Intragenus (Campylomormyrus) and intergenus hybrids in mormyrid fish: Physiological and histological investigations of the electric organ ontogeny.

Science.gov (United States)

Kirschbaum, Frank; Nguyen, Linh; Baumgartner, Stephanie; Chi, Hiu Wan Linda; Wolfart, Rene; Elarbani, Khouloud; Eppenstein, Hari; Korniienko, Yevheniia; Guido-Böhm, Lilian; Mamonekene, Victor; Vater, Marianne; Tiedemann, Ralph

2016-10-01

African weakly electric mormyrid fish show a high diversity of their electric organ discharge (EOD) both across and within genera. Thanks to a recently developed technique of artificial reproduction in mormyrid fish, we were able to perform hybridizations between different genera and within one genus (Campylomormyrus). The hybrids of intergenus hybridizations exhibited different degrees of reduced survival related to the phylogenetic distance of the parent species: hybrids of the crosses between C. rhynchophorus and its sister genus Gnathonemus survived and developed normally. Hybrids between C. rhynchophorus and a Mormyrus species (a more basal clade compared to Campylomormyrus s) survived up to 42days and developed many malformations, e.g., at the level of the unpaired fins. Hybrids between C. numenius and Hippopotamyrus pictus (a derived clade, only distantly related to Campylomormyrus) only survived for two days during embryological development. Eight different hybrid combinations among five Campylomormyrus species (C. tamandua, C. compressirostris, C. tshokwe, C. rhynchophorus, C. numenius) were performed. The aim of the hybridizations was to combine species with (1) either caudal or rostral position of the main stalk innervating the electrocytes in the electric organ and (2) short, median or long duration of their EOD. The hybrids, though they are still juveniles, show very interesting features concerning electrocyte geometry as well as EOD form and duration: the caudal position of the stalk is prevailing over the rostral position, and the penetration of the stalk is dominant over the non-penetrating feature (in the Campylomormyrus hybrids); in the hybrid between C. rhynchophorus and Gnathonemus petersii it is the opposite. When crossing species with long and short EODs, it is always the long duration EOD that is expressed in the hybrids. The F1-Hybrids of the cross C. tamandua×C. compressirostris are fertile: viable F2-fish could be obtained with artificial

Metaphase FISH on a Chip: Miniaturized Microfluidic Device for Fluorescence in situ Hybridization

Directory of Open Access Journals (Sweden)

Niels Tommerup

2010-11-01

Full Text Available Fluorescence in situ Hybridization (FISH is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA-FISH, the process continues to be a manual, labour intensive, expensive and time consuming technique, often taking over 3–5 days, even in dedicated labs. We have developed a novel microFISH device to perform metaphase FISH on a chip which overcomes many shortcomings of the current laboratory protocols. This work also introduces a novel splashing device for preparing metaphase spreads on a microscope glass slide, followed by a rapid adhesive tape-based bonding protocol leading to rapid fabrication of the microFISH device. The microFISH device allows for an optimized metaphase FISH protocol on a chip with over a 20-fold reduction in the reagent volume. This is the first demonstration of metaphase FISH on a microfluidic device and offers a possibility of automation and significant cost reduction of many routine diagnostic tests of genetic anomalies.

Does interspecies hybridization affect the host specificity of parasites in cyprinid fish?

Czech Academy of Sciences Publication Activity Database

Šimková, A.; Dávidová, M.; Papoušek, Ivo; Vetešník, Lukáš

2013-01-01

Roč. 6, č. 95 (2013), s. 95 ISSN 1756-3305 R&D Projects: GA ČR GAP505/12/0375 Institutional support: RVO:68081766 Keywords : Cyprinid fish * Interspecies hybridization * Metazoan parasites * Monogenea * Host specificity Subject RIV: EG - Zoology Impact factor: 3.251, year: 2013

Single DNA denaturation and bubble dynamics

DEFF Research Database (Denmark)

Metzler, Ralf; Ambjörnsson, Tobias; Hanke, Andreas

2009-01-01

While the Watson-Crick double-strand is the thermodynamically stable state of DNA in a wide range of temperature and salt conditions, even at physiological conditions local denaturation bubbles may open up spontaneously due to thermal activation. By raising the ambient temperature, titration......, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA occurs. Based on the Poland-Scheraga model we investigate both the equilibrium transition of DNA denaturation and the dynamics of the denaturation bubbles with respect to recent single DNA chain experiments...... for situations below, at, and above the denaturation transition. We also propose a new single molecule setup based on DNA constructs with two bubble zones to measure the bubble coalescence and extract the physical parameters relevant to DNA breathing. Finally we consider the interplay between denaturation...

Fusion-Fission hybrid reactors and nonproliferation

International Nuclear Information System (INIS)

Greenspan, E.

1984-09-01

New options for the development of the nuclear energy economy which might become available by a successful development of fusion-breeders or fusion-fission hybrid power reactors, identified and their nonproliferative attributes are discussed. The more promising proliferation-resistance ettributes identified include: (1) Justification for a significant delay in the initiation of fuel processing, (2) Denaturing the plutonium with 238 Pu before its use in power reactors of any kind, and (3) Making practical the development of denatured uranium fuel cycles and, in particular, denaturing the uranium with 232 U. Fuel resource utilization, time-table and economic considerations associated with the use of fusion-breeders are also discussed. It is concluded that hybrid reactors may enable developing a nuclear energy economy which is more proliferation resistant than possible otherwise, whileat the same time, assuring high utilization of t he uranium and thorium resources in an economically acceptable way. (author)

Single DNA denaturation and bubble dynamics

International Nuclear Information System (INIS)

Metzler, Ralf; Ambjoernsson, Tobias; Hanke, Andreas; Fogedby, Hans C

2009-01-01

While the Watson-Crick double-strand is the thermodynamically stable state of DNA in a wide range of temperature and salt conditions, even at physiological conditions local denaturation bubbles may open up spontaneously due to thermal activation. By raising the ambient temperature, titration, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA occurs. Based on the Poland-Scheraga model we investigate both the equilibrium transition of DNA denaturation and the dynamics of the denaturation bubbles with respect to recent single DNA chain experiments for situations below, at, and above the denaturation transition. We also propose a new single molecule setup based on DNA constructs with two bubble zones to measure the bubble coalescence and extract the physical parameters relevant to DNA breathing. Finally we consider the interplay between denaturation bubbles and selectively single-stranded DNA binding proteins.

Design of Hybrid Solar and Wind Energy Harvester for Fishing Boat

Science.gov (United States)

Banjarnahor, D. A.; Hanifan, M.; Budi, E. M.

2017-07-01

In southern beach of West Java, Indonesia, there are many villagers live as fishermen. They use small boats for fishing, in one to three days. Therefore, they need a fish preservation system. Fortunately, the area has high potential of solar and wind energy. This paper presents the design of a hybrid solar and wind energy harvester to power a refrigerator in the fishing boat. The refrigerator should keep the fish in 2 - 4 °C. The energy needed is 720 Wh daily. In the area, the daily average wind velocity is 4.27 m/s and the sun irradiation is 672 W/m2. The design combined two 100W solar panels and a 300W wind turbine. The testing showed that the solar panels can harvest 815 - 817 Wh of energy, while the wind turbine can harvest 43 - 62 Wh of energy daily. Therefore, the system can fulfil the energy requirement in fishing boat, although the solar panels were more dominant. To install the wind turbine on the fishing-boat, a computational design had been conducted. The boat hydrostatic dimension was measured to determine its stability condition. To reach a stable equilibrium condition, the wind turbine should be installed no more than 1.7 m of height.

mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization.

Science.gov (United States)

Yilmaz, L Safak; Parnerkar, Shreyas; Noguera, Daniel R

2011-02-01

Mathematical models of RNA-targeted fluorescence in situ hybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc.edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design.

Application of Fluorescence In Situ Hybridization (FISH) Technique for the Detection of Genetic Aberration in Medical Science

Science.gov (United States)

Ratan, Zubair Ahmed; Zaman, Sojib Bin; Haidere, Mohammad Faisal; Runa, Nusrat Jahan; Akter, Nasrin

2017-01-01

Fluorescence in situ hybridization (FISH) is a macromolecule recognition technique, which is considered as a new advent in the field of cytology. Initially, it was developed as a physical mapping tool to delineate genes within chromosomes. The accuracy and versatility of FISH were subsequently capitalized upon in biological and medical research. This visually appealing technique provides an intermediate degree of resolution between DNA analysis and chromosomal investigations. FISH consists of a hybridizing DNA probe, which can be labeled directly or indirectly. In the case of direct labeling, fluorescent nucleotides are used, while indirect labeling is incorporated with reporter molecules that are subsequently detected by fluorescent antibodies or other affinity molecules. FISH is applied to detect genetic abnormalities that include different characteristic gene fusions or the presence of an abnormal number of chromosomes in a cell or loss of a chromosomal region or a whole chromosome. It is also applied in different research applications, such as gene mapping or the identification of novel oncogenes. This article reviews the concept of FISH, its application, and its advantages in medical science.  PMID:28690958

Influence of environment and mitochondrial heritage on the ecological characteristics of fish in a hybrid zone.

