4 isolates was similar to that of V. cholerae strain 2010 EL-1786, identified during the epidemic in Haiti in 2010. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Sequencing, Characterization, and Comparative Analyses of the Plastome of Caragana rosea var. rosea

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Mei Jiang

2018-05-01

Full Text Available To exploit the drought-resistant Caragana species, we performed a comparative study of the plastomes from four species: Caragana rosea, C. microphylla, C. kozlowii, and C. Korshinskii. The complete plastome sequence of the C. rosea was obtained using the next generation DNA sequencing technology. The genome is a circular structure of 133,122 bases and it lacks inverted repeat. It contains 111 unique genes, including 76 protein-coding, 30 tRNA, and four rRNA genes. Repeat analyses obtained 239, 244, 258, and 246 simple sequence repeats in C. rosea, C. microphylla, C. kozlowii, and C. korshinskii, respectively. Analyses of sequence divergence found two intergenic regions: trnI-CAU-ycf2 and trnN-GUU-ycf1, exhibiting a high degree of variations. Phylogenetic analyses showed that the four Caragana species belong to a monophyletic clade. Analyses of Ka/Ks ratios revealed that five genes: rpl16, rpl20, rps11, rps7, and ycf1 and several sites having undergone strong positive selection in the Caragana branch. The results lay the foundation for the development of molecular markers and the understanding of the evolutionary process for drought-resistant characteristics.

Metagenomic analyses reveal the involvement of syntrophic consortia in methanol/electricity conversion in microbial fuel cells.

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Yamamuro, Ayaka; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

2014-01-01

Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs) have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m(-2) (based on the projected area of the anode). In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors.

Lagrangian Coherent Structure Analysis of Terminal Winds: Three-Dimensionality, Intramodel Variations, and Flight Analyses

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Brent Knutson

2015-01-01

Full Text Available We present a study of three-dimensional Lagrangian coherent structures (LCS near the Hong Kong International Airport and relate to previous developments of two-dimensional (2D LCS analyses. The LCS are contrasted among three independent models and against 2D coherent Doppler light detection and ranging (LIDAR data. Addition of the velocity information perpendicular to the LIDAR scanning cone helps solidify flow structures inferred from previous studies; contrast among models reveals the intramodel variability; and comparison with flight data evaluates the performance among models in terms of Lagrangian analyses. We find that, while the three models and the LIDAR do recover similar features of the windshear experienced by a landing aircraft (along the landing trajectory, their Lagrangian signatures over the entire domain are quite different—a portion of each numerical model captures certain features resembling those LCS extracted from independent 2D LIDAR analyses based on observations.

Transcriptomic and Functional Analyses Reveal That PpGLK1 Regulates Chloroplast Development in Peach (Prunus persica

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Min Chen

2018-01-01

Full Text Available Peach is an ideal species for fruit tree research because of its small, fully sequenced genome. Chloroplast development is dependent on the tight cooperation between the nuclear and plastid genomes, and is regulated by GLK transcription factors. In this work, the pigment content was monitored and the chloroplast-to-chromoplast conversion during the fruit ripening was visualized by transmission electron microscopy. Localization and expression analyses showed that PpGLK1 was located in the nucleus and expressed mainly in the leaves and fruit skin. A transcriptome analysis showed that PpGLK1 and its target genes were significantly differentially expressed in ripening peach fruit skin. PpGLK1 silencing affected chlorophyll accumulation in peach leaves and fruits. Overexpression of PpGLK1 rescued the phenotypes of the Arabidopsis Atglk1Atglk2 double mutant and the tomato uniform ripening mutant. The results of a yeast two-hybrid analysis showed that PpGLK1 is autoactivated and that PpGLK1 (301-542 a.a. interacted with PpARF5. Together, our results indicate that PpGLK1 regulates chloroplast development in green tissues in peach. Therefore, it may be a promising target gene for improving the production and quality of peach by genetic engineering and breeding approaches.

Transcriptome-wide mega-analyses reveal joint dysregulation of immunologic genes and transcription regulators in brain and blood in schizophrenia.

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Hess, Jonathan L; Tylee, Daniel S; Barve, Rahul; de Jong, Simone; Ophoff, Roel A; Kumarasinghe, Nishantha; Tooney, Paul; Schall, Ulrich; Gardiner, Erin; Beveridge, Natalie Jane; Scott, Rodney J; Yasawardene, Surangi; Perera, Antionette; Mendis, Jayan; Carr, Vaughan; Kelly, Brian; Cairns, Murray; Tsuang, Ming T; Glatt, Stephen J

2016-10-01

The application of microarray technology in schizophrenia research was heralded as paradigm-shifting, as it allowed for high-throughput assessment of cell and tissue function. This technology was widely adopted, initially in studies of postmortem brain tissue, and later in studies of peripheral blood. The collective body of schizophrenia microarray literature contains apparent inconsistencies between studies, with failures to replicate top hits, in part due to small sample sizes, cohort-specific effects, differences in array types, and other confounders. In an attempt to summarize existing studies of schizophrenia cases and non-related comparison subjects, we performed two mega-analyses of a combined set of microarray data from postmortem prefrontal cortices (n=315) and from ex-vivo blood tissues (n=578). We adjusted regression models per gene to remove non-significant covariates, providing best-estimates of transcripts dysregulated in schizophrenia. We also examined dysregulation of functionally related gene sets and gene co-expression modules, and assessed enrichment of cell types and genetic risk factors. The identities of the most significantly dysregulated genes were largely distinct for each tissue, but the findings indicated common emergent biological functions (e.g. immunity) and regulatory factors (e.g., predicted targets of transcription factors and miRNA species across tissues). Our network-based analyses converged upon similar patterns of heightened innate immune gene expression in both brain and blood in schizophrenia. We also constructed generalizable machine-learning classifiers using the blood-based microarray data. Our study provides an informative atlas for future pathophysiologic and biomarker studies of schizophrenia. Published by Elsevier B.V.

Leveraging Algal Omics to Reveal Potential Targets for Augmenting TAG Accumulation

Energy Technology Data Exchange (ETDEWEB)

Guarnieri, Michael T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Pienkos, Philip T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Arora, Neha [Indian Institute of Technology Roorkee; Pruthi, Vikas [Indian Institute of Technology Roorkee; Poluri, Krishna Mohan [Indian Institute of Technology Roorkee

2018-04-18

Ongoing global efforts to commercialize microalgal biofuels have expedited the use of multi-omics techniques to gain insights into lipid biosynthetic pathways. Functional genomics analyses have recently been employed to complement existing sequence-level omics studies, shedding light on the dynamics of lipid synthesis and its interplay with other cellular metabolic pathways, thus revealing possible targets for metabolic engineering. Here, we review the current status of algal omics studies to reveal potential targets to augment TAG accumulation in various microalgae. This review specifically aims to examine and catalog systems level data related to stress-induced TAG accumulation in oleaginous microalgae and inform future metabolic engineering strategies to develop strains with enhanced bioproductivity, which could pave a path for sustainable green energy.

Direct visualization of epidermal growth factor receptors (EGFR) in A431 and placental cell membrane by western blot with 125I-EGF

International Nuclear Information System (INIS)

Lin, P.H.; Selinfreund, R.; Wharton, W.

1986-01-01

Using the western blot technique, they have devised a new procedure that allowed the direct visualization of both the 150KD and the 170KD forms of EGFR by its natural ligand, 125 I-EGF. A431, and placental plasmalemma were purified and solubilized in either SDS-PAGE buffer (without DTT, EDTA) or Triton X-100 (0.5%), resolved on PAGE and electrophoretically transferred onto nitrocellulose (NC) paper. In the absence of boiling, SDS did not denature the EGFR. Although EGER band can be detected after hybridization with 125 I-EGF, the receptor signal was considerably improved with the addition of 0.1% Tween-20. The binding of 125 I-EGF to the both the 150KD and the 170KD bands of the EGFR was specific, reversible and increased with the amount of membrane protein present. The direct visualization of the EGFR using its natural ligand eliminated the necessity for the time consuming antibody preparation. Presently, they are using this technique to identify specific receptors for other ligands

Los complejos Chaperonina(MSP63inducen anticuerpos de reacciones cruzadas, bactericidas y opsonofagocítica.

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Juan Marzoa

2009-08-01

Full Text Available Alteration of the native structure of antigens can lead to the loss of protective epitopes. Our previous results showed that separation of the meningococcal outer membrane proteins in native conditions revealed the existence of protein complexes that could be relevant for the development of new vaccine formulations. The aim of this work was to analyse the immunogenic characteristics of a highly conserved 700 kDa chaperonin complex (CxChap detected and purified by using high resolution clear native electrophoresis. Analysis of the anti-CxChap serum by Western-blotting revealed the presence of antibodies against the MSP63 but also against the macrophage infectivity potentiator-like protein (MIP, which is coopurified with the chaperonin complex. Antibodies raised by immunisation with CxChap chaperonin complex show bactericidal and opsonophagocytic activity.

Comparative biochemical analyses of venous blood and peritoneal fluid from horses with colic using a portable analyser and an in-house analyser.

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Saulez, M N; Cebra, C K; Dailey, M

2005-08-20

Fifty-six horses with colic were examined over a period of three months. The concentrations of glucose, lactate, sodium, potassium and chloride, and the pH of samples of blood and peritoneal fluid, were determined with a portable clinical analyser and with an in-house analyser and the results were compared. Compared with the in-house analyser, the portable analyser gave higher pH values for blood and peritoneal fluid with greater variability in the alkaline range, and lower pH values in the acidic range, lower concentrations of glucose in the range below 8.3 mmol/l, and lower concentrations of lactate in venous blood in the range below 5 mmol/l and in peritoneal fluid in the range below 2 mmol/l, with less variability. On average, the portable analyser underestimated the concentrations of lactate and glucose in peritoneal fluid in comparison with the in-house analyser. Its measurements of the concentrations of sodium and chloride in peritoneal fluid had a higher bias and were more variable than the measurements in venous blood, and its measurements of potassium in venous blood and peritoneal fluid had a smaller bias and less variability than the measurements made with the in-house analyser.

Molecular systematics of Indian Alysicarpus (Fabaceae) based on analyses of nuclear ribosomal DNA sequences.

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Gholami, Akram; Subramaniam, Shweta; Geeta, R; Pandey, Arun K

2017-06-01

Alysicarpus Necker ex Desvaux (Fabaceae, Desmodieae) consists of ~30 species that are distributed in tropical and subtropical regions of theworld. In India, the genus is represented by ca. 18 species, ofwhich seven are endemic. Sequences of the nuclear Internal transcribed spacer from38 accessions representing 16 Indian specieswere subjected to phylogenetic analyses. The ITS sequence data strongly support the monophyly of the genus Alysicarpus. Analyses revealed four major well-supported clades within Alysicarpus. Ancestral state reconstructions were done for two morphological characters, namely calyx length in relation to pod (macrocalyx and microcalyx) and pod surface ornamentation (transversely rugose and nonrugose). The present study is the first report on molecular systematics of Indian Alysicarpus.

Landscape genetic analyses reveal fine-scale effects of forest fragmentation in an insular tropical bird.

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Khimoun, Aurélie; Peterman, William; Eraud, Cyril; Faivre, Bruno; Navarro, Nicolas; Garnier, Stéphane

2017-10-01

Within the framework of landscape genetics, resistance surface modelling is particularly relevant to explicitly test competing hypotheses about landscape effects on gene flow. To investigate how fragmentation of tropical forest affects population connectivity in a forest specialist bird species, we optimized resistance surfaces without a priori specification, using least-cost (LCP) or resistance (IBR) distances. We implemented a two-step procedure in order (i) to objectively define the landscape thematic resolution (level of detail in classification scheme to describe landscape variables) and spatial extent (area within the landscape boundaries) and then (ii) to test the relative role of several landscape features (elevation, roads, land cover) in genetic differentiation in the Plumbeous Warbler (Setophaga plumbea). We detected a small-scale reduction of gene flow mainly driven by land cover, with a negative impact of the nonforest matrix on landscape functional connectivity. However, matrix components did not equally constrain gene flow, as their conductivity increased with increasing structural similarity with forest habitat: urban areas and meadows had the highest resistance values whereas agricultural areas had intermediate resistance values. Our results revealed a higher performance of IBR compared to LCP in explaining gene flow, reflecting suboptimal movements across this human-modified landscape, challenging the common use of LCP to design habitat corridors and advocating for a broader use of circuit theory modelling. Finally, our results emphasize the need for an objective definition of landscape scales (landscape extent and thematic resolution) and highlight potential pitfalls associated with parameterization of resistance surfaces. © 2017 John Wiley & Sons Ltd.

Hemoglobin analyses in the Netherlands reveal more than 80 different variants including six novel ones.

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van Zwieten, Rob; Veldthuis, Martijn; Delzenne, Barend; Berghuis, Jeffrey; Groen, Joke; Ait Ichou, Fatima; Clifford, Els; Harteveld, Cornelis L; Stroobants, An K

2014-01-01

More than 20,000 blood samples of individuals living in The Netherlands and suspected of hemolytic anemia or diabetes were analyzed by high resolution cation exchange high performance liquid chromatography (HPLC). Besides common disease-related hemoglobins (Hbs), rare variants were also detected. The variant Hbs were retrospectively analyzed by capillary zone electrophoresis (CZE) and by isoelectric focusing (IEF). For unambiguous identification, the globin genes were sequenced. Most of the 80 Hb variants detected by initial screening on HPLC were also separated by capillary electrophoresis (CE), but a few variants were only detectable with one of these methods. Some variants were unstable, had thalassemic properties or increased oxygen affinity, and some interfered with Hb A2 measurement, detection of sickle cell Hb or Hb A1c quantification. Two of the six novel variants, Hb Enschede (HBA2: c.308G  > A, p.Ser103Asn) and Hb Weesp (HBA1: c.301C > T, p.Leu101Phe), had no clinical consequences. In contrast, two others appeared clinically significant: Hb Ede (HBB: c.53A > T, p.Lys18Met) caused thalassemia and Hb Waterland (HBB: c.428C > T, pAla143Val) was related to mild polycytemia. Hb A2-Venlo (HBD: c.193G > A, p.Gly65Ser) and Hb A2-Rotterdam (HBD: c.38A > C, p.Asn13Thr) interfered with Hb A2 quantification. This survey shows that HPLC analysis followed by globin gene sequencing of rare variants is an effective method to reveal Hb variants.