Directory of Open Access Journals (Sweden)

Nicolas Stolzenberg

2009-06-01

Full Text Available Ecological characteristics (growth, morphology, reproduction arise from the interaction between environmental factors and genetics. Genetic analysis of individuals' life history traits might be used to improve our understanding of mechanisms that form and maintain a hybrid zone.A fish hybrid zone was used to characterize the process of natural selection. Data were collected during two reproductive periods (2001 and 2002 and 1117 individuals (nase, Chondrostama nasus nasus, sofie C. toxostoma toxostoma and hybrids were sampled. Reproductive dates of the two parental species overlapped at sympatric sites. The nase had an earlier reproductive period than the sofie; males had longer reproductive periods for both species. Hybridisation between female nase and male sofie was the most likely. Hybrids had a reproductive period similar to the inherited parental mitochondrial type. Growth and reproductive information from different environments has been synthesised following a bayesian approach of the von Bertalanffy model. Hybrid life history traits appear to link with maternal heritage. Hybrid size from the age of two and size at first maturity appeared to be closer to the size of the maternal origin species (nase or sofie. Median growth rates for hybrids were similar and intermediate between those of the parental species. We observed variable life history traits for hybrids and pure forms in the different parts of the hybrid zone. Geometrical analysis of the hybrid fish shape gave evidence of two main morphologies with a link to maternal heritage.Selective mating seemed to be the underlying process which, with mitochondrial heritage, could explain the evolution of the studied hybrid zone. More generally, we showed the importance of studies on hybrid zones and specifically the study of individuals' ecological characteristics, to improve our understanding of speciation.

Influence of environment and mitochondrial heritage on the ecological characteristics of fish in a hybrid zone.

Science.gov (United States)

Stolzenberg, Nicolas; Nguyen The, Bénédicte; Salducci, Marie Dominique; Cavalli, Laurent

2009-06-18

Ecological characteristics (growth, morphology, reproduction) arise from the interaction between environmental factors and genetics. Genetic analysis of individuals' life history traits might be used to improve our understanding of mechanisms that form and maintain a hybrid zone. A fish hybrid zone was used to characterize the process of natural selection. Data were collected during two reproductive periods (2001 and 2002) and 1117 individuals (nase, Chondrostama nasus nasus, sofie C. toxostoma toxostoma and hybrids) were sampled. Reproductive dates of the two parental species overlapped at sympatric sites. The nase had an earlier reproductive period than the sofie; males had longer reproductive periods for both species. Hybridisation between female nase and male sofie was the most likely. Hybrids had a reproductive period similar to the inherited parental mitochondrial type. Growth and reproductive information from different environments has been synthesised following a bayesian approach of the von Bertalanffy model. Hybrid life history traits appear to link with maternal heritage. Hybrid size from the age of two and size at first maturity appeared to be closer to the size of the maternal origin species (nase or sofie). Median growth rates for hybrids were similar and intermediate between those of the parental species. We observed variable life history traits for hybrids and pure forms in the different parts of the hybrid zone. Geometrical analysis of the hybrid fish shape gave evidence of two main morphologies with a link to maternal heritage. Selective mating seemed to be the underlying process which, with mitochondrial heritage, could explain the evolution of the studied hybrid zone. More generally, we showed the importance of studies on hybrid zones and specifically the study of individuals' ecological characteristics, to improve our understanding of speciation.

Application of Fluorescence In Situ Hybridization (FISH) Technique for the Detection of Genetic Aberration in Medical Science

OpenAIRE

Ratan, Zubair Ahmed; Zaman, Sojib Bin; Mehta, Varshil; Haidere, Mohammad Faisal; Runa, Nusrat Jahan; Akter, Nasrin

2017-01-01

Fluorescence in situ hybridization (FISH) is a macromolecule recognition technique, which is considered as a new advent in the field of cytology.?Initially, it was developed as a physical mapping tool to delineate genes within chromosomes. The accuracy and versatility of FISH were subsequently capitalized upon in biological and medical research. This visually appealing technique provides an intermediate degree of resolution between DNA analysis and chromosomal investigations. FISH consists of...

A novel, simple and rapid nondenaturing FISH (ND-FISH) technique for the detection of plant telomeres. Potential used and possible target structures detected.

Science.gov (United States)

Cuadrado, Angeles; Golczyk, Hieronim; Jouve, Nicolás

2009-01-01

We report a new technique-nondenaturing FISH (ND-FISH)-for the rapid detection of plant telomeres without the need for prior denaturation of the chromosomes. In its development, two modified, synthetic oligonucleotides, 21 nt in length, fluorescently labelled at their 5' and 3' ends and complementary to either the cytidine-rich (C(3)TA(3)) or guanosine-rich (T(3)AG(3)) telomeric DNA strands, were used as probes. The high binding affinity of these probes and the short hybridization time required allows the visualization of plant telomeres in less than an hour. In tests, both probes gave strong signals visualized as double spots at both chromosome ends; this was true of both the mitotic and meiotic chromosomes of barley, wheat, rye, maize, Brachypodium distachyon and Rhoeo spathacea. They were also able to detect telomere motifs at certain intercalary sites in the chromosomes of R. spathacea. To investigate the nature of the target structures detected, the chromosomes were treated with RNase A and single strand-specific nuclease S1 before ND-FISH experiments. Signal formation was resistant to standard enzymatic treatment, but sensitive when much higher enzyme concentrations were used. The results are discussed in relation to current knowledge of telomere structure.

Fluorescence In Situ Hybridization (FISH Signal Analysis Using Automated Generated Projection Images

Directory of Open Access Journals (Sweden)

Xingwei Wang

2012-01-01

Full Text Available Fluorescence in situ hybridization (FISH tests provide promising molecular imaging biomarkers to more accurately and reliably detect and diagnose cancers and genetic disorders. Since current manual FISH signal analysis is low-efficient and inconsistent, which limits its clinical utility, developing automated FISH image scanning systems and computer-aided detection (CAD schemes has been attracting research interests. To acquire high-resolution FISH images in a multi-spectral scanning mode, a huge amount of image data with the stack of the multiple three-dimensional (3-D image slices is generated from a single specimen. Automated preprocessing these scanned images to eliminate the non-useful and redundant data is important to make the automated FISH tests acceptable in clinical applications. In this study, a dual-detector fluorescence image scanning system was applied to scan four specimen slides with FISH-probed chromosome X. A CAD scheme was developed to detect analyzable interphase cells and map the multiple imaging slices recorded FISH-probed signals into the 2-D projection images. CAD scheme was then applied to each projection image to detect analyzable interphase cells using an adaptive multiple-threshold algorithm, identify FISH-probed signals using a top-hat transform, and compute the ratios between the normal and abnormal cells. To assess CAD performance, the FISH-probed signals were also independently visually detected by an observer. The Kappa coefficients for agreement between CAD and observer ranged from 0.69 to 1.0 in detecting/counting FISH signal spots in four testing samples. The study demonstrated the feasibility of automated FISH signal analysis that applying a CAD scheme to the automated generated 2-D projection images.

27 CFR 19.456 - Adding denaturants.

Science.gov (United States)

2010-04-01

... proprietor shall submit a flow diagram of the intended process or method of adding denaturants. (Sec. 201... OF THE TREASURY LIQUORS DISTILLED SPIRITS PLANTS Denaturing Operations and Manufacture of Articles...

Automated processing of fluorescence in-situ hybridization slides for HER2 testing in breast and gastro-esophageal carcinomas.

Science.gov (United States)

Tafe, Laura J; Allen, Samantha F; Steinmetz, Heather B; Dokus, Betty A; Cook, Leanne J; Marotti, Jonathan D; Tsongalis, Gregory J

2014-08-01

HER2 fluorescence in-situ hybridization (FISH) is used in breast and gastro-esophageal carcinoma for determining HER2 gene amplification and patients' eligibility for HER2 targeted therapeutics. Traditional manual processing of the FISH slides is labor intensive because of multiple steps that require hands on manipulation of the slides and specifically timed intervals between steps. This highly manual processing also introduces inter-run and inter-operator variability that may affect the quality of the FISH result. Therefore, we sought to incorporate an automated processing instrument into our FISH workflow. Twenty-six cases including breast (20) and gastro-esophageal (6) cancer comprising 23 biopsies and three excision specimens were tested for HER2 FISH (Pathvysion, Abbott) using the Thermobrite Elite (TBE) system (Leica). Up to 12 slides can be run simultaneously. All cases were previously tested by the Pathvysion HER2 FISH assay with manual preparation. Twenty cells were counted by two observers for each case; five cases were tested on three separate runs by different operators to evaluate the precision and inter-operator variability. There was 100% concordance in the scoring between the manual and TBE methods as well as among the five cases that were tested on three runs. Only one case failed due to poor probe hybridization. In total, seven cases were positive for HER2 amplification (HER2:CEP17 ratio >2.2) and the remaining 19 were negative (HER2:CEP17 ratio <1.8) utilizing the 2007 ASCO/CAP scoring criteria. Due to the automated denaturation and hybridization, for each run, there was a reduction in labor of 3.5h which could then be dedicated to other lab functions. The TBE is a walk away pre- and post-hybridization system that automates FISH slide processing, improves work flow and consistency and saves approximately 3.5h of technologist time. The instrument has a small footprint thus occupying minimal counter space. TBE processed slides performed

"Cooperative collapse" of the denatured state revealed through Clausius-Clapeyron analysis of protein denaturation phase diagrams.