Analyses of Evolutionary Characteristics of the Hemagglutinin-Esterase Gene of Influenza C Virus during a Period of 68 Years Reveals Evolutionary Patterns Different from Influenza A and B Viruses

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Yuki Furuse

2016-11-01

Full Text Available Infections with the influenza C virus causing respiratory symptoms are common, particularly among children. Since isolation and detection of the virus are rarely performed, compared with influenza A and B viruses, the small number of available sequences of the virus makes it difficult to analyze its evolutionary dynamics. Recently, we reported the full genome sequence of 102 strains of the virus. Here, we exploited the data to elucidate the evolutionary characteristics and phylodynamics of the virus compared with influenza A and B viruses. Along with our data, we obtained public sequence data of the hemagglutinin-esterase gene of the virus; the dataset consists of 218 unique sequences of the virus collected from 14 countries between 1947 and 2014. Informatics analyses revealed that (1 multiple lineages have been circulating globally; (2 there have been weak and infrequent selective bottlenecks; (3 the evolutionary rate is low because of weak positive selection and a low capability to induce mutations; and (4 there is no significant positive selection although a few mutations affecting its antigenicity have been induced. The unique evolutionary dynamics of the influenza C virus must be shaped by multiple factors, including virological, immunological, and epidemiological characteristics.

Conventional and advanced exergetic analyses applied to a combined cycle power plant

International Nuclear Information System (INIS)

Petrakopoulou, Fontina; Tsatsaronis, George; Morosuk, Tatiana; Carassai, Anna

2012-01-01

Conventional exergy-based methods pinpoint components and processes with high irreversibilities. However, they lack certain insight. For a given advanced technological state, there is a minimum level of exergy destruction related to technological and/or economic constraints that is unavoidable. Furthermore, in any thermodynamic system, exergy destruction stems from both component interactions (exogenous) and component inefficiencies (endogenous). To overcome the limitations of the conventional analyses and to increase our knowledge about a plant, advanced exergy-based analyses have been developed. In this paper, a combined cycle power plant is analyzed using both conventional and advanced exergetic analyses. Except for the expander of the gas turbine system and the high-pressure steam turbine, most of the exergy destruction in the plant components is unavoidable. This unavoidable part is constrained by internal technological limitations, i.e. each component’s endogenous exergy destruction. High levels of endogenous exergy destruction show that component interactions do not contribute significantly to the thermodynamic inefficiencies. In addition, these inefficiencies are unavoidable to a large extent. With the advanced analysis, new improvement strategies are revealed that could not otherwise be found. -- Highlights: ► This is the first application of a complete advanced exergetic analysis to a complex power plant. ► In the three-pressure-level combined cycle power plant studied here, the improvement potential of the majority of the components is low, since most of the exergy destruction is unavoidable. ► Component interactions are generally of lower importance for the considered plant. ► Splitting the exogenous exergy destruction reveals one-to-one component interactions and improvement strategies. ► The advanced exergetic analysis is a necessary supplement to the conventional analysis in improving a complex system.

Biostratigraphic and morphometric analyses of specimens from the calcareous nannofossil genus Tribrachiatus

Science.gov (United States)

Self-Trail, Jean; Seefelt, Ellen L.; Shepherd, Claire L.; Martin, Victoria A.

2017-01-01

Biostratigraphic and morphometric analyses of calcareous nannofossil assemblages from one outcrop and two cored sections of lower Eocene sediments reveal the presence of two new species: Tribrachiatus lunatus sp. nov., and Tribrachiatus absidatus sp. nov. Differences between the new species and Tribrachiatus orthostylus are discussed. The first occurrence of the two new species is just below the calcareous nannofossil Zone NP11/NP12 boundary, close to the Chron 24r/23n boundary, and thus they are globally useful biostratigraphic markers.

Comprehensive logic based analyses of Toll-like receptor 4 signal transduction pathway.

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Mahesh Kumar Padwal

Full Text Available Among the 13 TLRs in the vertebrate systems, only TLR4 utilizes both Myeloid differentiation factor 88 (MyD88 and Toll/Interleukin-1 receptor (TIR-domain-containing adapter interferon-β-inducing Factor (TRIF adaptors to transduce signals triggering host-protective immune responses. Earlier studies on the pathway combined various experimental data in the form of one comprehensive map of TLR signaling. But in the absence of adequate kinetic parameters quantitative mathematical models that reveal emerging systems level properties and dynamic inter-regulation among the kinases/phosphatases of the TLR4 network are not yet available. So, here we used reaction stoichiometry-based and parameter independent logical modeling formalism to build the TLR4 signaling network model that captured the feedback regulations, interdependencies between signaling kinases and phosphatases and the outcome of simulated infections. The analyses of the TLR4 signaling network revealed 360 feedback loops, 157 negative and 203 positive; of which, 334 loops had the phosphatase PP1 as an essential component. The network elements' interdependency (positive or negative dependencies in perturbation conditions such as the phosphatase knockout conditions revealed interdependencies between the dual-specific phosphatases MKP-1 and MKP-3 and the kinases in MAPK modules and the role of PP2A in the auto-regulation of Calmodulin kinase-II. Our simulations under the specific kinase or phosphatase gene-deficiency or inhibition conditions corroborated with several previously reported experimental data. The simulations to mimic Yersinia pestis and E. coli infections identified the key perturbation in the network and potential drug targets. Thus, our analyses of TLR4 signaling highlights the role of phosphatases as key regulatory factors in determining the global interdependencies among the network elements; uncovers novel signaling connections; identifies potential drug targets for

Dynamic Analyses of Alternative Polyadenylation from RNA-Seq Reveal 3′-UTR Landscape Across 7 Tumor Types

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Xia, Zheng; Donehower, Lawrence A; Cooper, Thomas A.; Neilson, Joel R.; Wheeler, David A.; Wagner, Eric J.; Li, Wei

2015-01-01

Alternative polyadenylation (APA) is a pervasive mechanism in the regulation of most human genes, and its implication in diseases including cancer is only beginning to be appreciated. Since conventional APA profiling has not been widely adopted, global cancer APA studies are very limited. Here we develop a novel bioinformatics algorithm (DaPars) for the de novo identification of dynamic APAs from standard RNA-seq. When applied to 358 TCGA Pan-Cancer tumor/normal pairs across 7 tumor types, DaPars reveals 1,346 genes with recurrent and tumor-specific APAs. Most APA genes (91%) have shorter 3′ UTRs in tumors that can avoid miRNA-mediated repression, including glutaminase (GLS), a key metabolic enzyme for tumor proliferation. Interestingly, selected APA events add strong prognostic power beyond common clinical and molecular variables, suggesting their potential as novel prognostic biomarkers. Finally, our results implicate CstF64, an essential polyadenylation factor, as a master regulator of 3′ UTR shortening across multiple tumor types. PMID:25409906

Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

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Marangoni Sérgio

2009-04-01

Full Text Available Abstract Background Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area – BA22p identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6 and glial fibrillary acidic protein (GFAP were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

Metagenomic analyses reveal the involvement of syntrophic consortia in methanol/electricity conversion in microbial fuel cells.

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Ayaka Yamamuro

Full Text Available Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m(-2 (based on the projected area of the anode. In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors.

Water deficit mechanisms in perennial shrubs Cerasus humilis leaves revealed by physiological and proteomic analyses.

Science.gov (United States)

Yin, Zepeng; Ren, Jing; Zhou, Lijuan; Sun, Lina; Wang, Jiewan; Liu, Yulong; Song, Xingshun

2016-01-01

Drought (Water deficit, WD) poses a serious threat to extensively economic losses of trees throughout the world. Chinese dwarf cherry ( Cerasus humilis ) is a good perennial plant for studying the physiological and sophisticated molecular network under WD. The aim of this study is to identify the effect of WD on C. humilis through physiological and global proteomics analysis and improve understanding of the WD resistance of plants. Currently, physiological parameters were applied to investigate C. humilis response to WD. Moreover, we used two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in C. humilis leaves subjected to WD (24 d). Furthermore, we also examined the correlation between protein and transcript levels. Several physiological parameters, including relative water content and Pn were reduced by WD. In addition, the malondialdehyde (MDA), relative electrolyte leakage (REL), total soluble sugar, and proline were increased in WD-treated C. humilis . Comparative proteomic analysis revealed 46 protein spots (representing 43 unique proteins) differentially expressed in C. humilis leaves under WD. These proteins were mainly involved in photosynthesis, ROS scavenging, carbohydrate metabolism, transcription, protein synthesis, protein processing, and nitrogen and amino acid metabolisms, respectively. WD promoted the CO 2 assimilation by increase light reaction and Calvin cycle, leading to the reprogramming of carbon metabolism. Moreover, the accumulation of osmolytes (i.e., proline and total soluble sugar) and enhancement of ascorbate-glutathione cycle and glutathione peroxidase/glutathione s-transferase pathway in leaves could minimize oxidative damage of membrane and other molecules under WD. Importantly, the regulation role of carbohydrate metabolisms (e. g. glycolysis, pentose phosphate pathways, and TCA) was enhanced. These findings provide key candidate proteins for genetic improvement of perennial plants metabolism under

2-tiered antibody testing for early and late Lyme disease using only an immunoglobulin G blot with the addition of a VlsE band as the second-tier test.

Science.gov (United States)

Branda, John A; Aguero-Rosenfeld, Maria E; Ferraro, Mary Jane; Johnson, Barbara J B; Wormser, Gary P; Steere, Allen C

2010-01-01

Standard 2-tiered immunoglobulin G (IgG) testing has performed well in late Lyme disease (LD), but IgM testing early in the illness has been problematic. IgG VlsE antibody testing, by itself, improves early sensitivity, but may lower specificity. We studied whether elements of the 2 approaches could be combined to produce a second-tier IgG blot that performs well throughout the infection. Separate serum sets from LD patients and control subjects were tested independently at 2 medical centers using whole-cell enzyme immunoassays and IgM and IgG immunoblots, with recombinant VlsE added to the IgG blots. The results from both centers were combined, and a new second-tier IgG algorithm was developed. With standard 2-tiered IgM and IgG testing, 31% of patients with active erythema migrans (stage 1), 63% of those with acute neuroborreliosis or carditis (stage 2), and 100% of those with arthritis or late neurologic involvement (stage 3) had positive results. Using new IgG criteria, in which only the VlsE band was scored as a second-tier test among patients with early LD (stage 1 or 2) and 5 of 11 IgG bands were required in those with stage 3 LD, 34% of patients with stage 1, 96% of those with stage 2, and 100% of those with stage 3 infection had positive responses. Both new and standard testing achieved 100% specificity. Compared with standard IgM and IgG testing, the new IgG algorithm (with VlsE band) eliminates the need for IgM testing; it provides comparable or better sensitivity, and it maintains high specificity.

Phylogenetic and evolutionary analyses of dengue viruses isolated in Jakarta, Indonesia.

Science.gov (United States)

Lestari, C S Whinie; Yohan, Benediktus; Yunita, Anisa; Meutiawati, Febrina; Hayati, Rahma Fitri; Trimarsanto, Hidayat; Sasmono, R Tedjo

2017-12-01

Dengue has affected Indonesia for the last five decades and become a major health problem in many cities in the country. Jakarta, the capital of Indonesia, reports dengue cases annually, with several outbreaks documented. To gain information on the dynamic and evolutionary history of dengue virus (DENV) in Jakarta, we conducted phylogenetic and evolutionary analyses of DENV isolated in 2009. Three hundred thirty-three dengue-suspected patients were recruited. Our data revealed that dengue predominantly affected young adults, and the majority of cases were due to secondary infection. A total of 171 virus isolates were successfully serotyped. All four DENV serotypes were circulating in the city, and DENV-1 was the predominant serotype. The DENV genotyping of 17 isolates revealed the presence of Genotypes I and IV in DENV-1, while DENV-2 isolates were grouped into the Cosmopolitan genotype. The grouping of isolates into Genotype I and II was seen for DENV-3 and DENV-4, respectively. Evolutionary analysis revealed the relatedness of Jakarta isolates with other isolates from other cities in Indonesia and isolates from imported cases in other countries. We revealed the endemicity of DENV and the role of Jakarta as the potential source of imported dengue cases in other countries. Our study provides genetic information regarding DENV from Jakarta, which will be useful for upstream applications, such as the study of DENV epidemiology and evolution and transmission dynamics.

Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta.

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Kui Shi

Full Text Available Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs and proteins (DEPs, respectively. In total 4,588 DEGs including 1,980 up- and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up- and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-to-protein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.

Quantifying Shapes: Mathematical Techniques for Analysing Visual Representations of Sound and Music

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Genevieve L. Noyce

2013-12-01

Full Text Available Research on auditory-visual correspondences has a long tradition but innovative experimental paradigms and analytic tools are sparse. In this study, we explore different ways of analysing real-time visual representations of sound and music drawn by both musically-trained and untrained individuals. To that end, participants' drawing responses captured by an electronic graphics tablet were analysed using various regression, clustering, and classification techniques. Results revealed that a Gaussian process (GP regression model with a linear plus squared-exponential covariance function was able to model the data sufficiently, whereas a simpler GP was not a good fit. Spectral clustering analysis was the best of a variety of clustering techniques, though no strong groupings are apparent in these data. This was confirmed by variational Bayes analysis, which only fitted one Gaussian over the dataset. Slight trends in the optimised hyperparameters between musically-trained and untrained individuals allowed for the building of a successful GP classifier that differentiated between these two groups. In conclusion, this set of techniques provides useful mathematical tools for analysing real-time visualisations of sound and can be applied to similar datasets as well.

Restriction fragment polymorphism (RFLP) of a "new" HLA-DP specificity, CDP-HEI

DEFF Research Database (Denmark)

Hyldig-Nielsen, J J; Ødum, Niels; Morling, Niels

1988-01-01

Southern blotting with a DP beta cDNA probe of MspI digested DNA from 83 healthy unrelated individuals revealed a 1.8 kb fragment present in all four individuals (and no others) possessing the newly determined DP specificity, CDP-HEI.......Southern blotting with a DP beta cDNA probe of MspI digested DNA from 83 healthy unrelated individuals revealed a 1.8 kb fragment present in all four individuals (and no others) possessing the newly determined DP specificity, CDP-HEI....

Multicollinearity in spatial genetics: separating the wheat from the chaff using commonality analyses.

Science.gov (United States)

Prunier, J G; Colyn, M; Legendre, X; Nimon, K F; Flamand, M C

2015-01-01

Direct gradient analyses in spatial genetics provide unique opportunities to describe the inherent complexity of genetic variation in wildlife species and are the object of many methodological developments. However, multicollinearity among explanatory variables is a systemic issue in multivariate regression analyses and is likely to cause serious difficulties in properly interpreting results of direct gradient analyses, with the risk of erroneous conclusions, misdirected research and inefficient or counterproductive conservation measures. Using simulated data sets along with linear and logistic regressions on distance matrices, we illustrate how commonality analysis (CA), a detailed variance-partitioning procedure that was recently introduced in the field of ecology, can be used to deal with nonindependence among spatial predictors. By decomposing model fit indices into unique and common (or shared) variance components, CA allows identifying the location and magnitude of multicollinearity, revealing spurious correlations and thus thoroughly improving the interpretation of multivariate regressions. Despite a few inherent limitations, especially in the case of resistance model optimization, this review highlights the great potential of CA to account for complex multicollinearity patterns in spatial genetics and identifies future applications and lines of research. We strongly urge spatial geneticists to systematically investigate commonalities when performing direct gradient analyses. © 2014 John Wiley & Sons Ltd.

Antibody Banding Patterns of the Enzyme-Linked Immunoelectrotransfer Blot and Brain Imaging Findings in Patients With Neurocysticercosis.

Science.gov (United States)

Arroyo, Gianfranco; Rodriguez, Silvia; Lescano, Andres G; Alroy, Karen A; Bustos, Javier A; Santivañez, Saul; Gonzales, Isidro; Saavedra, Herbert; Pretell, E Javier; Gonzalez, Armando E; Gilman, Robert H; Tsang, Victor C W; Garcia, Hector H

2018-01-06

The enzyme-linked immunoelectrotransfer blot (EITB) assay is the reference serological test for neurocysticercosis (NCC). A positive result on EITB does not always correlate with the presence of active infections in the central nervous system (CNS), and patients with a single viable brain cyst may be EITB negative. Nonetheless, EITB antibody banding patterns appears to be related with the expression of 3 protein families of Taenia solium, and in turn with the characteristics of NCC in the CNS (type, stage, and burden of viable cysts). We evaluated EITB antibody banding patterns and brain imaging findings of 548 NCC cases. Similar banding patterns were grouped into homogeneous classes using latent class analysis. The association between classes and brain imaging findings was assessed. Four classes were identified. Class 1 (patients negative or only positive to the GP50 band, related to the protein family of the same name) was associated with nonviable or single viable parenchymal cysticerci; class 2 (patients positive to bands GP42-39 and GP24, related to the T24-42 protein family, with or without anti-GP50 antibodies) was associated with intraparenchymal viable and nonviable infections; classes 3 and 4 (positive to GP50, GP42-39, and GP24 but also responding to low molecular weight bands GP21, GP18, GP14, and GP13, related to the 8 kDa protein family) were associated with extraparenchymal and intraparenchymal multiple viable cysticerci. EITB antibody banding patterns correlate with brain imaging findings and complement imaging information for the diagnosis of NCC and for staging NCC patients. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Novel cancer gene variants and gene fusions of triple-negative breast cancers (TNBCs) reveal their molecular diversity conserved in the patient-derived xenograft (PDX) model.

Science.gov (United States)

Jung, Jaeyun; Jang, Kiwon; Ju, Jung Min; Lee, Eunji; Lee, Jong Won; Kim, Hee Jung; Kim, Jisun; Lee, Sae Byul; Ko, Beom Seok; Son, Byung Ho; Lee, Hee Jin; Gong, Gyungyup; Ahn, Sei Yeon; Choi, Jung Kyoon; Singh, Shree Ram; Chang, Suhwan

2018-04-20

Despite the improved 5-year survival rate of breast cancer, triple-negative breast cancer (TNBC) remains a challenge due to lack of effective targeted therapy and higher recurrence and metastasis than other subtypes. To identify novel druggable targets and to understand its unique biology, we tried to implement 24 patient-derived xenografts (PDXs) of TNBC. The overall success rate of PDX implantation was 45%, much higher than estrogen receptor (ER)-positive cases. Immunohistochemical analysis revealed conserved ER/PR/Her2 negativity (with two exceptions) between the original and PDX tumors. Genomic analysis of 10 primary tumor-PDX pairs with Ion AmpliSeq CCP revealed high degree of variant conservation (85.0% to 96.9%) between primary and PDXs. Further analysis showed 44 rare variants with a predicted high impact in 36 genes including Trp53, Pten, Notch1, and Col1a1. Among them, we confirmed frequent Notch1 variant. Furthermore, RNA-seq analysis of 24 PDXs revealed 594 gene fusions, of which 163 were in-frame, including AZGP1-GJC3 and NF1-AARSD1. Finally, western blot analysis of oncogenic signaling proteins supporting molecular diversity of TNBC PDXs. Overall, our report provides a molecular basis for the usefulness of the TNBC PDX model in preclinical study. Copyright © 2018. Published by Elsevier B.V.

Genome-wide-analyses of Listeria monocytogenes from food-processing plants reveal clonal diversity and date the emergence of persisting sequence types.

Science.gov (United States)

Knudsen, Gitte M; Nielsen, Jesper Boye; Marvig, Rasmus L; Ng, Yin; Worning, Peder; Westh, Henrik; Gram, Lone

2017-08-01

Whole genome sequencing is increasing used in epidemiology, e.g. for tracing outbreaks of food-borne diseases. This requires in-depth understanding of pathogen emergence, persistence and genomic diversity along the food production chain including in food processing plants. We sequenced the genomes of 80 isolates of Listeria monocytogenes sampled from Danish food processing plants over a time-period of 20 years, and analysed the sequences together with 10 public available reference genomes to advance our understanding of interplant and intraplant genomic diversity of L. monocytogenes. Except for three persisting sequence types (ST) based on Multi Locus Sequence Typing being ST7, ST8 and ST121, long-term persistence of clonal groups was limited, and new clones were introduced continuously, potentially from raw materials. No particular gene could be linked to the persistence phenotype. Using time-based phylogenetic analyses of the persistent STs, we estimate the L. monocytogenes evolutionary rate to be 0.18-0.35 single nucleotide polymorphisms/year, suggesting that the persistent STs emerged approximately 100 years ago, which correlates with the onset of industrialization and globalization of the food market. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Job Demands, Burnout, and Teamwork in Healthcare Professionals Working in a General Hospital that Was Analysed At Two Points in Time

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Dragan Mijakoski

2018-04-01

CONCLUSION: Actual longitudinal study revealed significantly higher mean values of emotional exhaustion and depersonalization in 2014 that could be explained by significantly increased job demands between analysed points in time.

Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

Science.gov (United States)

Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

2013-01-01

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns.

Science.gov (United States)

Alvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M

2013-01-01

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.

The Effect of Pleistocene Climate Fluctuations on Distribution of European Abalone (Haliotis tuberculata), Revealed by Combined Mitochondrial and Nuclear Marker Analyses.

Science.gov (United States)

Roussel, Valérie; Van Wormhoudt, Alain

2017-04-01

The genetic differentiation among the populations of the European abalone Haliotis tuberculata was investigated using different markers to better understand the evolutionary history and exchanges between populations. Three markers were used: mitochondrial cytochrome oxidase I (COI), the sperm lysin nuclear gene, and eight nuclear microsatellites. These markers present different characteristics concerning mutation rate and inheritance, which provided complementary information about abalone history and gene diversity. Genetic diversity and relationships among subspecies were calculated from a sample of approximately 500 individuals, collected from 17 different locations in the north-eastern Atlantic Ocean, Macaronesia, and Mediterranean Sea. COI marker was used to explore the phylogeny of the species with a network analysis and two phylogenetic methods. The analysis revealed 18 major haplotypes grouped into two distinct clades with a pairwise sequence divergence up to 3.5 %. These clades do not correspond to subspecies but revealed many contacts along Atlantic coast during the Pleistocene interglaciations. The sperm lysin gene analysis separated two different subtaxa: one associated to Macaronesian islands, and the other to all other populations. Moreover, a small population of the northern subtaxon was isolated in the Adriatic Sea-probably before the separation of the two lineages-and evolved independently. Microsatellites were analyzed by different genetics methods, including the Bayesian clustering method and migration patterns analysis. It revealed genetically distinct microsatellite patterns among populations from Mediterranean Sea, Brittany and Normandy, Morocco, and Canary and Balearic islands. Gene flow is asymmetric among the regions; the Azores and the Canary Islands are particularly isolated and have low effective population sizes. Our results support the hypothesis that climate changes since the Pleistocene glaciations have played a major role in the

Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

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NUNES Cáris Maroni

1997-01-01

Full Text Available Visceral larva migrans (VLM is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA using the larval excretory-secretory antigen of T. canis (TES, the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa. Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

Molecular Analyses Reveal Inflammatory Mediators in the Solid Component and Cyst Fluid of Human Adamantinomatous Craniopharyngioma.

Science.gov (United States)

Donson, Andrew M; Apps, John; Griesinger, Andrea M; Amani, Vladimir; Witt, Davis A; Anderson, Richard C E; Niazi, Toba N; Grant, Gerald; Souweidane, Mark; Johnston, James M; Jackson, Eric M; Kleinschmidt-DeMasters, Bette K; Handler, Michael H; Tan, Aik-Choon; Gore, Lia; Virasami, Alex; Gonzalez-Meljem, Jose Mario; Jacques, Thomas S; Martinez-Barbera, Juan Pedro; Foreman, Nicholas K; Hankinson, Todd C

2017-09-01

Pediatric adamantinomatous craniopharyngioma (ACP) is a highly solid and cystic tumor, often causing substantial damage to critical neuroendocrine structures such as the hypothalamus, pituitary gland, and optic apparatus. Paracrine signaling mechanisms driving tumor behavior have been hypothesized, with IL-6R overexpression identified as a potential therapeutic target. To identify potential novel therapies, we characterized inflammatory and immunomodulatory factors in ACP cyst fluid and solid tumor components. Cytometric bead analysis revealed a highly pro-inflammatory cytokine pattern in fluid from ACP compared to fluids from another cystic pediatric brain tumor, pilocytic astrocytoma. Cytokines and chemokines with particularly elevated concentrations in ACPs were IL-6, CXCL1 (GRO), CXCL8 (IL-8) and the immunosuppressive cytokine IL-10. These data were concordant with solid tumor compartment transcriptomic data from a larger cohort of ACPs, other pediatric brain tumors and normal brain. The majority of receptors for these cytokines and chemokines were also over-expressed in ACPs. In addition to IL-10, the established immunosuppressive factor IDO-1 was overexpressed by ACPs at the mRNA and protein levels. These data indicate that ACP cyst fluids and solid tumor components are characterized by an inflammatory cytokine and chemokine expression pattern. Further study regarding selective cytokine blockade may inform novel therapeutic interventions. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

Energy Technology Data Exchange (ETDEWEB)

Merchant, Sabeeha S

2007-04-09

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Culture-Independent Analyses Reveal Novel Anaerolineaceae as Abundant Primary Fermenters in Anaerobic Digesters Treating Waste Activated Sludge

Directory of Open Access Journals (Sweden)

Simon J. McIlroy

2017-06-01

Full Text Available Anaerobic digestion for biogas production is reliant on the tightly coupled synergistic activities of complex microbial consortia. Members of the uncultured A6 phylotype, within the phylum Chloroflexi, are among the most abundant genus-level-taxa of mesophilic anaerobic digester systems treating primary and surplus sludge from wastewater treatment plants, yet are known only by their 16S rRNA gene sequence. This study applied metagenomics to obtain a complete circular genome (2.57 Mbp from a representative of the A6 taxon. Preliminary annotation of the genome indicates these organisms to be anaerobic chemoorganoheterotrophs with a fermentative metabolism. Given their observed abundance, they are likely important primary fermenters in digester systems. Application of fluorescence in situ hybridisation probes designed in this study revealed their morphology to be short filaments present within the flocs. The A6 were sometimes co-located with the filamentous Archaea Methanosaeta spp. suggesting potential undetermined synergistic relationships. Based on its genome sequence and morphology we propose the species name Brevefilum fermentans gen. nov. sp. nov.

Genomic Analyses Reveal Demographic History and Temperate Adaptation of the Newly Discovered Honey Bee Subspecies Apis mellifera sinisxinyuan n. ssp.

Science.gov (United States)

Chen, Chao; Liu, Zhiguang; Pan, Qi; Chen, Xiao; Wang, Huihua; Guo, Haikun; Liu, Shidong; Lu, Hongfeng; Tian, Shilin; Li, Ruiqiang; Shi, Wei

2016-05-01

Studying the genetic signatures of climate-driven selection can produce insights into local adaptation and the potential impacts of climate change on populations. The honey bee (Apis mellifera) is an interesting species to study local adaptation because it originated in tropical/subtropical climatic regions and subsequently spread into temperate regions. However, little is known about the genetic basis of its adaptation to temperate climates. Here, we resequenced the whole genomes of ten individual bees from a newly discovered population in temperate China and downloaded resequenced data from 35 individuals from other populations. We found that the new population is an undescribed subspecies in the M-lineage of A. mellifera (Apis mellifera sinisxinyuan). Analyses of population history show that long-term global temperature has strongly influenced the demographic history of A. m. sinisxinyuan and its divergence from other subspecies. Further analyses comparing temperate and tropical populations identified several candidate genes related to fat body and the Hippo signaling pathway that are potentially involved in adaptation to temperate climates. Our results provide insights into the demographic history of the newly discovered A. m. sinisxinyuan, as well as the genetic basis of adaptation of A. mellifera to temperate climates at the genomic level. These findings will facilitate the selective breeding of A. mellifera to improve the survival of overwintering colonies. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Structural, Bioinformatic, and In Vivo Analyses of Two Treponema pallidum Lipoproteins Reveal a Unique TRAP Transporter

Energy Technology Data Exchange (ETDEWEB)

Deka, Ranjit K.; Brautigam, Chad A.; Goldberg, Martin; Schuck, Peter; Tomchick, Diana R.; Norgard, Michael V. (NIH); (UTSMC)

2012-05-25

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of 'tetratricopeptide repeat' (TPR) motifs. The crystal structure of recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).

Amplification and chromosomal dispersion of human endogenous retroviral sequences

International Nuclear Information System (INIS)

Steele, P.E.; Martin, M.A.; Rabson, A.B.; Bryan, T.; O'Brien, S.J.

1986-01-01

Endogenous retroviral sequences have undergone amplification events involving both viral and flanking cellular sequences. The authors cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked

Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting

DEFF Research Database (Denmark)

Söderling, Ann-Sofi; Hultman, Lena; Delbro, Dick

2007-01-01

We noted differences in the antibody response to 3-nitrotyrosine (NO(2)Tyr) in fixed and non-fixed tissues, and studied therefore potential problems associated with non-fixed tissues in Western blot analyses. Three different monoclonal anti-nitrotyrosine antibodies in Western blot analysis of inf...... is not detected by anti-NO(2)Tyr antibodies. Western blot analysis may therefore underestimate the level of tissue nitration, and factors causing a reduction of NO(2)Tyr during sample preparation might conceal the actual nitration of proteins....

Constraints on genome dynamics revealed from gene distribution among the Ralstonia solanacearum species.

Directory of Open Access Journals (Sweden)

Pierre Lefeuvre

Full Text Available Because it is suspected that gene content may partly explain host adaptation and ecology of pathogenic bacteria, it is important to study factors affecting genome composition and its evolution. While recent genomic advances have revealed extremely large pan-genomes for some bacterial species, it remains difficult to predict to what extent gene pool is accessible within or transferable between populations. As genomes bear imprints of the history of the organisms, gene distribution pattern analyses should provide insights into the forces and factors at play in the shaping and maintaining of bacterial genomes. In this study, we revisited the data obtained from a previous CGH microarrays analysis in order to assess the genomic plasticity of the R. solanacearum species complex. Gene distribution analyses demonstrated the remarkably dispersed genome of R. solanacearum with more than half of the genes being accessory. From the reconstruction of the ancestral genomes compositions, we were able to infer the number of gene gain and loss events along the phylogeny. Analyses of gene movement patterns reveal that factors associated with gene function, genomic localization and ecology delineate gene flow patterns. While the chromosome displayed lower rates of movement, the megaplasmid was clearly associated with hot-spots of gene gain and loss. Gene function was also confirmed to be an essential factor in gene gain and loss dynamics with significant differences in movement patterns between different COG categories. Finally, analyses of gene distribution highlighted possible highways of horizontal gene transfer. Due to sampling and design bias, we can only speculate on factors at play in this gene movement dynamic. Further studies examining precise conditions that favor gene transfer would provide invaluable insights in the fate of bacteria, species delineation and the emergence of successful pathogens.