Science.gov (United States)

Tischer, Alexander; Machha, Venkata R; Rösgen, Jörg; Auton, Matthew

2018-02-19

Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how ΔH and the urea m-value interconvert through the slope of c m versus T, (∂cm/∂T)=ΔH/(mT). This relationship permits the calculation of ΔH at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from ΔH obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of ΔH and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall

Hybrid striped bass feeds based on fish oil, beef tallow, and eicosapentaenoic acid/docosahexaenoic acid supplements: Insight regarding fish oil sparing and demand for -3 long-chain polyunsaturated fatty acids.

Science.gov (United States)

Bowzer, J; Jackson, C; Trushenski, J

2016-03-01

Previous research suggests that saturated (SFA) and monounsaturated fatty acid (MUFA) rich lipids, including beef tallow, can make utilization or diet-to-tissue transfer of long-chain polyunsaturated fatty acids (LC-PUFA) more efficient. We hypothesized that using beef tallow as an alternative to fish oil may effectively reduce the LC-PUFA demand of hybrid striped bass × and allow for greater fish oil sparing. Accordingly, we evaluated growth performance and tissue fatty acid profiles of juvenile fish (23.7 ± 0.3 g) fed diets containing menhaden fish oil (considered an ideal source of LC-PUFA for this taxon), beef tallow (BEEF ONLY), or beef tallow amended with purified sources of eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) to achieve levels corresponding to 50 or 100% of those observed in the FISH ONLY feed. Diets were randomly assigned to quadruplicate tanks of fish ( = 4; 10 fish/tank), and fish were fed assigned diets to apparent satiation once daily for 10 wk. Survival (98-100%) was equivalent among treatments, but weight gain (117-180%), specific growth rate (1.1-1.5% BW/d), feed intake (1.4-1.8% BW/d), thermal growth coefficient (0.50-0.70), and feed conversion ratio (FCR; 1.1-1.4, DM basis) varied. Except for FCR, no differences were observed between the FISH ONLY and BEEF ONLY treatments, but performance was generally numerically superior among fish fed the diets containing beef tallow supplemented with DHA at the 100% or both EPA and DHA at the 50% or 100% level. Tissue fatty acid composition was significantly distorted in favor among fish fed the beef tallow-based feeds; however, profile distortion was most overt in peripheral tissues. Results suggest that beef tallow may be used as a primary lipid source in practical diets for hybrid striped bass, but performance may be improved by supplementation with LC-PUFA, particularly DHA. Furthermore, our results suggest that -3 LC-PUFA requirements reported for hybrid striped bass may not be

Guanidinium-induced denaturation by breaking of salt bridges

NARCIS (Netherlands)

Meuzelaar, H.; Panman, M.R.; Woutersen, S.

2015-01-01

Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm+) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm+ can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm+-​induced denaturation of

Denatured plutonium: a study of deterrent action. Final report

International Nuclear Information System (INIS)

Hutchins, B.A.

1975-07-01

The safeguarding of nuclear reactor fuel includes physical security methods as well as technological process options. The purpose of this study was to provide a preliminary evaluation of a technological option; the introduction of denaturing as a deterrent to illicit plutonium diversion. Denaturing is accomplished by coextracting some highly-radioactive fission products with the plutonium during reprocessing of spent fuel. The radioactive denaturant is always in companion with the plutonium through all subsequent fuel cycle steps - and serves as a deterrent to diversion or illicit usage of this fissile source. In concept the denaturing approach is simple and straightforward. This report provides a preliminary analysis of denaturing which can be achieved within the framework of present reprocessing technology. The impact of denaturing is indicated by comparison to a conventional (i.e., non-denatured) light water reacter cycle approach

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

Energy Technology Data Exchange (ETDEWEB)

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen (Germany); Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany)

2016-07-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

International Nuclear Information System (INIS)

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

2016-01-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

Guanidinium-Induced Denaturation by Breaking of Salt Bridges.

Science.gov (United States)

Meuzelaar, Heleen; Panman, Matthijs R; Woutersen, Sander

2015-12-07

Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm(+) ) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm(+) can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm(+) -induced denaturation of a series of peptides containing Arg/Glu and Lys/Glu salt bridges that either stabilize or destabilize the folded conformation. The peptides containing stabilizing salt bridges are found to be denatured much more efficiently by Gdm(+) than the peptides containing destabilizing salt bridges. Complementary 2D-infrared measurements suggest a denaturation mechanism in which Gdm(+) binds to side-chain carboxylate groups involved in salt bridges. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hybridization of plant virus ssRNAs Transferred to Hybond N membrane

International Nuclear Information System (INIS)

Kudela, O.; Kudelova, K.; Plaschke-Jakubik, K.

1998-01-01

In this paper we present a protocol for the non-denaturating agarose gel electrophoresis of plant virus ssRNAs, their blotting onto Hybond N membrane, and hybridization with [alpha 32 P]dNTP-labelled cDNA probe. The protocol is not pretentious on technical equipment, omits denaturation and neutralization steps and some chemical required in other modifications. (authors)

When biogeographical provinces collide: Hybridization of reef fishes at the crossroads of marine biogeographical provinces in the Arabian Sea

KAUST Repository

DiBattista, Joseph

2015-04-01

Aim: Suture zones are areas where closely related species from different biogeographical regions come into contact and interbreed. This concept originated from the study of terrestrial ecosystems but it remains unclear whether a similar phenomenon occurs in the marine environment. Here we investigate a potential suture zone from a previously unknown hybrid hotspot at the Socotra Archipelago (Yemen), located in the Arabian Sea, where fauna from the Red Sea, Gulf of Aden, Arabian Sea, western Indian Ocean and greater Indo-Polynesian Province intersect. Location: Red Sea, Gulf of Aden, Arabian Sea and Indian Ocean. Methods: Putative hybrid reef fish were identified based on intermediate coloration and morphology. Underwater observations and collections were conducted to determine: (1) whether parent species form heterospecific social groups or breeding pairs; (2) the sex and reproductive status of morphologically intermediate individuals; and (3) whether parent species were forming mixed species associations owing to a dearth of conspecific partners. To support hybrid status, morphologically intermediate and parental individuals were genotyped using mitochondrial DNA cytochrome c oxidase subunit I (COI), nuclear recombination-activating gene 2 (RAG2) and the nuclear TMO-4C4 (TMO) gene. Results: We observed putative hybrids involving 14 species from four reef fish families at Socotra. Most cases involved a parental species with a restricted distribution (e.g. Red Sea or Arabian Sea) and a broadly distributed Indo-Pacific species. In most cases, at least one of the parent species was rare at Socotra. Hybrid gene flow was largely unidirectional, and although introgression was rare, we found evidence that some butterflyfish and surgeonfish hybrids were fertile and formed breeding groups with parental species. Main conclusions: The rate of hybrid discovery at Socotra is much greater than that recorded elsewhere in the marine environment and involved both allopatric and

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

Science.gov (United States)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

Science.gov (United States)

Kiyose, Shinichiro; Igarashi, Hisaki; Nagura, Kiyoko; Kamo, Takaharu; Kawane, Kazunori; Mori, Hiroki; Ozawa, Takachika; Maeda, Matsuyoshi; Konno, Keisuke; Hoshino, Hideaki; Konno, Hiroyuki; Ogura, Hiroyuki; Shinmura, Kazuya; Hattori, Naohiko; Sugimura, Haruhiko

2012-11-01

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples. © 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH).

Science.gov (United States)

Kubota, Kengo; Ohashi, Akiyoshi; Imachi, Hiroyuki; Harada, Hideki

2006-09-01

Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.

Reversibility of partial denaturation of DNA

International Nuclear Information System (INIS)

Acuna, M.I.; Mingot, F.; Davila, C.A.