Comparative sequence analyses of the major quantitative trait locus phosphorus uptake 1 (Pup1) reveal a complex genetic structure.

Science.gov (United States)

Heuer, Sigrid; Lu, Xiaochun; Chin, Joong Hyoun; Tanaka, Juan Pariasca; Kanamori, Hiroyuki; Matsumoto, Takashi; De Leon, Teresa; Ulat, Victor Jun; Ismail, Abdelbagi M; Yano, Masahiro; Wissuwa, Matthias

2009-06-01

The phosphorus uptake 1 (Pup1) locus was identified as a major quantitative trait locus (QTL) for tolerance of phosphorus deficiency in rice. Near-isogenic lines with the Pup1 region from tolerant donor parent Kasalath typically show threefold higher phosphorus uptake and grain yield in phosphorus-deficient field trials than the intolerant parent Nipponbare. In this study, we report the fine mapping of the Pup1 locus to the long arm of chromosome 12 (15.31-15.47 Mb). Genes in the region were initially identified on the basis of the Nipponbare reference genome, but did not reveal any obvious candidate genes related to phosphorus uptake. Kasalath BAC clones were therefore sequenced and revealed a 278-kbp sequence significantly different from the syntenic regions in Nipponbare (145 kb) and in the indica reference genome of 93-11 (742 kbp). Size differences are caused by large insertions or deletions (INDELs), and an exceptionally large number of retrotransposon and transposon-related elements (TEs) present in all three sequences (45%-54%). About 46 kb of the Kasalath sequence did not align with the entire Nipponbare genome, and only three Nipponbare genes (fatty acid alpha-dioxygenase, dirigent protein and aspartic proteinase) are highly conserved in Kasalath. Two Nipponbare genes (expressed proteins) might have evolved by at least three TE integrations in an ancestor gene that is still present in Kasalath. Several predicted Kasalath genes are novel or unknown genes that are mainly located within INDEL regions. Our results highlight the importance of sequencing QTL regions in the respective donor parent, as important genes might not be present in the current reference genomes.

Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure

International Nuclear Information System (INIS)

Soreq, H.; Ben-Aziz, R.; Prody, C.A.; Seidman, S.; Gnatt, A.; Neville, L.; Lieman-Hurwitz, J.; Lev-Lehman, E.; Ginzberg, D.; Lapidot-Lifson, Y.; Zakut, H.

1990-01-01

To study the primary structure of human acetylcholinesterase and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with ≥50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase. However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the AcChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification

Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

Science.gov (United States)

Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

2016-02-01

Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

IgG western blot for confirmatory diagnosis of equivocal cases of toxoplasmosis by EIA-IgG and fluorescent antibody test.

Science.gov (United States)

Khammari, Imen; Saghrouni, Fatma; Yaacoub, Alia; Gaied Meksi, Sondoss; Ach, Hinda; Garma, Lamia; Fathallah, Akila; Ben Saïd, Moncef

2013-08-01

The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.

Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

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Takuji Oyama

Full Text Available CpMan5B is a glycoside hydrolase (GH family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196 in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

Science.gov (United States)

Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

2013-01-01

CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

ALBEDO PATTERN RECOGNITION AND TIME-SERIES ANALYSES IN MALAYSIA

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S. A. Salleh

2012-07-01

Full Text Available Pattern recognition and time-series analyses will enable one to evaluate and generate predictions of specific phenomena. The albedo pattern and time-series analyses are very much useful especially in relation to climate condition monitoring. This study is conducted to seek for Malaysia albedo pattern changes. The pattern recognition and changes will be useful for variety of environmental and climate monitoring researches such as carbon budgeting and aerosol mapping. The 10 years (2000–2009 MODIS satellite images were used for the analyses and interpretation. These images were being processed using ERDAS Imagine remote sensing software, ArcGIS 9.3, the 6S code for atmospherical calibration and several MODIS tools (MRT, HDF2GIS, Albedo tools. There are several methods for time-series analyses were explored, this paper demonstrates trends and seasonal time-series analyses using converted HDF format MODIS MCD43A3 albedo land product. The results revealed significance changes of albedo percentages over the past 10 years and the pattern with regards to Malaysia's nebulosity index (NI and aerosol optical depth (AOD. There is noticeable trend can be identified with regards to its maximum and minimum value of the albedo. The rise and fall of the line graph show a similar trend with regards to its daily observation. The different can be identified in term of the value or percentage of rises and falls of albedo. Thus, it can be concludes that the temporal behavior of land surface albedo in Malaysia have a uniform behaviours and effects with regards to the local monsoons. However, although the average albedo shows linear trend with nebulosity index, the pattern changes of albedo with respects to the nebulosity index indicates that there are external factors that implicates the albedo values, as the sky conditions and its diffusion plotted does not have uniform trend over the years, especially when the trend of 5 years interval is examined, 2000 shows high

The secondary stress analyses in the fuel pin cladding due to the swelling gradient across the wall thickness

International Nuclear Information System (INIS)

Uwaba, Tomoyuki; Ukai, Shigeharu

2002-01-01

Irradiation deformation analyses of FBR fuel cladding were made by using the finite element method. In these analyses the history of the stress occurred in the cladding was evaluated paying attention to the secondary stress induced by the swelling difference across the wall thickness. It was revealed that the difference of the swelling incubation dose in the direction of the thickness and the irradiation creep deformation play an important role in the history of the secondary stress. The effect of the stress-enhanced swelling was also analyzed in this study

Analysing E-Services and Mobile Applications with Companied Conjoint Analysis and fMRI Technique

OpenAIRE

Heinonen, Jarmo

2015-01-01

Previous research has shown that neuromarketing and conjoint analysis have been used in many areas of consumer research, and to provide for further understanding of consumer behaviour. Together these two methods may reveal more information about hidden desires, expectations and restrains of consumers’ brain. This paper attempts to examine these two research methods together as a companied analysis. More specifically this study utilizes fMRI and conjoint analysis is a tool for analysing consum...

Isotropy analyses of the Planck convergence map

Science.gov (United States)

Marques, G. A.; Novaes, C. P.; Bernui, A.; Ferreira, I. S.

2018-01-01

The presence of matter in the path of relic photons causes distortions in the angular pattern of the cosmic microwave background (CMB) temperature fluctuations, modifying their properties in a slight but measurable way. Recently, the Planck Collaboration released the estimated convergence map, an integrated measure of the large-scale matter distribution that produced the weak gravitational lensing (WL) phenomenon observed in Planck CMB data. We perform exhaustive analyses of this convergence map calculating the variance in small and large regions of the sky, but excluding the area masked due to Galactic contaminations, and compare them with the features expected in the set of simulated convergence maps, also released by the Planck Collaboration. Our goal is to search for sky directions or regions where the WL imprints anomalous signatures to the variance estimator revealed through a χ2 analyses at a statistically significant level. In the local analysis of the Planck convergence map, we identified eight patches of the sky in disagreement, in more than 2σ, with what is observed in the average of the simulations. In contrast, in the large regions analysis we found no statistically significant discrepancies, but, interestingly, the regions with the highest χ2 values are surrounding the ecliptic poles. Thus, our results show a good agreement with the features expected by the Λ cold dark matter concordance model, as given by the simulations. Yet, the outliers regions found here could suggest that the data still contain residual contamination, like noise, due to over- or underestimation of systematic effects in the simulation data set.

YY1 binding association with sex-biased transcription revealed through X-linked transcript levels and allelic binding analyses.

Science.gov (United States)

Chen, Chih-Yu; Shi, Wenqiang; Balaton, Bradley P; Matthews, Allison M; Li, Yifeng; Arenillas, David J; Mathelier, Anthony; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Brown, Carolyn J; Wasserman, Wyeth W

2016-11-18

Sex differences in susceptibility and progression have been reported in numerous diseases. Female cells have two copies of the X chromosome with X-chromosome inactivation imparting mono-allelic gene silencing for dosage compensation. However, a subset of genes, named escapees, escape silencing and are transcribed bi-allelically resulting in sexual dimorphism. Here we conducted in silico analyses of the sexes using human datasets to gain perspectives into such regulation. We identified transcription start sites of escapees (escTSSs) based on higher transcription levels in female cells using FANTOM5 CAGE data. Significant over-representations of YY1 transcription factor binding motif and ChIP-seq peaks around escTSSs highlighted its positive association with escapees. Furthermore, YY1 occupancy is significantly biased towards the inactive X (Xi) at long non-coding RNA loci that are frequent contacts of Xi-specific superloops. Our study suggests a role for YY1 in transcriptional activity on Xi in general through sequence-specific binding, and its involvement at superloop anchors.

Genome sequencing reveals metabolic and cellular interdependence in an amoeba-kinetoplastid symbiosis

Czech Academy of Sciences Publication Activity Database

Tanifuji, G.; Cenci, U.; Moog, D.; Dean, S.; Nakayama, T.; David, Vojtěch; Fiala, Ivan; Curtis, B.A.; Sibbald, S. J.; Onodera, N. T.; Colp, M.; Flegontov, Pavel; Johnson-MacKinnon, J.; McPhee, M.; Inagaki, Y.; Hashimoto, T.; Kelly, S.; Gull, K.; Lukeš, Julius; Archibald, J.M.

2017-01-01

Roč. 7, SEP 15 (2017), č. článku 11688. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA14-23986S; GA MŠk LL1601 Institutional support: RVO:60077344 Keywords : trypanosoma-brucei reveals * hidden markov model * neoparamoeba-pemaquidensis * gill disease * phylogenetic analyses * ichthyobodo-necator * gene prediction * host control * evolution * proteomics Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.259, year: 2016

Correlation analyses revealed global microRNA-mRNA expression associations in human peripheral blood mononuclear cells.

Science.gov (United States)

Wang, Lan; Zhu, Jiang; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Xiao-Wei; Xia, Wei; Xie, Fang-Fei; He, Pei; Bing, Peng-Fei; Qiu, Ying-Hua; Lin, Xiang; Lu, Xin; Zhang, Lei; Yi, Neng-Jun; Zhang, Yong-Hong; Lei, Shu-Feng

2018-02-01

MicroRNAs (miRNAs) can regulate gene expression through binding to complementary sites in the 3'-untranslated regions of target mRNAs, which will lead to existence of correlation in expression between miRNA and mRNA. However, the miRNA-mRNA correlation patterns are complex and remain largely unclear yet. To establish the global correlation patterns in human peripheral blood mononuclear cells (PBMCs), multiple miRNA-mRNA correlation analyses and expression quantitative trait locus (eQTL) analysis were conducted in this study. We predicted and achieved 861 miRNA-mRNA pairs (65 miRNAs, 412 mRNAs) using multiple bioinformatics programs, and found global negative miRNA-mRNA correlations in PBMC from all 46 study subjects. Among the 861 pairs of correlations, 19.5% were significant (P correlation network was complex and highlighted key miRNAs/genes in PBMC. Some miRNAs, such as hsa-miR-29a, hsa-miR-148a, regulate a cluster of target genes. Some genes, e.g., TNRC6A, are regulated by multiple miRNAs. The identified genes tend to be enriched in molecular functions of DNA and RNA binding, and biological processes such as protein transport, regulation of translation and chromatin modification. The results provided a global view of the miRNA-mRNA expression correlation profile in human PBMCs, which would facilitate in-depth investigation of biological functions of key miRNAs/mRNAs and better understanding of the pathogenesis underlying PBMC-related diseases.

Microprobe analyses of uranium and thorium in uraninite from the Witwatersrand, South Africa, and Blind River, Ontario, Canada

International Nuclear Information System (INIS)

Grandstaff, D.E.

1981-01-01

Microprobe analyses of uranium and thorium in uraninite grains from the Witwatersrand, South Africa, and Blind River, Ontario, reveal that although individual grains are fairly homogeneous, the assemblage of grains is quite heterogeneous. This heterogeneity appears to favor genetic concepts advocating a detrital, placer origin for the uraninite

18F-FDG PET Reveals Fronto-temporal Dysfunction in Children with Fever-Induced Refractory Epileptic Encephalopathy

International Nuclear Information System (INIS)

Mazzuca, M.; Dulac, O.; Chiron, C.; Jambaque, I.; Hertz-Pannier, L.; Bouilleret, V.; Archambaud, F.; Rodrigo, S.; Dulac, O.; Chiron, C.; Jambaque, I.; Hertz-Pannier, L.; Bouilleret, V.; Archambaud, F.; Rodrigo, S.; Chiron, C.; Hertz-Pannier, L.; Rodrigo, S.; Dulac, O.; Chiron, C.; Caviness, V.

2011-01-01

Fever-induced refractory epileptic encephalopathy in school-age children (FIRES) is a recently described epileptic entity whose etiology remains unknown. Brain abnormalities shown by MRI are usually limited to mesial-temporal structures and do not account for the catastrophic neuro-psychologic findings. Methods: We conducted FIRES studies in 8 patients, aged 6-13 y, using 18 F-FDG PET to disclose eventual neo-cortical dysfunction. Voxel-based analyses of cerebral glucose metabolism were performed using statistical parametric mapping and an age-matched control group. Results: Group analysis revealed a widespread inter-ictal hypo-metabolic network including the temporo-parietal and orbito-frontal cortices bilaterally. The individual analyses in patients identified hypo-metabolic areas corresponding to the predominant electroencephalograph foci and neuro-psychologic deficits involving language, behavior, and memory. Conclusion: Despite clinical heterogeneity, 18 F-FDG PET reveals a common network dysfunction in patients with sequelae due to fever-induced refractory epileptic encephalopathy. (authors)

Molecular cytogenetic analyses of Epinephelus bruneus and Epinephelus moara (Perciformes, Epinephelidae

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Minglan Guo

2014-06-01

Full Text Available Genus Epinephelus (Perciformes, Epinephelidae, commonly known as groupers, are usually difficult in species identification for the lack and/or change of morphological specialization. In this study, molecular cytogenetic analyses were firstly performed to identify the closely related species Epinephelus bruneus and E. moara in this genus. The species-specific differences of both fish species showed in karyotype, chromosomal distribution of nucleolar organizer regions (NORs and localization of 18S rDNA. The heterochromatin (interstitial C-bands and distribution pattern of telomere (TTAGGGn in E. bruneus revealed the chromosomal rearrangements and different karyotypic evolutionary characteristics compared to those in E. moara. The cytogenetic data suggested that the lineages of E. bruneus and E. moara were recently derived within the genus Epinephelus, and E. moara exhibited more plesiomorphic features than E. bruneus. All results confirmed that E. moara, which has long been considered a synonym of E. bruneus, is a distinct species in the family Epinephelidae. In addition, molecular cytogenetic analyses are useful in species differentiation and phylogenetic reconstruction in groupers.

A Formal Model to Analyse the Firewall Configuration Errors

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T. T. Myo

2015-01-01

Full Text Available The firewall is widely known as a brandmauer (security-edge gateway. To provide the demanded security, the firewall has to be appropriately adjusted, i.e. be configured. Unfortunately, when configuring, even the skilled administrators may make mistakes, which result in decreasing level of a network security and network infiltration undesirable packages.The network can be exposed to various threats and attacks. One of the mechanisms used to ensure network security is the firewall.The firewall is a network component, which, using a security policy, controls packages passing through the borders of a secured network. The security policy represents the set of rules.Package filters work in the mode without inspection of a state: they investigate packages as the independent objects. Rules take the following form: (condition, action. The firewall analyses the entering traffic, based on the IP address of the sender and recipient, the port number of the sender and recipient, and the used protocol. When the package meets rule conditions, the action specified in the rule is carried out. It can be: allow, deny.The aim of this article is to develop tools to analyse a firewall configuration with inspection of states. The input data are the file with the set of rules. It is required to submit the analysis of a security policy in an informative graphic form as well as to reveal discrepancy available in rules. The article presents a security policy visualization algorithm and a program, which shows how the firewall rules act on all possible packages. To represent a result in an intelligible form a concept of the equivalence region is introduced.Our task is the program to display results of rules action on the packages in a convenient graphic form as well as to reveal contradictions between the rules. One of problems is the large number of measurements. As it was noted above, the following parameters are specified in the rule: Source IP address, appointment IP

Nomadic lifestyle of Lactobacillus plantarum revealed by comparative genomics of 54 strains isolated from different habitats.

Science.gov (United States)

Martino, Maria Elena; Bayjanov, Jumamurat R; Caffrey, Brian E; Wels, Michiel; Joncour, Pauline; Hughes, Sandrine; Gillet, Benjamin; Kleerebezem, Michiel; van Hijum, Sacha A F T; Leulier, François

2016-12-01

The ability of bacteria to adapt to diverse environmental conditions is well-known. The process of bacterial adaptation to a niche has been linked to large changes in the genome content, showing that many bacterial genomes reflect the constraints imposed by their habitat. However, some highly versatile bacteria are found in diverse habitats that almost share nothing in common. Lactobacillus plantarum is a lactic acid bacterium that is found in a large variety of habitat. With the aim of unravelling the link between evolution and ecological versatility of L. plantarum, we analysed the genomes of 54 L. plantarum strains isolated from different environments. Comparative genome analysis identified a high level of genomic diversity and plasticity among the strains analysed. Phylogenomic and functional divergence studies coupled with gene-trait matching analyses revealed a mixed distribution of the strains, which was uncoupled from their environmental origin. Our findings revealed the absence of specific genomic signatures marking adaptations of L. plantarum towards the diverse habitats it is associated with. This suggests fundamentally similar trends of genome evolution in L. plantarum, which occur in a manner that is apparently uncoupled from ecological constraint and reflects the nomadic lifestyle of this species. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Distribution-analytical techniques in the study of AD/HD: Delta plot analyses reveal deficits in response inhibition that are eliminated by methylphenidate treatment

NARCIS (Netherlands)

Ridderinkhof, K.R.; Scheres, A.; Oosterlaan, J.; Sergeant, J.A.

2005-01-01

The authors highlight the utility of distribution-analytical techniques in the study of individual differences and clinical disorders. Cognitive deficits associated with attention-deficit/hyperactivity disorder (AD/HD) were examined by using delta-plot analyses of performance data (reaction time and

Trophic structure of mesopelagic fishes in the Gulf of Mexico revealed by gut content and stable isotope analyses

Science.gov (United States)

McClain-Counts, Jennifer P.; Demopoulos, Amanda W.J.; Ross, Steve W.

2017-01-01

Mesopelagic fishes represent an important component of the marine food web due to their global distributions, high abundances and ability to transport organic material throughout a large part of the water column. This study combined stable isotope (SIAs) and gut content analyses (GCAs) to characterize the trophic structure of mesopelagic fishes in the North-Central Gulf of Mexico. Additionally, this study examined whether mesopelagic fishes utilized chemosynthetic energy from cold seeps. Specimens were collected (9–25 August 2007) over three deep (>1,000 m) cold seeps at discrete depths (surface to 1,503 m) over the diurnal cycle. GCA classified 31 species (five families) of mesopelagic fishes into five feeding guilds: piscivores, large crustacean consumers, copepod consumers, generalists and mixed zooplanktivores. However, these guilds were less clearly defined based on stable isotope mixing model (MixSIAR) results, suggesting diets may be more mixed over longer time periods (weeks–months) and across co-occurring species. Copepods were likely important for the majority of mesopelagic fishes, consistent with GCA (this study) and previous literature. MixSIAR results also identified non-crustacean prey items, including salps and pteropods, as potentially important prey items for mesopelagic fishes, including those fishes not analysed in GCA (Sternoptyx spp. and Melamphaidae). Salps and other soft-bodied species are often missed in GCAs. Mesopelagic fishes had δ13C results consistent with particulate organic matter serving as the baseline organic carbon source, fueling up to three trophic levels. Fishes that undergo diel vertical migration were depleted in 15N relative to weak migrators, consistent with depth-specific isotope trends in sources and consumers, and assimilation of 15N-depleted organic matter in surface waters. Linear correlations between fish size and δ15N values suggested ontogenetic changes in fish diets for several species. While there was

Tomographic local 2D analyses of the WISExSuperCOSMOS all-sky galaxy catalogue

Science.gov (United States)

Novaes, C. P.; Bernui, A.; Xavier, H. S.; Marques, G. A.

2018-05-01

The recent progress in obtaining larger and deeper galaxy catalogues is of fundamental importance for cosmological studies, especially to robustly measure the large scale density fluctuations in the Universe. The present work uses the Minkowski Functionals (MF) to probe the galaxy density field from the WISExSuperCOSMOS (WSC) all-sky catalogue by performing tomographic local analyses in five redshift shells (of thickness δz = 0.05) in the total range of 0.10 methodology reveals 1 - 3 regions of the GNC maps in each redshift shell with an uncommon behaviour (extreme regions), i.e., p-value < 1.4%. Indeed, the resulting MF curves show signatures that suggest the uncommon behaviour to be associated with the presence of over- or under-densities there, but contamination due to residual foregrounds is not discarded. Additionally, even though our analyses indicate a good agreement among data and simulations, we identify 1 highly extreme region, seemingly associated to a large clustered distribution of galaxies. Our results confirm the usefulness of the MF to analyse GNC maps from photometric galaxy datasets.

Microscopic and Spectroscopic Analyses of Chlorhexidine Tolerance in Delftia acidovorans Biofilms

Science.gov (United States)

Rema, Tara; Lawrence, John R.; Dynes, James J.; Hitchcock, Adam P.

2014-01-01

The physicochemical responses of Delftia acidovorans biofilms exposed to the commonly used antimicrobial chlorhexidine (CHX) were examined in this study. A CHX-sensitive mutant (MIC, 1.0 μg ml−1) was derived from a CHX-tolerant (MIC, 15.0 μg ml−1) D. acidovorans parent strain using transposon mutagenesis. D. acidovorans mutant (MT51) and wild-type (WT15) strain biofilms were cultivated in flow cells and then treated with CHX at sub-MIC and inhibitory concentrations and examined by confocal laser scanning microscopy (CLSM), scanning transmission X-ray microscopy (STXM), and infrared (IR) spectroscopy. Specific morphological, structural, and chemical compositional differences between the CHX-treated and -untreated biofilms of both strains were observed. Apart from architectural differences, CLSM revealed a negative effect of CHX on biofilm thickness in the CHX-sensitive MT51 biofilms relative to those of the WT15 strain. STXM analyses showed that the WT15 biofilms contained two morphochemical cell variants, whereas only one type was detected in the MT51 biofilms. The cells in the MT51 biofilms bioaccumulated CHX to a similar extent as one of the cell types found in the WT15 biofilms, whereas the other cell type in the WT15 biofilms did not bioaccumulate CHX. STXM and IR spectral analyses revealed that CHX-sensitive MT51 cells accumulated the highest levels of CHX. Pretreating biofilms with EDTA promoted the accumulation of CHX in all cells. Thus, it is suggested that a subpopulation of cells that do not accumulate CHX appear to be responsible for greater CHX resistance in D. acidovorans WT15 biofilm in conjunction with the possible involvement of bacterial membrane stability. PMID:25022584

Herbal medicine Guan Chang Fu Fang enhances 5-fluorouracil cytotoxicity and affects drug-associated genes in human colorectal carcinoma cells.

Science.gov (United States)

Yu, Chen; Liu, Shen-Lin; Qi, Ming-Hao; Zou, Xi; Wu, Jian; Zhang, Jing

2015-02-01

Guan Chang Fu Fang (GCFF) is a natural compound, which is extracted from three medicinal plants, Agrimonia pilosa Ledeb ., Patrinia scabiosaefolia and Solanum nigrum L . GCFF has demonstrated clinical efficacy in the treatment of colon cancer. At present, 5-fluorouracil (5-FU) is the primary active chemotherapeutic agent used for treating colon cancer. Using median-effect and apoptosis analyses, fluorescence microscopy and western blotting, the present study analyzed the association between GCFF and 5-FU in the human colon adenocarcinoma LoVo cell line. The effect of GCFF on the expression of chemotherapeutic agent-associated genes was also investigated. The results of the synergistic analysis revealed that GCFF exhibited a significant effect upon 5-FU-associated cytotoxicity within the LoVo cell line. This effect was observed over a broad dose-inhibition range (5-95%), but was particularly significant in the lower concentrations. The flow cytometry results revealed that low doses of GCFF or 5-FU induced S-phase arrest, as did a low-dose combination of the two drugs. After 48 h, GCFF significantly suppressed the expression levels of the chemotherapeutic agent resistance-associated genes within the colon cancer cells. The western blot analysis revealed that the combined effects of 5-FU and GCFF were due to a regulation of the B-cell lymphoma-2 family of proteins. The findings of the present study suggested that GCFF, when combined with 5-FU, has the potential to be a novel, chemotherapeutic compound for the treatment of colon cancer.

Genetic Markers Analyses and Bioinformatic Approaches to Distinguish Between Olive Tree (Olea europaea L.) Cultivars.

Science.gov (United States)

Ben Ayed, Rayda; Ben Hassen, Hanen; Ennouri, Karim; Rebai, Ahmed

2016-12-01

The genetic diversity of 22 olive tree cultivars (Olea europaea L.) sampled from different Mediterranean countries was assessed using 5 SNP markers (FAD2.1; FAD2.3; CALC; SOD and ANTHO3) located in four different genes. The genotyping analysis of the 22 cultivars with 5 SNP loci revealed 11 alleles (average 2.2 per allele). The dendrogram based on cultivar genotypes revealed three clusters consistent with the cultivars classification. Besides, the results obtained with the five SNPs were compared to those obtained with the SSR markers using bioinformatic analyses and by computing a cophenetic correlation coefficient, indicating the usefulness of the UPGMA method for clustering plant genotypes. Based on principal coordinate analysis using a similarity matrix, the first two coordinates, revealed 54.94 % of the total variance. This work provides a more comprehensive explanation of the diversity available in Tunisia olive cultivars, and an important contribution for olive breeding and olive oil authenticity.

Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases

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Seyyed Javad SEYYEDTABAEI

2017-06-01

Full Text Available Background: Visceral leishmaniasis (VL and cutaneous leishmaniasis (CL are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB compared with indirect immunofluorescence test (IFAT to serodiagnosis of leishmaniasis.Methods: This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. Results: WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups.Conclusion: WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

Proteomic signatures of infertile men with clinical varicocele and their validation studies reveal mitochondrial dysfunction leading to infertility

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Ashok Agarwal

2016-01-01

Full Text Available To study the major differences in the distribution of spermatozoa proteins in infertile men with varicocele by comparative proteomics and validation of their level of expression. The study-specific estimates for each varicocele outcome were combined to identify the proteins involved in varicocele-associated infertility in men irrespective of stage and laterality of their clinical varicocele. Expression levels of 5 key proteins (PKAR1A, AK7, CCT6B, HSPA2, and ODF2 involved in stress response and sperm function including molecular chaperones were validated by Western blotting. Ninety-nine proteins were differentially expressed in the varicocele group. Over 87% of the DEP involved in major energy metabolism and key sperm functions were underexpressed in the varicocele group. Key protein functions affected in the varicocele group were spermatogenesis, sperm motility, and mitochondrial dysfunction, which were further validated by Western blotting, corroborating the proteomics analysis. Varicocele is essentially a state of energy deprivation, hypoxia, and hyperthermia due to impaired blood supply, which is corroborated by down-regulation of lipid metabolism, mitochondrial electron transport chain, and Krebs cycle enzymes. To corroborate the proteomic analysis, expression of the 5 identified proteins of interest was validated by Western blotting. This study contributes toward establishing a biomarker "fingerprint" to assess sperm quality on the basis of molecular parameters.

Altered Brain Activity in Unipolar Depression Revisited: Meta-analyses of Neuroimaging Studies.

Science.gov (United States)

Müller, Veronika I; Cieslik, Edna C; Serbanescu, Ilinca; Laird, Angela R; Fox, Peter T; Eickhoff, Simon B

2017-01-01

major depressive disorder. For meta-analyses with a minimum of 17 experiments available, separate analyses were performed for increases and decreases. In total, 57 studies with 99 individual neuroimaging experiments comprising in total 1058 patients were included; 34 of them tested cognitive and 65 emotional processing. Overall analyses across cognitive processing experiments (P > .29) and across emotional processing experiments (P > .47) revealed no significant results. Similarly, no convergence was found in analyses investigating positive (all P > .15), negative (all P > .76), or memory (all P > .48) processes. Analyses that restricted inclusion of confounds (eg, medication, comorbidity, age) did not change the results. Inconsistencies exist across individual experiments investigating aberrant brain activity in UD and replication problems across previous neuroimaging meta-analyses. For individual experiments, these inconsistencies may relate to use of uncorrected inference procedures, differences in experimental design and contrasts, or heterogeneous clinical populations; meta-analytically, differences may be attributable to varying inclusion and exclusion criteria or rather liberal statistical inference approaches.

Transcriptomics and physiological analyses reveal co-ordinated alteration of metabolic pathways in Jatropha curcas drought tolerance.