1976-01-01

The recovery of hypochromicity in a partially denatured DNA sample when salt concentration is suddenly increased at a intermediate stage of the thermal transition is studied. The results of CsCl gradient analysis, PEG/DEX partition analysis, behaviour in a new thermal transition hydrodynamic properties and transforming ability, support the view that the process is an intramolecular double chain denaturation. The degree of denaturation irreversibility is dependent on single chain molecular weight of DNA (discontinuities denisty) and upon the helicity value at which salt concentration jump is performed. Both dependences are formally interpreted according to Elton's model for base distribution in DNA. Kinetically the process behaves as being an hydrodynamically limited rewinding. (author)

Evaluation of the damage in fish spermatozoa cryopreservation

Science.gov (United States)

Li, Jun; Liu, Qinghua; Zhang, Shicui

2006-12-01

Cryodamages occur during sperm cryopreservation. Cryopreservation of fish sperm usually results in marked decrease in sperm quality, such as swelling or disruption of the plasma membrane, mitochondrial dysfunction, diminished sperm motility, impaired velocity, shorter motility period, denaturation, and release of some enzymes from spermatozoa. In this paper, damages in morphology, physiology, biochemistry and metabolism, and genetic integrity of fish semen after cryopreservation are discussed. New approaches in assessment of fish thawed sperm quality such as computer assisted sperm analysis, flow cytometic analysis combined with fluorescent probes and single cell gel electrophoresis are also briefly reviewed.

Thermal denaturation of type I collagen vitrified gels

International Nuclear Information System (INIS)

Xia, Zhiyong; Calderon-Colon, Xiomara; Trexler, Morgana; Elisseeff, Jennifer; Guo, Qiongyu

2012-01-01

Highlights: ► We analyzed the denaturation of vitrigels synthesized under different conditions. ► Overall denaturation kinetics consisted of both reversible and irreversible steps. ► More stable vitrigels were formed under high level of vitrification. - Abstract: The denaturation kinetics of type I collagen vitrigels synthesized under different vitrification time and temperature were analyzed by the classical Kissinger approach and the advanced model free kinetics (AMFK) using the Vyazovkin algorithm. The AMFK successfully elucidated the overall denaturation into reversible and irreversible processes. Depending on vitrification conditions, the activation energy for the irreversible process ranged from 100 to 200 kJ/mol, and the reversible enthalpy ranged from 250 to 300 kJ/mol. All of these values increased with the vitrification time and temperature, indicating that a more stable and complex structure formed with increased vitrification. The classical Kissinger method predicted the presence of a critical temperate of approximately 60 °C for the transition between reversible and irreversible processes. Scanning electron microscopy revealed the presence of fibril structures in vitrigels both before and after full denaturation; however the fibrils had became thicker and rougher after denaturation.

Insights into signal transduction by a hybrid FixL: Denaturation study of on and off states of a multi-domain oxygen sensor.

Science.gov (United States)

Guimarães, Wellinson G; Gondim, Ana C S; Costa, Pedro Mikael da Silva; Gilles-Gonzalez, Marie-Alda; Lopes, Luiz G F; Carepo, Marta S P; Sousa, Eduardo H S

2017-07-01

FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(Fe III ) and inactive cyanomet-FixL (Fe III -CN - ) form was monitored by UV-visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV-visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔG H2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8kJmol -1 ). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2kJmol -1 ). For the three-state mechanism exhibited by fluorescence, the ΔG H2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors. Copyright © 2017 Elsevier Inc. All rights reserved.

A simple protocol for attenuating the auto-fluorescence of cyanobacteria for optimized fluorescence in situ hybridization (FISH) imaging.

Science.gov (United States)

Zeller, Perrine; Ploux, Olivier; Méjean, Annick

2016-03-01

Cyanobacteria contain pigments, which generate auto-fluorescence that interferes with fluorescence in situ hybridization (FISH) imaging of cyanobacteria. We describe simple chemical treatments using CuSO4 or H2O2 that significantly reduce the auto-fluorescence of Microcystis strains. These protocols were successfully applied in FISH experiments using 16S rRNA specific probes and filamentous cyanobacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of software and modification of Q-FISH protocol for estimation of individual telomere length in immunopathology.

Science.gov (United States)

Barkovskaya, M Sh; Bogomolov, A G; Knauer, N Yu; Rubtsov, N B; Kozlov, V A

2017-04-01

Telomere length is an important indicator of proliferative cell history and potential. Decreasing telomere length in the cells of an immune system can indicate immune aging in immune-mediated and chronic inflammatory diseases. Quantitative fluorescent in situ hybridization (Q-FISH) of a labeled (C 3 TA[Formula: see text] peptide nucleic acid probe onto fixed metaphase cells followed by digital image microscopy allows the evaluation of telomere length in the arms of individual chromosomes. Computer-assisted analysis of microscopic images can provide quantitative information on the number of telomeric repeats in individual telomeres. We developed new software to estimate telomere length. The MeTeLen software contains new options that can be used to solve some Q-FISH and microscopy problems, including correction of irregular light effects and elimination of background fluorescence. The identification and description of chromosomes and chromosome regions are essential to the Q-FISH technique. To improve the quality of cytogenetic analysis after Q-FISH, we optimized the temperature and time of DNA-denaturation to get better DAPI-banding of metaphase chromosomes. MeTeLen was tested by comparing telomere length estimations for sister chromatids, background fluorescence estimations, and correction of nonuniform light effects. The application of the developed software for analysis of telomere length in patients with rheumatoid arthritis was demonstrated.

Diagnostic and Prognostic Utility of Fluorescence In situ Hybridization (FISH) Analysis in Acute Myeloid Leukemia.

Science.gov (United States)

Gonzales, Patrick R; Mikhail, Fady M

2017-12-01

Acute myeloid leukemia (AML) is a hematologic neoplasia consisting of incompletely differentiated hematopoietic cells of the myeloid lineage that proliferate in the bone marrow, blood, and/or other tissues. Clinical implementation of fluorescence in situ hybridization (FISH) in cytogenetic laboratories allows for high-resolution analysis of recurrent structural chromosomal rearrangements specific to AML, especially in AML with normal karyotypes, which comprises approximately 33-50% of AML-positive specimens. Here, we review the use of several FISH probe strategies in the diagnosis of AML. We also review the standards and guidelines currently in place for use by clinical cytogenetic laboratories in the evaluation of AML. Updated standards and guidelines from the WHO, ACMG, and NCCN have further defined clinically significant, recurring cytogenetic anomalies in AML that are detectable by FISH. FISH continues to be a powerful technique in the diagnosis of AML, with higher resolution than conventional cytogenetic analysis, rapid turnaround time, and a considerable diagnostic and prognostic utility.

Thermally responsive silicon nanowire arrays for native/denatured-protein separation

International Nuclear Information System (INIS)

Wang Hongwei; Wang Yanwei; Yuan Lin; Wang Lei; Yang Weikang; Wu Zhaoqiang; Li Dan; Chen Hong

2013-01-01

We present our findings of the selective adsorption of native and denatured proteins onto thermally responsive, native-protein resistant poly(N-isopropylacrylamide) (PNIPAAm) decorated silicon nanowire arrays (SiNWAs). The PNIPAAm–SiNWAs surface, which shows very low levels of native-protein adsorption, favors the adsorption of denatured proteins. The amount of denatured-protein adsorption is higher at temperatures above the lower critical solution temperature (LCST) of PNIPAAm. Temperature cycling surrounding the LCST, which ensures against thermal denaturation of native proteins, further increases the amount of denatured-protein adsorption. Moreover, the PNIPAAm–SiNWAs surface is able to selectively adsorb denatured protein even from mixtures of different protein species; meanwhile, the amount of native proteins in solution is kept nearly at its original level. It is believed that these results will not only enrich current understanding of protein interactions with PNIPAAm-modified SiNWAs surfaces, but may also stimulate applications of PNIPAAm–SiNWAs surfaces for native/denatured protein separation. (paper)

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

Energy Technology Data Exchange (ETDEWEB)

Cui, Yi; Hu, Dehong; Markillie, Lye Meng; Chrisler, William B.; Gaffrey, Matthew J.; Ansong, Charles; Sussel, Lori; Orr, Galya

2017-10-04

Quantitative gene expression analysis in intact single cells can be achieved using single molecule- based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple FISH sub-probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific binding of sub-probes and tissue autofluorescence, limiting its accuracy. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on blinking frequencies of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses blinking frequency patterns, emitted from a transcript bound to multiple sub-probes, which are distinct from blinking patterns emitted from partial or nonspecifically bound sub-probes and autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2, and their ratio (Nkx2-2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct blinking frequency patterns, laying the foundation for reliable single-cell transcriptomics.

Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)

International Nuclear Information System (INIS)

Park, June-Woo; Tompsett, Amber; Zhang, Xiaowei; Newsted, John L.; Jones, Paul D.; Au, Doris; Kong, Richard; Wu, Rudolf S.S.; Giesy, John P.; Hecker, Markus

2008-01-01

The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 μg/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells

27 CFR 19.464 - Denatured spirits inventories.

Science.gov (United States)

2010-04-01

... of Articles Inventories § 19.464 Denatured spirits inventories. Each proprietor shall take a physical inventory of all denatured spirits in the processing account at the close of each calendar quarter and at... inventories. 19.464 Section 19.464 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE...