Science.gov (United States)

Sapeta, Helena; Lourenço, Tiago; Lorenz, Stefan; Grumaz, Christian; Kirstahler, Philipp; Barros, Pedro M; Costa, Joaquim Miguel; Sohn, Kai; Oliveira, M Margarida

2016-02-01

Jatropha curcas, a multipurpose plant attracting a great deal of attention due to its high oil content and quality for biofuel, is recognized as a drought-tolerant species. However, this drought tolerance is still poorly characterized. This study aims to contribute to uncover the molecular background of this tolerance, using a combined approach of transcriptional profiling and morphophysiological characterization during a period of water-withholding (49 d) followed by rewatering (7 d). Morphophysiological measurements showed that J. curcas plants present different adaptation strategies to withstand moderate and severe drought. Therefore, RNA sequencing was performed for samples collected under moderate and severe stress followed by rewatering, for both roots and leaves. Jatropha curcas transcriptomic analysis revealed shoot- and root-specific adaptations across all investigated conditions, except under severe stress, when the dramatic transcriptomic reorganization at the root and shoot level surpassed organ specificity. These changes in gene expression were clearly shown by the down-regulation of genes involved in growth and water uptake, and up-regulation of genes related to osmotic adjustments and cellular homeostasis. However, organ-specific gene variations were also detected, such as strong up-regulation of abscisic acid synthesis in roots under moderate stress and of chlorophyll metabolism in leaves under severe stress. Functional validation further corroborated the differential expression of genes coding for enzymes involved in chlorophyll metabolism, which correlates with the metabolite content of this pathway. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Molecular cytogenetic and genomic analyses reveal new insights into the origin of the wheat B genome.

Science.gov (United States)

Zhang, Wei; Zhang, Mingyi; Zhu, Xianwen; Cao, Yaping; Sun, Qing; Ma, Guojia; Chao, Shiaoman; Yan, Changhui; Xu, Steven S; Cai, Xiwen

2018-02-01

This work pinpointed the goatgrass chromosomal segment in the wheat B genome using modern cytogenetic and genomic technologies, and provided novel insights into the origin of the wheat B genome. Wheat is a typical allopolyploid with three homoeologous subgenomes (A, B, and D). The donors of the subgenomes A and D had been identified, but not for the subgenome B. The goatgrass Aegilops speltoides (genome SS) has been controversially considered a possible candidate for the donor of the wheat B genome. However, the relationship of the Ae. speltoides S genome with the wheat B genome remains largely obscure. The present study assessed the homology of the B and S genomes using an integrative cytogenetic and genomic approach, and revealed the contribution of Ae. speltoides to the origin of the wheat B genome. We discovered noticeable homology between wheat chromosome 1B and Ae. speltoides chromosome 1S, but not between other chromosomes in the B and S genomes. An Ae. speltoides-originated segment spanning a genomic region of approximately 10.46 Mb was detected on the long arm of wheat chromosome 1B (1BL). The Ae. speltoides-originated segment on 1BL was found to co-evolve with the rest of the B genome. Evidently, Ae. speltoides had been involved in the origin of the wheat B genome, but should not be considered an exclusive donor of this genome. The wheat B genome might have a polyphyletic origin with multiple ancestors involved, including Ae. speltoides. These novel findings will facilitate genome studies in wheat and other polyploids.

Comparative analyses of reproductive structures in harvestmen (opiliones reveal multiple transitions from courtship to precopulatory antagonism.

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Mercedes M Burns

Full Text Available Explaining the rapid, species-specific diversification of reproductive structures and behaviors is a long-standing goal of evolutionary biology, with recent research tending to attribute reproductive phenotypes to the evolutionary mechanisms of female mate choice or intersexual conflict. Progress in understanding these and other possible mechanisms depends, in part, on reconstructing the direction, frequency and relative timing of phenotypic evolution of male and female structures in species-rich clades. Here we examine evolution of reproductive structures in the leiobunine harvestmen or "daddy long-legs" of eastern North America, a monophyletic group that includes species in which males court females using nuptial gifts and other species that are equipped for apparent precopulatory antagonism (i.e., males with long, hardened penes and females with sclerotized pregenital barriers. We used parsimony- and Bayesian likelihood-based analyses to reconstruct character evolution in categorical reproductive traits and found that losses of ancestral gift-bearing penile sacs are strongly associated with gains of female pregenital barriers. In most cases, both events occur on the same internal branch of the phylogeny. These coevolutionary changes occurred at least four times, resulting in clade-specific designs in the penis and pregenital barrier. The discovery of convergent origins and/or enhancements of apparent precopulatory antagonism among closely related species offers an unusual opportunity to investigate how major changes in reproductive morphology have occurred. We propose new hypotheses that attribute these enhancements to changes in ecology or life history that reduce the duration of breeding seasons, an association that is consistent with female choice, sexual conflict, and/or an alternative evolutionary mechanism.

Plant dynamics analyses of fast reactor concept: RAPID-A without any control rod

International Nuclear Information System (INIS)

Kambe, Mitsuru

1996-01-01

Plant dynamics analyses of a fast reactor concept RAPID-A without any control rod have been demonstrated in case of reactor startup and sudden change of the primary flow rate. RAIP-A concept involves Lithium Expansion Module (LEM) for inherent reactivity feedback, Lithium Injection Module (LIM) for inherent ultimate shutdown and Lithium Release Module (LRM) for automated reactor startup. LEM consists of Quick-LEM and Slow-LEM. Slow-LEM provides with moderate reactivity addition as decreasing temperature. Quick-LEM assures quick negative reactivity feedback as increasing temperature. Plant dynamics analyses revealed that reactor power is nearly proportional to the primary flow rate even if the flow rate increases suddenly. Fully automated reactor startup from the subcritical condition has been attempted by inserting reactivity at a constant rate by LRM. Allowable rate of reactivity addition has been obtained in respect to Quick-LEM reactivity worth. (author)

Northern Bobwhite (Colinus virginianus Mitochondrial Population Genomics Reveals Structure, Divergence, and Evidence for Heteroplasmy.

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Yvette A Halley

Full Text Available Herein, we evaluated the concordance of population inferences and conclusions resulting from the analysis of short mitochondrial fragments (i.e., partial or complete D-Loop nucleotide sequences versus complete mitogenome sequences for 53 bobwhites representing six ecoregions across TX and OK (USA. Median joining (MJ haplotype networks demonstrated that analyses performed using small mitochondrial fragments were insufficient for estimating the true (i.e., complete mitogenome haplotype structure, corresponding levels of divergence, and maternal population history of our samples. Notably, discordant demographic inferences were observed when mismatch distributions of partial (i.e., partial D-Loop versus complete mitogenome sequences were compared, with the reduction in mitochondrial genomic information content observed to encourage spurious inferences in our samples. A probabilistic approach to variant prediction for the complete bobwhite mitogenomes revealed 344 segregating sites corresponding to 347 total mutations, including 49 putative nonsynonymous single nucleotide variants (SNVs distributed across 12 protein coding genes. Evidence of gross heteroplasmy was observed for 13 bobwhites, with 10 of the 13 heteroplasmies involving one moderate to high frequency SNV. Haplotype network and phylogenetic analyses for the complete bobwhite mitogenome sequences revealed two divergent maternal lineages (dXY = 0.00731; FST = 0.849; P < 0.05, thereby supporting the potential for two putative subspecies. However, the diverged lineage (n = 103 variants almost exclusively involved bobwhites geographically classified as Colinus virginianus texanus, which is discordant with the expectations of previous geographic subspecies designations. Tests of adaptive evolution for functional divergence (MKT, frequency distribution tests (D, FS and phylogenetic analyses (RAxML provide no evidence for positive selection or hybridization with the sympatric scaled quail

NMR spectroscopic and bioinformatic analyses of the LTBP1 C-terminus reveal a highly dynamic domain organisation.

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Ian B Robertson

Full Text Available Proteins from the LTBP/fibrillin family perform key structural and functional roles in connective tissues. LTBP1 forms the large latent complex with TGFβ and its propeptide LAP, and sequesters the latent growth factor to the extracellular matrix. Bioinformatics studies suggest the main structural features of the LTBP1 C-terminus are conserved through evolution. NMR studies were carried out on three overlapping C-terminal fragments of LTBP1, comprising four domains with characterised homologues, cbEGF14, TB3, EGF3 and cbEGF15, and three regions with no homology to known structures. The NMR data reveal that the four domains adopt canonical folds, but largely lack the interdomain interactions observed with homologous fibrillin domains; the exception is the EGF3-cbEGF15 domain pair which has a well-defined interdomain interface. (15N relaxation studies further demonstrate that the three interdomain regions act as flexible linkers, allowing a wide range of motion between the well-structured domains. This work is consistent with the LTBP1 C-terminus adopting a flexible "knotted rope" structure, which may facilitate cell matrix interactions, and the accessibility to proteases or other factors that could contribute to TGFβ activation.

Kan du blot blive buddreng..

DEFF Research Database (Denmark)

Henriksen, Carsten

2011-01-01

ADHD? Adfærdsvanskelig? Diagnoserne står i kø, når vi i dag skal forklare, hvorfor Emil ikke kan følge med i skolen. Men startskuddet til den videnskabelige opdeling af de børn, der ikke følger flertallets læringskurve, lød allerede for 100 år siden.......ADHD? Adfærdsvanskelig? Diagnoserne står i kø, når vi i dag skal forklare, hvorfor Emil ikke kan følge med i skolen. Men startskuddet til den videnskabelige opdeling af de børn, der ikke følger flertallets læringskurve, lød allerede for 100 år siden....

Phylogenetic analyses and nitrate-reducing activity of fungal cultures isolated from the permanent, oceanic oxygen minimum zone of the Arabian Sea

Digital Repository Service at National Institute of Oceanography (India)

Manohar, C.S; Menezes, L.D.; Ramasamy, K.P.; Meena, R.M.

of the oxygen-depleted environments is less than that of mangrove regions and deep-sea habitats. Phylogenetic analyses of 18S rRNA sequences revealed a few divergent isolates that clustered with environmental sequences previously obtained by others...

Trend analyses of transformer problems in the U.S. nuclear power plants

International Nuclear Information System (INIS)

Shimada, Yoshio

2009-01-01

Up to 2007, the authors have conducted the trend analyses of trouble events related to main generators, emergency diesel generators, breakers and motors, which are more likely to cause problems than other electric equipments in nuclear power plants. The frequency of trouble events in transformers in domestic nuclear power plants at present is approximately one third of the publicly reported cases in the U.S. However, as the situation of maintenance in Japan in the future will become similar to those in the U.S. if the operating period is extended or the maintenance method is to be shifted from preventive maintenance to condition based maintenance, there is a concern that the frequency of transformer events in Japan will increase in Japan, also. Thus, trend analyses were conducted on transformers events which had not been subject to such analyses, from among electrical equipments which are likely to cause problems. The trend analyses were performed on 23 transformer events which had occurred in the U.S. nuclear power plants in five years from 2003 through 2007 among events reported in the Licensee Event Reports (LERs: event reports submitted to NRC by U.S. nuclear power plants) which have been registered in the nuclear information database of the Institute of Nuclear Safety System, Incorporated's (INSS), as well as 8 events registered in the Nuclear Information Archives (NUCIA), which had occurred in domestic nuclear power plants in five from 2003 through 2007. Lessons learned from the trend analyses of the transformer trouble events in the U.S. revealed that for transformers in general, the maintenance management of tap changers is important, while for the main transformers which are most likely to cause problems, it is vital to prevent the deterioration of insulation and insulating oil. (author)

Gradual aridification of the Sahara during the last 11,000 years revealed by plant wax δD analyses of Lake Yoa (Chad)

Science.gov (United States)

Rethemeyer, Janet; Kröpelin, Stefan; Karls, Jens; Thienemann, Matthias; Melles, Martin; Schefuß, Enno

2014-05-01

It is still an ongoing debate whether the transition of the last 'green Sahara' period to today's large desert during the Holocene, the African Humid Period (AHP), was a progressive or an abrupt change in hydrological conditions. Several climate records mainly from East Africa suggest a rapid decline of moisture availability at the end of the AHP including new data from a marine sequence off the Horn of Africa (Tierney & deMenocal, 2013). Other archives including sedimentological, geochemical and palynological data from the central North African Lakes Chad and Lake Yoa point to a gradual rather than an abrupt transition near 5,000 years ago (Amaral et al., 2013; Kröpelin et al., 2008). The discrepancy of the available paleo-hydrological reconstructions underline the importance of proxy parameters directly related to hydrological conditions for accurate assessment of continental rainfall changes. Here, we present the first molecular-isotopic data from Lake Yoa documenting the hydrologic evolution over the entire Holocene. Compound-specific carbon and hydrogen isotope analyses were performed on long-chain n-alkanes. Our data indicate relative high but variable contributions of plant-derived long-chain n-alkanes carrying a distinct leaf-wax signature, i.e., a high Carbon Preference Index (CPI). A trend towards higher CPI values since 7,300 years ago suggests declining soil degradation and vegetation cover under increasingly drier conditions. In parallel, the average-chain-length of the long-chain n-alkanes increases gradually towards the present implying higher relative contributions from grasses. Compound-specific carbon isotope data confirm this finding, indicating a mixed C3/C4 contribution in the early and mid-Holocene changing towards a C4-grass dominated vegetation in the late Holocene. Most importantly, compound-specific hydrogen isotope data reveal a continuous increase from 8,100 years ago towards the present, reflecting a gradual aridification. The large

Towards equitable access to medicines for the rural poor: analyses of insurance claims reveal rural pharmacy initiative triggers price competition in Kyrgyzstan.

Science.gov (United States)

Waning, Brenda; Maddix, Jason; Tripodis, Yorghos; Laing, Richard; Leufkens, Hubert Gm; Gokhale, Manjusha

2009-12-14

A rural pharmacy initiative (RPI) designed to increase access to medicines in rural Kyrgyzstan created a network of 12 pharmacies using a revolving drug fund mechanism in 12 villages where no pharmacies previously existed. The objective of this study was to determine if the establishment of the RPI resulted in the unforeseen benefit of triggering medicine price competition in pre-existing (non-RPI) private pharmacies located in the region. We conducted descriptive and multivariate analyses on medicine insurance claims data from Kyrgyzstan's Mandatory Health Insurance Fund for the Jumgal District of Naryn Province from October 2003 to December 2007. We compared average quarterly medicine prices in competitor pharmacies before and after the introduction of the rural pharmacy initiative in October 2004 to determine the RPI impact on price competition. Descriptive analyses suggest competitors reacted to RPI prices for 21 of 30 (70%) medicines. Competitor medicine prices from the quarter before RPI introduction to the end of the study period decreased for 17 of 30 (57%) medicines, increased for 4 of 30 (13%) medicines, and remained unchanged for 9 of 30 (30%) medicines. Among the 9 competitor medicines with unchanged prices, five initially decreased in price but later reverted back to baseline prices. Multivariate analyses on 19 medicines that met sample size criteria confirm these findings. Fourteen of these 19 (74%) competitor medicines changed significantly in price from the quarter before RPI introduction to the quarter after RPI introduction, with 9 of 19 (47%) decreasing in price and 5 of 19 (26%) increasing in price. The RPI served as a market driver, spurring competition in medicine prices in competitor pharmacies, even when they were located in different villages. Initiatives designed to increase equitable access to medicines in rural regions of developing and transitional countries should consider the potential to leverage medicine price competition as a means

Towards equitable access to medicines for the rural poor: analyses of insurance claims reveal rural pharmacy initiative triggers price competition in Kyrgyzstan

Directory of Open Access Journals (Sweden)

Leufkens Hubert GM

2009-12-01

Full Text Available Abstract Background A rural pharmacy initiative (RPI designed to increase access to medicines in rural Kyrgyzstan created a network of 12 pharmacies using a revolving drug fund mechanism in 12 villages where no pharmacies previously existed. The objective of this study was to determine if the establishment of the RPI resulted in the unforeseen benefit of triggering medicine price competition in pre-existing (non-RPI private pharmacies located in the region. Methods We conducted descriptive and multivariate analyses on medicine insurance claims data from Kyrgyzstan's Mandatory Health Insurance Fund for the Jumgal District of Naryn Province from October 2003 to December 2007. We compared average quarterly medicine prices in competitor pharmacies before and after the introduction of the rural pharmacy initiative in October 2004 to determine the RPI impact on price competition. Results Descriptive analyses suggest competitors reacted to RPI prices for 21 of 30 (70% medicines. Competitor medicine prices from the quarter before RPI introduction to the end of the study period decreased for 17 of 30 (57% medicines, increased for 4 of 30 (13% medicines, and remained unchanged for 9 of 30 (30% medicines. Among the 9 competitor medicines with unchanged prices, five initially decreased in price but later reverted back to baseline prices. Multivariate analyses on 19 medicines that met sample size criteria confirm these findings. Fourteen of these 19 (74% competitor medicines changed significantly in price from the quarter before RPI introduction to the quarter after RPI introduction, with 9 of 19 (47% decreasing in price and 5 of 19 (26% increasing in price. Conclusions The RPI served as a market driver, spurring competition in medicine prices in competitor pharmacies, even when they were located in different villages. Initiatives designed to increase equitable access to medicines in rural regions of developing and transitional countries should consider the

Effect of laser power and specimen temperature on atom probe analyses of magnesium alloys

International Nuclear Information System (INIS)

Oh-ishi, K.; Mendis, C.L.; Ohkubo, T.; Hono, K.

2011-01-01

The influence of laser power, wave length, and specimen temperature on laser assisted atom probe analyses for Mg alloys was investigated. Higher laser power and lower specimen temperature led to improved mass and spatial resolutions. Background noise and mass resolutions were degraded with lower laser power and higher specimen temperature. By adjusting the conditions for laser assisted atom probe analyses, atom probe results with atomic layer resolutions were obtained from all the Mg alloys so far investigated. Laser assisted atom probe investigations revealed detailed chemical information on Guinier-Preston zones in Mg alloys. -- Research highlights: → We study performance of UV laser assisted atom probe analysis for Mg alloys. → There is an optimized range of laser power and specimen temperature. → Optimized UV laser enables atom probe data of Mg alloys with high special resolution.

Phylogenetic Analysis of Staphylococcus aureus CC398 Reveals a Sub-Lineage Epidemiologically Associated with Infections in Horses

DEFF Research Database (Denmark)

Abdelbary, Mohamed M. H.; Wittenberg, Anne; Cuny, Christiane

2014-01-01

-allelic polymorphisms, and phylogenetic analyses revealed that an epidemic sub-clone within CC398 (dubbed 'clade (C)') has spread within and between equine hospitals, where it causes nosocomial infections in horses and colonises the personnel. While clade (C) was strongly associated with S. aureus from horses...

Comparative analyses reveal discrepancies among results of commonly used methods for Anopheles gambiaemolecular form identification

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Pinto João

2011-08-01

Full Text Available Abstract Background Anopheles gambiae M and S molecular forms, the major malaria vectors in the Afro-tropical region, are ongoing a process of ecological diversification and adaptive lineage splitting, which is affecting malaria transmission and vector control strategies in West Africa. These two incipient species are defined on the basis of single nucleotide differences in the IGS and ITS regions of multicopy rDNA located on the X-chromosome. A number of PCR and PCR-RFLP approaches based on form-specific SNPs in the IGS region are used for M and S identification. Moreover, a PCR-method to detect the M-specific insertion of a short interspersed transposable element (SINE200 has recently been introduced as an alternative identification approach. However, a large-scale comparative analysis of four widely used PCR or PCR-RFLP genotyping methods for M and S identification was never carried out to evaluate whether they could be used interchangeably, as commonly assumed. Results The genotyping of more than 400 A. gambiae specimens from nine African countries, and the sequencing of the IGS-amplicon of 115 of them, highlighted discrepancies among results obtained by the different approaches due to different kinds of biases, which may result in an overestimation of MS putative hybrids, as follows: i incorrect match of M and S specific primers used in the allele specific-PCR approach; ii presence of polymorphisms in the recognition sequence of restriction enzymes used in the PCR-RFLP approaches; iii incomplete cleavage during the restriction reactions; iv presence of different copy numbers of M and S-specific IGS-arrays in single individuals in areas of secondary contact between the two forms. Conclusions The results reveal that the PCR and PCR-RFLP approaches most commonly utilized to identify A. gambiae M and S forms are not fully interchangeable as usually assumed, and highlight limits of the actual definition of the two molecular forms, which might

Multiple Spectral Ratio Analyses Reveal Earthquake Source Spectra of Small Earthquakes and Moment Magnitudes of Microearthquakes

Science.gov (United States)

Uchide, T.; Imanishi, K.

2016-12-01

Spectral studies for macroscopic earthquake source parameters are helpful for characterizing earthquake rupture process and hence understanding earthquake source physics and fault properties. Those studies require us mute wave propagation path and site effects in spectra of seismograms to accentuate source effect. We have recently developed the multiple spectral ratio method [Uchide and Imanishi, BSSA, 2016] employing many empirical Green's function (EGF) events to reduce errors from the choice of EGF events. This method helps us estimate source spectra more accurately as well as moment ratios among reference and EGF events, which are useful to constrain the seismic moment of microearthquakes. First, we focus on earthquake source spectra. The source spectra have generally been thought to obey the omega-square model with single corner-frequency. However recent studies imply the existence of another corner frequency for some earthquakes. We analyzed small shallow inland earthquakes (3.5 multiple spectral ratio analyses. For 20000 microearthquakes in Fukushima Hamadori and northern Ibaraki prefecture area, we found that the JMA magnitudes (Mj) based on displacement or velocity amplitude are systematically below Mw. The slope of the Mj-Mw relation is 0.5 for Mj 5. We propose a fitting curve for the obtained relationship as Mw = (1/2)Mj + (1/2)(Mjγ + Mcorγ)1/γ+ c, where Mcor is a corner magnitude, γ determines the sharpness of the corner, and c denotes an offset. We obtained Mcor = 4.1, γ = 5.6, and c = -0.47 to fit the observation. The parameters are useful for characterizing the Mj-Mw relationship. This non-linear relationship affects the b-value of the Gutenberg-Richter law. Quantitative discussions on b-values are affected by the definition of magnitude to use.

GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

DEFF Research Database (Denmark)

Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan

2010-01-01

lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice...... and wild-type control animals revealed two novel components of the GABA(B1) complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA(B) receptors via the GABA(B2) subunit. In transfected hippocampal neurons, potassium...

Phylogenetic and morphologic analyses of a coastal fish reveals a marine biogeographic break of terrestrial origin in the southern Caribbean.

Directory of Open Access Journals (Sweden)

Ricardo Betancur-R

2010-07-01

Full Text Available Marine allopatric speciation involves interplay between intrinsic organismal properties and extrinsic factors. However, the relative contribution of each depends on the taxon under study and its geographic context. Utilizing sea catfishes in the Cathorops mapale species group, this study tests the hypothesis that both reproductive strategies conferring limited dispersal opportunities and an apparent geomorphologic barrier in the Southern Caribbean have promoted speciation in this group from a little studied area of the world.Mitochondrial gene sequences were obtained from representatives of the Cathorops mapale species group across its distributional range from Colombia to Venezuela. Morphometric and meristic analyses were also done to assess morphologic variation. Along a approximately 2000 km transect, two major lineages, Cathorops sp. and C. mapale, were identified by levels of genetic differentiation, phylogenetic reconstructions, and morphological analyses. The lineages are separated by approximately 150 km at the Santa Marta Massif (SMM in Colombia. The northward displacement of the SMM into the Caribbean in the early Pleistocene altered the geomorphology of the continental margin, ultimately disrupting the natural habitat of C. mapale. The estimated approximately 0.86 my divergence of the lineages from a common ancestor coincides with the timing of the SMM displacement at approximately 0.78 my.Results presented here support the hypothesis that organismal properties as well as extrinsic factors lead to diversification of the Cathorops mapale group along the northern coast of South America. While a lack of pelagic larval stages and ecological specialization are forces impacting this process, the identification of the SMM as contributing to allopatric speciation in marine organisms adds to the list of recognized barriers in the Caribbean. Comparative examination of additional Southern Caribbean taxa, particularly those with varying life

Complete genome of a European hepatitis C virus subtype 1g isolate: phylogenetic and genetic analyses.

Science.gov (United States)

Bracho, Maria A; Saludes, Verónica; Martró, Elisa; Bargalló, Ana; González-Candelas, Fernando; Ausina, Vicent

2008-06-05

Hepatitis C virus isolates have been classified into six main genotypes and a variable number of subtypes within each genotype, mainly based on phylogenetic analysis. Analyses of the genetic relationship among genotypes and subtypes are more reliable when complete genome sequences (or at least the full coding region) are used; however, so far 31 of 80 confirmed or proposed subtypes have at least one complete genome available. Of these, 20 correspond to confirmed subtypes of epidemic interest. We present and analyse the first complete genome sequence of a HCV subtype 1g isolate. Phylogenetic and genetic distance analyses reveal that HCV-1g is the most divergent subtype among the HCV-1 confirmed subtypes. Potential genomic recombination events between genotypes or subtype 1 genomes were ruled out. We demonstrate phylogenetic congruence of previously deposited partial sequences of HCV-1g with respect to our sequence. In light of this, we propose changing the current status of its subtype-specific designation from provisional to confirmed.

Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.

Science.gov (United States)

Liu, Xin; Yang, Lihua; Zhou, Xianyao; Zhou, Miaoping; Lu, Yan; Ma, Lingjian; Ma, Hongxiang; Zhang, Zengyan

2013-05-01

The disease take-all, caused by the fungus Gaeumannomyces graminis, is one of the most destructive root diseases of wheat worldwide. Breeding resistant cultivars is an effective way to protect wheat from take-all. However, little progress has been made in improving the disease resistance level in commercial wheat cultivars. MYB transcription factors play important roles in plant responses to environmental stresses. In this study, an R2R3-MYB gene in Thinopyrum intermedium, TiMYB2R-1, was cloned and characterized. The gene sequence includes two exons and an intron. The expression of TiMYB2R-1 was significantly induced following G. graminis infection. An in vitro DNA binding assay proved that TiMYB2R-1 protein could bind to the MYB-binding site cis-element ACI. Subcellular localization assays revealed that TiMYB2R-1 was localized in the nucleus. TiMYB2R-1 transgenic wheat plants were generated, characterized molecularly, and evaluated for take-all resistance. PCR and Southern blot analyses confirmed that TiMYB2R-1 was integrated into the genomes of three independent transgenic wheat lines by distinct patterns and the transgene was heritable. Reverse transcription-PCR and western blot analyses revealed that TiMYB2R-1 was highly expressed in the transgenic wheat lines. Based on disease response assessments for three successive generations, the significantly enhanced resistance to take-all was observed in the three TiMYB2R-1-overexpressing transgenic wheat lines. Furthermore, the transcript levels of at least six wheat defence-related genes were significantly elevated in the TiMYB2R-1 transgenic wheat lines. These results suggest that engineering and overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops.

Consensuses and discrepancies of basin-scale ocean heat content changes in different ocean analyses

Science.gov (United States)

Wang, Gongjie; Cheng, Lijing; Abraham, John; Li, Chongyin

2018-04-01

Inconsistent global/basin ocean heat content (OHC) changes were found in different ocean subsurface temperature analyses, especially in recent studies related to the slowdown in global surface temperature rise. This finding challenges the reliability of the ocean subsurface temperature analyses and motivates a more comprehensive inter-comparison between the analyses. Here we compare the OHC changes in three ocean analyses (Ishii, EN4 and IAP) to investigate the uncertainty in OHC in four major ocean basins from decadal to multi-decadal scales. First, all products show an increase of OHC since 1970 in each ocean basin revealing a robust warming, although the warming rates are not identical. The geographical patterns, the key modes and the vertical structure of OHC changes are consistent among the three datasets, implying that the main OHC variabilities can be robustly represented. However, large discrepancies are found in the percentage of basinal ocean heating related to the global ocean, with the largest differences in the Pacific and Southern Ocean. Meanwhile, we find a large discrepancy of ocean heat storage in different layers, especially within 300-700 m in the Pacific and Southern Oceans. Furthermore, the near surface analysis of Ishii and IAP are consistent with sea surface temperature (SST) products, but EN4 is found to underestimate the long-term trend. Compared with ocean heat storage derived from the atmospheric budget equation, all products show consistent seasonal cycles of OHC in the upper 1500 m especially during 2008 to 2012. Overall, our analyses further the understanding of the observed OHC variations, and we recommend a careful quantification of errors in the ocean analyses.

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

Science.gov (United States)

Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-08-01

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

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Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Taş, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

2015-01-01

The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below −10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

Perspectives for comprehensive biomechanical analyses in Mogul skiing.

Science.gov (United States)

Kurpiers, Nicolas; McAlpine, Paul R; Kersting, Uwe G

2009-01-01

The purpose of this study was to examine the effects of using a force measurement device on riding technique in mogul skiing. A mock-up version of such a device was positioned between ski boot and binding. Data on three-dimensional kinematics and perception were collected for eight subjects skiing down a mogul course. Parameters analysed were knee angle, side and forward lean of the trunk and hip, and the path of the body's centre of mass. A perception questionnaire was used on selective aspects to assess the skiers' perception of the performances. Perception ratings showed no significant detrimental effects. All assessed components showed a trend of improvement from the first to last run, thus suggesting familiarisation was achieved. Kinematic analysis revealed that no significant alterations occurred. In conclusion, it is intended to utilise a functional force plate similar to the one presented by Kiefmann et al. (2006) for future studies in freestyle skiing.