[HER-2 oncogene amplification assessment in invasive breast cancer by dual-color in situ hybridization (dc-CISH): a comparative study with fluorescent in situ hybridization (FISH)].

Science.gov (United States)

Akhdar, Abbas; Bronsard, Marc; Lemieux, Renald; Geha, Sameh

2011-12-01

The amplification of the gene encoding for the human epidermal growth factor receptor 2 (HER-2 oncogene), located on chromosome 17 (17q21-q22), or the overexpression of this receptor have prognostic and therapeutic implications in invasive breast cancer. An evaluation of the HER-2 status by immunohistochemistry (IHC) is performed on all invasive breast cancer cases. Fluorescent in situ hybridization (FISH) is considered as the gold standard for the detection of HER-2 gene amplification for IHC equivocal cases (score 2+). A more recent in situ hybridization technique, the dual-color chromogenic in situ hybridization (dc-CISH), has been proposed as an alternative to FISH. The aim of this study was to measure the correlation between dc-CISH and FISH for HER-2 oncogene amplification assessment in invasive breast cancer. We built four tissue micro-array (TMA) blocs with 100 breast invasive cancer cases that had been previously tested by IHC for HER-2 detection: 10 score 0 cases, 10 score 3+cases, 39 score 1+and 41 score 2+cases. Both FISH and dc-CISH techniques were applied on all TMA cases as well as on two additional slides serving as controls. Interpretation of dc-CISH was carried out by a pathologist using an optical microscope. For FISH, the interpretation was done by a professional from the medical genetics department using a fluorescent microscope linked to a computer system for image capturing and analysis. The interpretation of the HER-2/CEN-17 ratio for both tests was in accordance with the values of the updated recommendations from the Canadian National Consensus Meeting on HER-2/neu testing in breast cancer and from the ASCO/CAP. Among the 100 cases initially included in the study, eight were excluded from the analysis due to sampling or technical flaws. From the 92 remaining cases, we obtained a concordance of 97.8% (90/92 cases) between the two techniques (Kappa coefficient 0.97, 95% confidence interval). The correlation coefficient (rho) between ratios

Microwave-enhanced folding and denaturation of globular proteins

DEFF Research Database (Denmark)

Bohr, Henrik; Bohr, Jakob

2000-01-01

It is shown that microwave irradiation can affect the kinetics of the folding process of some globular proteins, especially beta-lactoglobulin. At low temperature the folding from the cold denatured phase of the protein is enhanced, while at a higher temperature the denaturation of the protein from...... its folded state is enhanced. In the latter case, a negative temperature gradient is needed for the denaturation process, suggesting that the effects of the microwaves are nonthermal. This supports the notion that coherent topological excitations can exist in proteins. The application of microwaves...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

DEFF Research Database (Denmark)

Ronander, Elena; Bengtsson, Dominique C; Joergensen, Louise

2012-01-01

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has...... been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors...... fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE(1). Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human...

Analyzing Protein Denaturation using Fast Differential Scanning Calorimetry

NARCIS (Netherlands)

Splinter, R.; Van Herwaarden, A.W.; Iervolino, E.; Vanden Poel, G.; Istrate, D.; Sarro, P.M.

2012-01-01

This paper investigates the possibility to measure protein denaturation with Fast Differential Scanning Calorimetry (FDSC). Cancer can be diagnosed by measuring protein denaturation in blood plasma using Differential Scanning Calorimetry (DSC). FDSC can reduce diagnosis time from hours to minutes,

Comparison of bacterial communities of tilapia fish from Cameroon ...

African Journals Online (AJOL)

Comparison of bacterial communities of tilapia fish from Cameroon and Vietnam using PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) ... The different PCR-DGGE 16S rDNA banding profiles obtained were analysed and results showed that there were specific bands for each geographical ...

Effects of Dehydration on Fish Muscles at Chilled Temperature

Science.gov (United States)

Miki, Hidemasa; Seto, Fuminori; Nishimoto, Motomi; Nishimoto, Junichi

Recently,new method of removing water from fish fillet at low temperature using dehydration sheet have been reported. The present study is concerned with the factors to affect the quality during dehydration of horse mackerel muscle at low temperature. The rate of dehydration at -3 °C was about two times faster than that at 0 °C. The rate of denaturation of fish muscle protein was kept less than about 10 % (ATPase activity) of the undenaturated initial values after removing free water content. Present results suggest the practical possibility of the dehydration at -3 °C for keeping quality of fish flesh.

Use of anionic denaturing detergents to purify insoluble proteins after overexpression

Directory of Open Access Journals (Sweden)

Schlager Benjamin

2012-12-01

Full Text Available Background Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification. Results Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC. Conclusion Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology.

Marine Fish Hybridization

KAUST Repository

He, Song

2017-01-01

for each hybrid offspring in each case, haploweb analysis on diagnostic markers (nuclear and/or mitochondrial) and the DAPC/PCA analysis on microsatellite data were used. By combining the genetic evidences, morphological traits, and ecological observations

Construction of gateway-compatible yeast two-hybrid vectors for ...

African Journals Online (AJOL)

USER

2010-03-01

Mar 1, 2010 ... vectors pBTM116GW and pVP16GW by introducing the gateway cassette ... Key words: Yeast two-hybrid, gateway cloning technology, protein interaction. .... cycling parameters were as follows: an initial denaturation step at.

Light water reactors with a denatured thorium fuel cycle

International Nuclear Information System (INIS)

1978-05-01

Discussed in this paper is the performance of denatured thorium fuel cycles in PWR plants of conventional design, such as those currently in operation or under construction. Although some improvement in U 3 O 8 utilization is anticipated in PWRs optimized explicitly for the denatured thorium fuel cycle, this paper is limited to a discussion of the performance of denatured thorium fuels in conventional PWRs and consequently the data presented is representative of the use of thorium fuel in existing PWRs or those presently under construction. In subsequent sections of this paper, the design of the PWR, its performance on the denatured thorium fuel cycle, safety, accident and environmental considerations, and technological status and R and D requirements are discussed

Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH).

Science.gov (United States)

Deloge-Abarkan, Magali; Ha, Thi-Lan; Robine, Enric; Zmirou-Navier, Denis; Mathieu, Laurence

2007-01-01

Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.

Lessons Learned on Bioaugmentation of DNAPL Source Zone Areas

Science.gov (United States)

2007-10-01

but rather have stringers, ganglia or blobs that can create an “effective pool length”. As the leading edge of these discontinuous DNAPL free-phases...terminal restriction fragment length polymorphism (T-RFLP), denaturing gradient gel electrophoresis (DGGE), and fluorescent in situ hybridization ( FISH ...question of interest (e.g. PCR, FISH , DGGE); (ii) sampling location(s); (iii) an appropriate sampling procedure; and (iv) an appropriate sample handling

Single-molecule denaturation mapping of DNA in nanofluidic channels

DEFF Research Database (Denmark)

Reisner, Walter; Larsen, Niels Bent; Silahtaroglu, Asli

2010-01-01

Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO (R)-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips...... and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence...

Fluorescence in situ hybridization (CARD-FISH) of microorganisms in hydrocarbon contaminated aquifer sediment samples.

Science.gov (United States)

Tischer, Karolin; Zeder, Michael; Klug, Rebecca; Pernthaler, Jakob; Schattenhofer, Martha; Harms, Hauke; Wendeberg, Annelie

2012-12-01

Groundwater ecosystems are the most important sources of drinking water worldwide but they are threatened by contamination and overexploitation. Petroleum spills account for the most common source of contamination and the high carbon load results in anoxia and steep geochemical gradients. Microbes play a major role in the transformation of petroleum hydrocarbons into less toxic substances. To investigate microbial populations at the single cell level, fluorescence in situ hybridization (FISH) is now a well-established technique. Recently, however, catalyzed reporter deposition (CARD)-FISH has been introduced for the detection of microbes from oligotrophic environments. Nevertheless, petroleum contaminated aquifers present a worst case scenario for FISH techniques due to the combination of high background fluorescence of hydrocarbons and the presence of small microbial cells caused by the low turnover rates characteristic of groundwater ecosystems. It is therefore not surprising that studies of microorganisms from such sites are mostly based on cultivation techniques, fingerprinting, and amplicon sequencing. However, to reveal the population dynamics and interspecies relationships of the key participants of contaminant degradation, FISH is an indispensable tool. In this study, a protocol for FISH was developed in combination with cell quantification using an automated counting microscope. The protocol includes the separation and purification of microbial cells from sediment particles, cell permeabilization and, finally, CARD-FISH in a microwave oven. As a proof of principle, the distribution of Archaea and Bacteria was shown in 60 sediment samples taken across the contaminant plume of an aquifer (Leuna, Germany), which has been heavily contaminated with several ten-thousand tonnes of petroleum hydrocarbons since World War II. Copyright © 2012 Elsevier GmbH. All rights reserved.

Automated DNA electrophoresis, hybridization and detection

International Nuclear Information System (INIS)

Zapolski, E.J.; Gersten, D.M.; Golab, T.J.; Ledley, R.S.

1986-01-01

A fully automated, computer controlled system for nucleic acid hybridization analysis has been devised and constructed. In practice, DNA is digested with restriction endonuclease enzyme(s) and loaded into the system by pipette; 32 P-labelled nucleic acid probe(s) is loaded into the nine hybridization chambers. Instructions for all the steps in the automated process are specified by answering questions that appear on the computer screen at the start of the experiment. Subsequent steps are performed automatically. The system performs horizontal electrophoresis in agarose gel, fixed the fragments to a solid phase matrix, denatures, neutralizes, prehybridizes, hybridizes, washes, dries and detects the radioactivity according to the specifications given by the operator. The results, printed out at the end, give the positions on the matrix to which radioactivity remains hybridized following stringent washing

NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

Science.gov (United States)

Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

2003-01-01

The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

BIOTECHNOLOGY OF THE FISH AQUACULTURE

Directory of Open Access Journals (Sweden)

L. P. Buchatsky

2013-12-01

Full Text Available The latest progress in biotechnology on fish aquaculture and different modern methods of investigations for increasing of fish productivity in aquaculture are analyzed. Except for the applied aspect, the use of modern biotechnological methods of investigations opens new possibilities for fundamental researches of sex-determining mechanisms, polyploidy, distant hybridization, and developmental biology of bony fishes. Review contains examples of utilizing modern biotechnology methods to obtain transgenic fishes with accelerated growth and for designing surrogate fishes. Methods for receiving unisexual shoals of salmon and sturgeon female fishes with the view of obtaining a large quantity of caviar, as well as receiving sterile (triploid fishes are analyzed. Great attention is given to androgenesis, particularly to disperm one, in connection with the problem of conserving rare and vanishing fish species using only sperm genetic material. Examples how distant hybrids may be obtained with the use of disperm androgenesis and alkylated DNA are given. Methods of obtaining fish primordium germ cells, recent developments in cultivation of fish stem cells and their use in biotechnology, as well as ones of transplantation of oogonium and spermatogonium to obtain surrogate fishes. The examples of successful experiments on spermatogonial xenotransplantation and characteristic of antifreezing fish proteins and also the prospect of their practical usage are given.

Hysteresis in pressure-driven DNA denaturation.

Directory of Open Access Journals (Sweden)

Enrique Hernández-Lemus

Full Text Available In the past, a great deal of attention has been drawn to thermal driven denaturation processes. In recent years, however, the discovery of stress-induced denaturation, observed at the one-molecule level, has revealed new insights into the complex phenomena involved in the thermo-mechanics of DNA function. Understanding the effect of local pressure variations in DNA stability is thus an appealing topic. Such processes as cellular stress, dehydration, and changes in the ionic strength of the medium could explain local pressure changes that will affect the molecular mechanics of DNA and hence its stability. In this work, a theory that accounts for hysteresis in pressure-driven DNA denaturation is proposed. We here combine an irreversible thermodynamic approach with an equation of state based on the Poisson-Boltzmann cell model. The latter one provides a good description of the osmotic pressure over a wide range of DNA concentrations. The resulting theoretical framework predicts, in general, the process of denaturation and, in particular, hysteresis curves for a DNA sequence in terms of system parameters such as salt concentration, density of DNA molecules and temperature in addition to structural and configurational states of DNA. Furthermore, this formalism can be naturally extended to more complex situations, for example, in cases where the host medium is made up of asymmetric salts or in the description of the (helical-like charge distribution along the DNA molecule. Moreover, since this study incorporates the effect of pressure through a thermodynamic analysis, much of what is known from temperature-driven experiments will shed light on the pressure-induced melting issue.

27 CFR 20.261 - Records of completely denatured alcohol.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Records of completely denatured alcohol. 20.261 Section 20.261 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM...

Utility and impact of early t(15;17) identification by Fluorescence In Situ Hybridization (FISH) in clinical decision making for patients in Acute Promyelocytic Leukemia (APL).

Science.gov (United States)

Kolhe, R; Mangaonkar, A; Mansour, J; Clemmons, A; Shaw, J; Dupont, B; Walczak, L; Mondal, A; Rojiani, A; Jillella, A; Kota, V

2015-08-01

Acute Promyelocytic Leukemia (APL) is a curable malignancy with studies showing above 90% survival. However, population-based studies looking at survival suggest that approximately 30% of patients with APL die during induction. Early demonstration of t(15;17) will lead to accurate decision making regarding treatment. The aim of this project was to validate earlier time frames for the Abbott Molecular Vysis LSI promyelocytic leukemia (PML)/ retinoic acid receptor alpha (RARA) fluorescence in situ hybridization (FISH) probe (ASR 6-16 h). Twenty patients (15 APL cases and five non-APL cases) were selected for validating various hybridization times for the FISH probe. Expected normal signal pattern was two red and two green signals (2R2G), and the most common expected abnormal signal pattern was two fusion (yellow) signals, one red and one green (2F1R1G) and/or one fusion, one red and one green (1F1R1G). The specificity of the probe ranged from 84% at 2 h, 86% at 4 h, 84% at 6 h, and 87% for overnight hybridization. The sensitivity increased from 79% at 2 h, 80% at 4 h, 81% at 6 h to 87% for overnight hybridization. Based on the validation studies, we recommend reading of FISH results at the 4-h incubation mark for a preliminary diagnosis and confirmation with overnight hybridization. © 2015 John Wiley & Sons Ltd.

27 CFR 20.144 - Packages of completely denatured alcohol.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Packages of completely denatured alcohol. 20.144 Section 20.144 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Sale...

Fluorescent in situ hybridization (FISH as a diagnostic tool for Williams-Beuren syndrome

Directory of Open Access Journals (Sweden)

Deise Helena de Souza

2007-01-01

Full Text Available Fluorescent in situ hybridization (FISH with commercial probes covering the elastin gene (ELN was used to determine the frequency of the 7q11.23 deletion in 18 children clinically diagnosed with Williams-Beuren syndrome (WBS. A de novo deletion was detected in 15 of the children (83%. Diagnostic investigation for WBS started late in childhood (median = 5.8 years. All the children showed facial features typical of the syndrome, mental retardation and developmental delay. Over-friendliness was observed in the majority of cases. Clinodactyly of the 5th finger (n = 13, cardiovascular disease (n = 9, loquacity (n = 9, low birthweight (n = 8, and failure to thrive (n = 9 were observed only in those children with the deletion. Respiratory problems (n = 9, though not previously reported in the literature, was a common finding in the group studied. Our results confirmed that FISH is useful in identifying 7q11.23 deletions in cases of WBS. Clinical manifestations were more evident in the deletion-positive children.

The generation of denatured reactor plutonium by different options of the fuel cycle

Energy Technology Data Exchange (ETDEWEB)

Broeders, C.H.M.; Kessler, G. [Inst. for Neutron Physics and Reactor Technology, Research Center Karlsruhe (Germany)

2006-11-15

Denatured (proliferation resistant) reactor plutonium can be generated in a number of different fuel cycle options. First denatured reactor plutonium can be obtained if, instead of low enriched U-235 PWR fuel, re-enriched U-235/U-236 from reprocessed uranium is used (fuel type A). Also the envisaged existing 2,500 t of reactor plutonium (being generated world wide up to the year 2010), mostly stored in intermediate fuel storage facilities at present, could be converted during a transition phase into denatured reactor plutonium by the options fuel type B and D. Denatured reactor plutonium could have the same safeguards standard as present low enriched (<20% U-235) LWR fuel. It could be incinerated by recycling once or twice in PWRs and subsequently by multi-recycling in FRs (CAPRA type or IFRs). Once denatured, such reactor plutonium could remain denatured during multiple recycling. In a PWR, e.g., denatured reactor plutonium could be destroyed at a rate of about 250 kg/GWey. While denatured reactor plutonium could be recycled and incinerated under relieved IAEA safeguards, neptunium would still have to be monitored by the IAEA in future for all cases in which considerable amounts of neptunium are produced. (orig.)

Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue

DEFF Research Database (Denmark)

Laub Petersen, Bodil; Zeuthen, Mette Christa; Pedersen, Sanni

2004-01-01

Fluorescence in situ hybridization (FISH) is widely used to study numerical and structural genetic abnormalities in both metaphase and interphase cells. The technique is based on the hybridization of labeled probes to complementary sequences in the DNA or RNA of the cells. Interphase FISH is most...... in time lapse between removal of tissue and fixation, duration of fixation, enzymatic pretreatment, hybridization conditions, and posthybridization washing conditions are important factors in the hybridization. In this study, we have listed the results of a systematic approach to improve FISH on isolated...