SNP analyses of growth factor genes EGF, TGFβ-1, and HGF reveal haplotypic association of EGF with autism

International Nuclear Information System (INIS)

Toyoda, Takao; Nakamura, Kazuhiko; Yamada, Kazuo; Thanseem, Ismail; Anitha, Ayyappan; Suda, Shiro; Tsujii, Masatsugu; Iwayama, Yoshimi; Hattori, Eiji; Toyota, Tomoko; Miyachi, Taishi; Iwata, Yasuhide; Suzuki, Katsuaki; Matsuzaki, Hideo; Kawai, Masayoshi; Sekine, Yoshimoto; Tsuchiya, Kenji; Sugihara, Gen-ichi; Ouchi, Yasuomi; Sugiyama, Toshiro; Takei, Nori; Yoshikawa, Takeo; Mori, Norio

2007-01-01

Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-β (TGFβ) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGFβ1, and HGF genes with autism, in a trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGFβ1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism

Comprehensive microRNA profiling reveals potential augmentation of the IL1 pathway in rheumatic heart valve disease.

Science.gov (United States)

Lu, Qiyu; Sun, Yi; Duan, Yuyin; Li, Bin; Xia, Jianming; Yu, Songhua; Zhang, Guimin

2018-03-16

Valvular heart disease is a leading cause of cardiovascular mortality, especially in China. More than a half of valvular heart diseases are caused by acute rheumatic fever. microRNA is involved in many physiological and pathological processes. However, the miRNA profile of the rheumatic valvular heart disease is unknown. This research is to discuss microRNAs and their target gene pathways involved in rheumatic heart valve disease. Serum miRNA from one healthy individual and four rheumatic heart disease patients were sequenced. Specific differentially expressed miRNAs were quantified by Q-PCR in 40 patients, with 20 low-to-moderate rheumatic mitral valve stenosis patients and 20 severe mitral valve stenosis patients. The target relationship between certain miRNA and predicted target genes were analysis by Luciferase reporter assay. The IL-1β and IL1R1 expression levels were analyzed by immunohistochemistry and western blot in the mitral valve from surgery of mitral valve replacement. The results showed that 13 and 91 miRNAs were commonly upregulated or downregulated in all four patients. Nine miRNAs, 1 upregulated and 8 downregulated, that had a similar fold change in all 4 patients were selected for quantitative PCR verification. The results showed similar results from miRNA sequencing. Within these 9 tested miRNAs, hsa-miR-205-3p and hsa-miR-3909 showed a low degree of dispersion between the members of each group. Hsa miR-205-3p and hsa-miR-3909 were predicted to target the 3'UTR of IL-1β and IL1R1 respectively. This was verified by luciferase reporter assays. Immunohistochemistry and Western blot results showed that the mitral valve from rheumatic valve heart disease showed higher levels of IL- 1β and IL1R1 expression compared with congenital heart valve disease. This suggested a difference between rheumatic heart valve disease and other types of heart valve diseases, with more inflammatory responses in the former. In the present study, by next generation

Multilocus sequence data reveal dozens of putative cryptic species in a radiation of endemic Californian mygalomorph spiders (Araneae, Mygalomorphae, Nemesiidae).

Science.gov (United States)

Leavitt, Dean H; Starrett, James; Westphal, Michael F; Hedin, Marshal

2015-10-01

We use mitochondrial and multi-locus nuclear DNA sequence data to infer both species boundaries and species relationships within California nemesiid spiders. Higher-level phylogenetic data show that the California radiation is monophyletic and distantly related to European members of the genus Brachythele. As such, we consider all California nemesiid taxa to belong to the genus Calisoga Chamberlin, 1937. Rather than find support for one or two taxa as previously hypothesized, genetic data reveal Calisoga to be a species-rich radiation of spiders, including perhaps dozens of species. This conclusion is supported by multiple mitochondrial barcoding analyses, and also independent analyses of nuclear data that reveal general genealogical congruence. We discovered three instances of sympatry, and genetic data indicate reproductive isolation when in sympatry. An examination of female reproductive morphology does not reveal species-specific characters, and observed male morphological differences for a subset of putative species are subtle. Our coalescent species tree analysis of putative species lays the groundwork for future research on the taxonomy and biogeographic history of this remarkable endemic radiation. Copyright © 2015 Elsevier Inc. All rights reserved.

Genealogical analyses in open populations: the case of three Arab-derived Spanish horse breeds.

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Cervantes, I; Gutiérrez, J P; Molina, A; Goyache, F; Valera, M

2009-10-01

This research assesses the genetic composition of three Arab-derived Spanish horse breeds as an example to highlight the major shortcomings related to genealogical analyses in open populations and to propose approaches useful to deal with this task. The studbooks of three Spanish Arab (SA)-derived horse breeds, Spanish Anglo-Arab (dAA), Hispano-Arab (dHA) and Spanish Sport Horse (dSSH) and those of their parental breeds SA, Spanish Purebred (SPB) and Thoroughbred (TB), totalling 211 754 individuals, were available. The genealogies of the dAA, dHA and dSSH were analysed not only using the corresponding studbook (breed exclusive dataset) but also including the genealogies of the founders from parental breeds (completed dataset). Coancestry analyses revealed that the present SA-derived populations share more genes with the Arab than with the other parental breeds. Effective population size was computed by accounting for migration rates to obtain an equivalent closed-population effective size ((eq)N(e)) of 39.2 for the dAA, 56.3 for dHA and 114.1 for dSSH. The essayed methodologies were useful for characterising populations involving migration. The consequences of the management of the analysed breeds are discussed. The results emphasize the need to include the complete genealogies of the individuals to attain reliable genealogical parameters.

Genomic and Phenotypic Analyses Reveal the Emergence of an Atypical Salmonella enterica Serovar Senftenberg Variant in China

KAUST Repository

Abd El Ghany, Moataz

2016-05-25

Human infections with Salmonella enterica subspecies enterica serovar Senftenberg are often associated with exposure to poultry flocks, farm environments, or contaminated food. The recent emergence of multidrug-resistant isolates has raised public health concerns. In this study, comparative genomics and phenotypic analysis were used to characterize 14 Salmonella Senftenberg clinical isolates recovered from multiple outbreaks in Shenzhen and Shanghai, China, between 2002 and 2011. Single-nucleotide polymorphism analyses identified two phylogenetically distinct clades of S. Senftenberg, designated SC1 and SC2, harboring variations in Salmonella pathogenicity island 1 (SPI-1) and SPI-2 and exhibiting distinct biochemical and phenotypic signatures. Although the two variants shared the same serotype, the SC2 isolates of sequence type 14 (ST14) harbored intact SPI-1 and -2 and hence were characterized by possessing efficient invasion capabilities. In contrast, the SC1 isolates had structural deletion patterns in both SPI-1 and -2 that correlated with an impaired capacity to invade cultured human cells and also the year of their isolation. These atypical SC1 isolates also lacked the capacity to produce hydrogen sulfide. These findings highlight the emergence of atypical Salmonella Senftenberg variants in China and provide genetic validation that variants lacking SPI-1 and regions of SPI-2, which leads to impaired invasion capacity, can still cause clinical disease. These data have identified an emerging public health concern and highlight the need to strengthen surveillance to detect the prevalence and transmission of nontyphoidal Salmonella species.

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

Science.gov (United States)

Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

2012-01-01

Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

Identification and expression analyses of two genes encoding putative low-affinity nitrate transporters from Nicotiana plumbaginifolia.

Science.gov (United States)

Fraisier, V; Dorbe, M F; Daniel-Vedele, F

2001-01-01

Higher plants have both high- and low-affinity nitrate uptake systems (HATS and LATS respectively). Here we report the isolation and characterization of two genes, NpNRT1.1 and NpNRT1.2, from Nicotiana plumbaginifolia whose structural features suggest that they both belong to the NRT1 gene family, which is involved in the LATS. Amino acid sequence alignment showed that the N. plumbaginifolia proteins have greater similarity to their corresponding tomato homologues than to each other. Genomic Southern blot analysis indicates that there are probably more than two members of this family in N. plumbaginifolia. Northern blot analysis shows that NpNRT1.2 expression is restricted strictly to roots, whereas NpNRT1.1, in addition to roots, is expressed at a basal level in all other plant organs. Likewise, differential expression in response to external treatments with various N sources was observed for these two genes: NpNRT1.1 can be considered as a constitutively expressed gene whereas NpNRT1.2 expression is dependent strictly on high nitrate concentrations. Finally, over-expression of a gene involved in the HATS does not lead to any modification of LATS gene expression.

Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2.

Science.gov (United States)

Witting, N; Duno, M; Vissing, J

2013-01-01

Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest that this unusual phenotype is caused by translation re-initiation downstream from the mutation site. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of Causes and Treatments for Chronic Pain in a Model of Gulf War Illness

Science.gov (United States)

2017-10-01

saline delivered. Euthanasia and necropsy at days 5 and 20 post acidic/normal saline, and tissue analysis performed ( ELISA , Western Blot, LC-MS...performed ( ELISA , Western Blot, LC-MS). Milestone 3: the association between GWI-induced musculoskeletal pain and neuroinflammation, as well as the...thresholds. Serum samples analysed by ELISA . Major Task 7: Acidic/normal saline administered 180 days after final DFP injection; pharmacological reversal

Comparative Genomics Reveals High Genomic Diversity in the Genus Photobacterium.

Science.gov (United States)

Machado, Henrique; Gram, Lone

2017-01-01

Vibrionaceae is a large marine bacterial family, which can constitute up to 50% of the prokaryotic population in marine waters. Photobacterium is the second largest genus in the family and we used comparative genomics on 35 strains representing 16 of the 28 species described so far, to understand the genomic diversity present in the Photobacterium genus. Such understanding is important for ecophysiology studies of the genus. We used whole genome sequences to evaluate phylogenetic relationships using several analyses (16S rRNA, MLSA, fur , amino-acid usage, ANI), which allowed us to identify two misidentified strains. Genome analyses also revealed occurrence of higher and lower GC content clades, correlating with phylogenetic clusters. Pan- and core-genome analysis revealed the conservation of 25% of the genome throughout the genus, with a large and open pan-genome. The major source of genomic diversity could be traced to the smaller chromosome and plasmids. Several of the physiological traits studied in the genus did not correlate with phylogenetic data. Since horizontal gene transfer (HGT) is often suggested as a source of genetic diversity and a potential driver of genomic evolution in bacterial species, we looked into evidence of such in Photobacterium genomes. Genomic islands were the source of genomic differences between strains of the same species. Also, we found transposase genes and CRISPR arrays that suggest multiple encounters with foreign DNA. Presence of genomic exchange traits was widespread and abundant in the genus, suggesting a role in genomic evolution. The high genetic variability and indications of genetic exchange make it difficult to elucidate genome evolutionary paths and raise the awareness of the roles of foreign DNA in the genomic evolution of environmental organisms.

The impact of parent-child interaction on brain structures: cross-sectional and longitudinal analyses.

Science.gov (United States)

Takeuchi, Hikaru; Taki, Yasuyuki; Hashizume, Hiroshi; Asano, Kohei; Asano, Michiko; Sassa, Yuko; Yokota, Susumu; Kotozaki, Yuka; Nouchi, Rui; Kawashima, Ryuta

2015-02-04

There is a vast amount of evidence from psychological studies that the amount of parent-child interaction affects the development of children's verbal skills and knowledge. However, despite the vast amount of literature, brain structural development associated with the amount of parent-child interaction has never been investigated. In the present human study, we used voxel-based morphometry to measure regional gray matter density (rGMD) and examined cross-sectional correlations between the amount of time spent with parents and rGMD among 127 boys and 135 girls. We also assessed correlations between the amount of time spent with parents and longitudinal changes that occurred a few years later among 106 boys and 102 girls. After correcting for confounding factors, we found negative effects of spending time with parents on rGMD in areas in the bilateral superior temporal gyrus (STG) via cross-sectional analyses as well as in the contingent areas of the right STG. We also confirmed positive effects of spending time with parents on the Verbal Comprehension score in cross-sectional and longitudinal analyses. rGMD in partly overlapping or contingent areas of the right STG was negatively correlated with age and the Verbal Comprehension score in cross-sectional analyses. Subsequent analyses revealed verbal parent-child interactions have similar effects on Verbal Comprehension scores and rGMD in the right STG in both cross-sectional and longitudinal analyses. These findings indicate that parent-child interactions affect the right STG, which may be associated with verbal skills. Copyright © 2015 the authors 0270-6474/15/352233-13$15.00/0.

Statistical reliability analyses of two wood plastic composite extrusion processes

International Nuclear Information System (INIS)

Crookston, Kevin A.; Mark Young, Timothy; Harper, David; Guess, Frank M.

2011-01-01

Estimates of the reliability of wood plastic composites (WPC) are explored for two industrial extrusion lines. The goal of the paper is to use parametric and non-parametric analyses to examine potential differences in the WPC metrics of reliability for the two extrusion lines that may be helpful for use by the practitioner. A parametric analysis of the extrusion lines reveals some similarities and disparities in the best models; however, a non-parametric analysis reveals unique and insightful differences between Kaplan-Meier survival curves for the modulus of elasticity (MOE) and modulus of rupture (MOR) of the WPC industrial data. The distinctive non-parametric comparisons indicate the source of the differences in strength between the 10.2% and 48.0% fractiles [3,183-3,517 MPa] for MOE and for MOR between the 2.0% and 95.1% fractiles [18.9-25.7 MPa]. Distribution fitting as related to selection of the proper statistical methods is discussed with relevance to estimating the reliability of WPC. The ability to detect statistical differences in the product reliability of WPC between extrusion processes may benefit WPC producers in improving product reliability and safety of this widely used house-decking product. The approach can be applied to many other safety and complex system lifetime comparisons.

The Influence of Orthographic Neighborhood Density and Word Frequency on Visual Word Recognition: Insights from RT Distributional Analyses

Directory of Open Access Journals (Sweden)

Stephen Wee Hun eLim

2016-03-01

Full Text Available The effects of orthographic neighborhood density and word frequency in visual word recognition were investigated using distributional analyses of response latencies in visual lexical decision. Main effects of density and frequency were observed in mean latencies. Distributional analyses, in addition, revealed a density x frequency interaction: for low-frequency words, density effects were mediated predominantly by distributional shifting whereas for high-frequency words, density effects were absent except at the slower RTs, implicating distributional skewing. The present findings suggest that density effects in low-frequency words reflect processes involved in early lexical access, while the effects observed in high-frequency words reflect late postlexical checking processes.

Cell apoptosis of taste buds in circumvallate papillae in diabetic rats.

Science.gov (United States)

Cheng, B; Pan, S; Liu, X; Zhang, S; Sun, X

2011-09-01

Diabetes mellitus may result in taste disturbance. The present study has revealed that cell apoptosis of taste buds in circumvallate papillae may contribute to the taste disturbance in a rat model of type2 diabetes. Type2 diabetes was induced in Wistar rats by feeding them with a high-fat diet (30% fat), and a single intraperitoneal injection of streptozotocin (30 mg/kg). The increased cell apoptosis of taste buds in circumvallate papilla sections was detected by TUNEL staining in diabetic rats, and the ultrastructure was further examined by transmission electronic microscopy. Immunohistochemical and Western blot analyses revealed the downregulation of Bcl-2, upregulation of Bax, and increased activation of caspase-9 and -3, in diabetic rats, indicating that the apoptosis of taste bud cells may be mediated via the intrinsic mitochondrial pathway in diabetics. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.