Heavy water reactors on the denatured thorium cycles

International Nuclear Information System (INIS)

1978-05-01

This paper presents preliminary technical and economic data to INFCE on the denatured U-233/Thorium fuel cycle for use in early comparisons of alternate nuclear systems. The once-through uranium fuel cycle is discussed in a companion paper. In presenting this preliminary information at this time, it is recognized that there are several other denatured thorium fuel cycles of potential interest, such as the U-235/thorium cycle which could be implemented at an earlier date. Information on these alternate cycles is currently being developed, and will be provided to INFCE when available

Irreversible denaturation of maltodextrin glucosidase studied by differential scanning calorimetry, circular dichroism, and turbidity measurements.

Science.gov (United States)

Goyal, Megha; Chaudhuri, Tapan K; Kuwajima, Kunihiro

2014-01-01

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5-1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).

Interim assessment of the denatured 233U fuel cycle: feasibility and nonproliferation characteristics

International Nuclear Information System (INIS)

Abbott, L.S.; Bartine, D.E.; Burns, T.J.

1979-12-01

A fuel cycle that employs 233 U denatured with 238 U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured 233 U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured 233 U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured 233 U fuel and are based on the energy center concept are evaluated

Detection of Chrysanthemum stunt viroid (CSV) by nucleic acid spot hybridization

Energy Technology Data Exchange (ETDEWEB)

Bothma, G C

1985-01-01

Chrysanthemum stunt viroid (CSV) was extracted from Chrysanthemum stunt viroid-infected Chrysanthemum plants. The CSV was then purified to homogenicity from this extract by one cycle of non-denaturating vertical slab gel electrophoresis and was followed by one cycle of denaturating vertical slab gel electrophoresis. No pure circular CSV was obtained. The 3' terminal of purified ASBV was polyadenylated using poly (A) polymerase. The polyadenylated ASBV was used as a template to synthesise radioactively labelled single stranded ASBV cDNA using reverse transcriptase. The radioactively labelled recombinant DNA was hybridised to crude extracts from avocado leaves and chrysanthemum leaves. Autoradiography was employed to detect cDNA-RNA hybrids.

Weakening Pin Bone Attachment in Fish Fillets Using High-Intensity Focused Ultrasound.

Science.gov (United States)

Skjelvareid, Martin H; Stormo, Svein Kristian; Þórarinsdóttir, Kristín Anna; Heia, Karsten

2017-09-18

High Intensity Focused Ultrasound (HIFU) can be used for the localized heating of biological tissue through the conversion of sound waves into heat. Although originally developed for human medicine, HIFU may also be used to weaken the attachment of pin bones in fish fillets to enable easier removal of such bones. This was shown in the present study, where a series of experiments were performed on HIFU phantoms and fillets of cod and salmon. In thin objects such as fish fillets, the heat is mainly dissipated at the surfaces. However, bones inside the fillet absorb ultrasound energy more efficiently than the surrounding tissue, resulting in a "self-focusing" heating of the bones. Salmon skin was found to effectively block the ultrasound, resulting in a significantly lower heating effect in fillets with skin. Cod skin partly blocked the ultrasound, but only to a small degree, enabling HIFU treatment through the skin. The treatment of fillets to reduce the pin bone attachment yielded an average reduction in the required pulling force by 50% in cod fillets with skin, with little muscle denaturation, and 72% in skinned fillets, with significant muscle denaturation. Salmon fillets were treated from the muscle side of the fillet to circumvent the need for penetration through skin. The treatment resulted in a 30% reduction in the peak pulling force and 10% reduction in the total pulling work, with a slight denaturation of the fillet surface.

Identification of the centromeric repeat in the threespine stickleback fish (Gasterosteus aculeatus).

Science.gov (United States)

Cech, Jennifer N; Peichel, Catherine L

2015-12-01

Centromere sequences exist as gaps in many genome assemblies due to their repetitive nature. Here we take an unbiased approach utilizing centromere protein A (CENP-A) chomatin immunoprecipitation followed by high-throughput sequencing to identify the centromeric repeat sequence in the threespine stickleback fish (Gasterosteus aculeatus). A 186-bp, AT-rich repeat was validated as centromeric using both fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on interphase nuclei and metaphase spreads. This repeat hybridizes strongly to the centromere on all chromosomes, with the exception of weak hybridization to the Y chromosome. Together, our work provides the first validated sequence information for the threespine stickleback centromere.

27 CFR 20.148 - Manufacture of articles with completely denatured alcohol.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Manufacture of articles with completely denatured alcohol. 20.148 Section 20.148 Alcohol, Tobacco Products and Firearms ALCOHOL... ALCOHOL AND RUM Sale and Use of Completely Denatured Alcohol § 20.148 Manufacture of articles with...

Transurethral radiofrequency collagen denaturation for the treatment of women with urinary incontinence.

Science.gov (United States)

Kang, Diana; Han, Julia; Neuberger, Molly M; Moy, M Louis; Wallace, Sheila A; Alonso-Coello, Pablo; Dahm, Philipp

2015-03-18

Transurethral radiofrequency collagen denaturation is a relatively novel, minimally invasive device-based intervention used to treat individuals with urinary incontinence (UI). No systematic review of the evidence supporting its use has been published to date. To evaluate the efficacy of transurethral radiofrequency collagen denaturation, compared with other interventions, in the treatment of women with UI.Review authors sought to compare the following.• Transurethral radiofrequency collagen denaturation versus no treatment/sham treatment.• Transurethral radiofrequency collagen denaturation versus conservative physical treatment.• Transurethral radiofrequency collagen denaturation versus mechanical devices (pessaries for UI).• Transurethral radiofrequency collagen denaturation versus drug treatment.• Transurethral radiofrequency collagen denaturation versus injectable treatment for UI.• Transurethral radiofrequency collagen denaturation versus other surgery for UI. We conducted a systematic search of the Cochrane Incontinence Group Specialised Register (searched 19 December 2014), EMBASE and EMBASE Classic (January 1947 to 2014 Week 50), Google Scholar and three trials registries in December 2014, along with reference checking. We sought to identify unpublished studies by handsearching abstracts of major gynaecology and urology meetings, and by contacting experts in the field and the device manufacturer. Randomised and quasi-randomised trials of transurethral radiofrequency collagen denaturation versus no treatment/sham treatment, conservative physical treatment, mechanical devices, drug treatment, injectable treatment for UI or other surgery for UI in women were eligible. We screened search results and selected eligible studies for inclusion. We assessed risk of bias and analysed dichotomous variables as risk ratios (RRs) with 95% confidence intervals (CIs) and continuous variables as mean differences (MDs) with 95% CIs. We rated the quality of

Detection of the clostridial hydrogenase gene activity as a bio-index in a molasses wastewater bio-hydrogen producing system by real time PCR and FISH/ flow cytometry

International Nuclear Information System (INIS)

Jui-Jen Chang; Ping-Chi Hsu; Chi-Wa Choi; Sian-Jhong Yu; Cheng-Yu Ho; Wei-En Chen; Jiunn-Jyi Lay; Chieh-Chen Huang; Fu-Shyan Wen

2006-01-01

Hydrogenase is a key enzyme that is used by obligate, anaerobic clostridial to produce hydrogen. In this study a fermentative system with molasses wastewater as nutrient was used to produce hydrogen. For establishing the relationship between the vicissitude of clostridial hydrogenase gene activity and the hydrogen production of this system during the culturing period, total cellular RNA isolated at different growing stages were subjected to real time PCR using primer pair, which were designed according to the conserved sequence of clostridial hydrogenase genes. Cell samples at corresponding growing stages were subjected to in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR) using the same primers and then to fluorescence in situ hybridization (FISH) using clostridial hydrogenase gene-specific DNA probe. Those clostridial cells expressed hydrogenase gene activity could be detected by fluorescence microscopy. This is the first time hydrogen-producing activity in a mixed culture could be successfully studied by means of FISH of hydrogenase mRNA. Besides, 16S rDNA was amplified from total cellular DNA analyzed by denaturing gradient gel electrophoresis (DGGE) to reveal the bacterial diversity in the fermentative system; FISH and flow cytometry aiming at 16S rRNA were also carried out to calculate the population of clostridia and total eubacteria in the system. (authors)

Preparation of denatured protein bone sterilized with gamma radiation

International Nuclear Information System (INIS)

Luna Z, D.

2005-01-01

The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

Mutation of charged residues to neutral ones accelerates urea denaturation of HP-35.

Science.gov (United States)

Wei, Haiyan; Yang, Lijiang; Gao, Yi Qin

2010-09-16

Following the studies of urea denaturation of β-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of α-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of β-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.

Interim assessment of the denatured 233U fuel cycle: feasibility and nonproliferation characteristics

International Nuclear Information System (INIS)

Abbott, L.S.; Bartine, D.E.; Burns, T.J.

1978-12-01

A fuel cycle that employs 233 U denatured with 238 U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured 233 U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured 233 U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured 233 U fuel and are based on the energy center concept are evaluated. Under this concept, dispersed power reactors fueled with denatured or low-enriched uranium fuel are supported by secure energy centers in which sensitive activities of the nuclear cycle are performed. These activities include 233 U production by Pu-fueled transmuters (thermal or fast reactors) and reprocessing. A summary chapter presents the most significant conclusions from the study and recommends areas for future work

Urea-temperature phase diagrams capture the thermodynamics of denatured state expansion that accompany protein unfolding

Science.gov (United States)

Tischer, Alexander; Auton, Matthew

2013-01-01

We have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea-induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea–temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two-state character. Global analysis of the urea–temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of and that define a complex temperature dependence of the m-value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea-denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions. PMID:23813497

Mesoscopic modeling of DNA denaturation rates: Sequence dependence and experimental comparison

Energy Technology Data Exchange (ETDEWEB)

Dahlen, Oda, E-mail: oda.dahlen@ntnu.no; Erp, Titus S. van, E-mail: titus.van.erp@ntnu.no [Department of Chemistry, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, Realfagbygget D3-117 7491 Trondheim (Norway)

2015-06-21

Using rare event simulation techniques, we calculated DNA denaturation rate constants for a range of sequences and temperatures for the Peyrard-Bishop-Dauxois (PBD) model with two different parameter sets. We studied a larger variety of sequences compared to previous studies that only consider DNA homopolymers and DNA sequences containing an equal amount of weak AT- and strong GC-base pairs. Our results show that, contrary to previous findings, an even distribution of the strong GC-base pairs does not always result in the fastest possible denaturation. In addition, we applied an adaptation of the PBD model to study hairpin denaturation for which experimental data are available. This is the first quantitative study in which dynamical results from the mesoscopic PBD model have been compared with experiments. Our results show that present parameterized models, although giving good results regarding thermodynamic properties, overestimate denaturation rates by orders of magnitude. We believe that our dynamical approach is, therefore, an important tool for verifying DNA models and for developing next generation models that have higher predictive power than present ones.

Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.

Science.gov (United States)

Goud, Kalal Iravathy; Dayakar, Seetha; Vijayalaxmi, Kolanupaka; Babu, Saidam Jangu; Reddy, P Vijay Anand

2012-03-01

Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value

Role of cyclobutane dimers in UV-denaturation of DNA

International Nuclear Information System (INIS)

Zavil'gel'skij, G.B.; Zuev, A.V.

1978-01-01

UV irradiation of double-stranded DNA produces local denatured regions. The evidence presented indicates that these single-stranded regions arise from photoproducts other than pyrimidine dimers. The irradiation of T2 DNA at 8x10 4 erg/mm 2 (254 nm) produces 6-8% thymine dimers, amd Tsub(mel) drops by 12-14 deg C, accompanied by a significant broadening of the transition profile. The kinetics of denatured region formation and lowering Tsub(mel) corresponds to that of formation of crosslinkages and differs markedly from the kinetics of formation of cyclobutane pyrimidine dimers. Treatment of UV-irradiated DNA with light in the presence of yeast photoreactivating enzyme monomerizes almost all thymine dimers but does not change the Tsub(mel). Local denatured regions are detected in UV-irradiated DNA and are absent from AcPhM-sensibilized DNA, which contains 20-25% thymine dimers, as determined by the accridine orange fluorescence technique. S1 nuclease from Aspergillis oryzae produces single-strand breaks in UV-irradiated DNA of phage PM2 but is not active on AcPhM-treated PM2 DNA, which contains about 50 thymine dimers. It is supposed that the formation of a cyclobutane dimer only weakens the hydrogen bonds in the AT base pair rather than breaks them. Local denatured regions are thought to arise from the accumulation in UV-irradiated DNA (254 nm) of the sufficient number of photoproducts with impaired ability to base pairing

Thermal denaturation of protein studied by terahertz time-domain spectroscopy

Science.gov (United States)

Fu, Xiuhua; Li, Xiangjun; Liu, Jianjun; Du, Yong; Hong, Zhi

2012-12-01

In this study, the absorption spectra of native or thermal protein were measured in 0.2-1.4THz using terahertz time-domain spectroscopy (THz-TDS) system at room temperature, their absorption spectra and the refractive spectra were obtained. Experimental results indicate that protein both has strong absorption but their characteristics were not distinct in the THz region, and the absorption decreased during thermal denatured state. In order to prove protein had been denatured, we used Differential scanning calorimeter (DSC) measured their denatured temperature, from their DSC heating traces, collagen Td=101℃, Bovine serum albumin Td=97℃. While we also combined the Fourier transform infrared spectrometer (FTIR) to investigate their secondary and tertiary structure before and after denatuation, but the results did not have the distinct changes. We turned the absorption spectra and the refractive spectra to the dielectric spectra, and used the one-stage Debye model simulated the terahertz dielectric spectra of protein before and after denaturation. This research proved that the terahertz spectrum technology is feasible in testing protein that were affected by temperature or other factors which can provide theoretical foundation in the further study about the THz spectrum of protein and peptide temperature stability.

Hydrolysates of Fish Skin Collagen: An Opportunity for Valorizing Fish Industry Byproducts.

Science.gov (United States)

Blanco, María; Vázquez, José Antonio; Pérez-Martín, Ricardo I; Sotelo, Carmen G

2017-05-05

During fish processing operations, such as skinning and filleting, the removal of collagen-containing materials can account for up to 30% of the total fish byproducts. Collagen is the main structural protein in skin, representing up to 70% of dry weight depending on the species, age and season. It has a wide range of applications including cosmetic, pharmaceutical, food industry, and medical. In the present work, collagen was obtained by pepsin extraction from the skin of two species of teleost and two species of chondrychtyes with yields varying between 14.16% and 61.17%. The storage conditions of the skins appear to influence these collagen extractions yields. Pepsin soluble collagen (PSC) was enzymatically hydrolyzed and the resultant hydrolysates were ultrafiltrated and characterized. Electrophoretic patterns showed the typical composition of type I collagen, with denaturation temperatures ranged between 23 °C and 33 °C. In terms of antioxidant capacity, results revealed significant intraspecific differences between hydrolysates, retentate, and permeate fractions when using β -Carotene and DPPH methods and also showed interspecies differences between those fractions when using DPPH and ABTS methods. Under controlled conditions, PSC hydrolysates from Prionace glauca , Scyliorhinus canicula , Xiphias gladius, and Thunnus albacares provide a valuable source of peptides with antioxidant capacities constituting a feasible way to efficiently upgrade fish skin biomass.

Genetic identification of F1 and post-F1 serrasalmid juvenile hybrids in Brazilian aquaculture.

Directory of Open Access Journals (Sweden)

Diogo Teruo Hashimoto

Full Text Available Juvenile fish trade monitoring is an important task on Brazilian fish farms. However, the identification of juvenile fish through morphological analysis is not feasible, particularly between interspecific hybrids and pure species individuals, making the monitoring of these individuals difficult. Hybrids can be erroneously identified as pure species in breeding facilities, which might reduce production on farms and negatively affect native populations due to escapes or stocking practices. In the present study, we used a multi-approach analysis (molecular and cytogenetic markers to identify juveniles of three serrasalmid species (Colossoma macropomum, Piaractus mesopotamicus and Piaractus brachypomus and their hybrids in different stocks purchased from three seed producers in Brazil. The main findings of this study were the detection of intergenus backcrossing between the hybrid ♀ patinga (P. mesopotamicus×P. brachypomus×♂ C. macropomum and the occurrence of one hybrid triploid individual. This atypical specimen might result from automixis, a mechanism that produces unreduced gametes in some organisms. Moreover, molecular identification indicated that hybrid individuals are traded as pure species or other types of interspecific hybrids, particularly post-F1 individuals. These results show that serrasalmid fish genomes exhibit high genetic heterogeneity, and multi-approach methods and regulators could improve the surveillance of the production and trade of fish species and their hybrids, thereby facilitating the sustainable development of fish farming.

The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs.

Science.gov (United States)

Liang, Pengfei; Jiang, Bimei; Lv, Chunliu; Huang, Xu; Sun, Li; Zhang, Pihong; Huang, Xiaoyuan

2013-10-01

The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burn model in Sprague-Dawley rats and the heat denatured cell model (52°C, 35s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin. Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs. The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF. We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis. Copyright © 2013 Elsevier B.V. All rights reserved.

Cation-Induced Stabilization and Denaturation of DNA Origami Nanostructures in Urea and Guanidinium Chloride.

Science.gov (United States)

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2017-11-01

The stability of DNA origami nanostructures under various environmental conditions constitutes an important issue in numerous applications, including drug delivery, molecular sensing, and single-molecule biophysics. Here, the effect of Na + and Mg 2+ concentrations on DNA origami stability is investigated in the presence of urea and guanidinium chloride (GdmCl), two strong denaturants commonly employed in protein folding studies. While increasing concentrations of both cations stabilize the DNA origami nanostructures against urea denaturation, they are found to promote DNA origami denaturation by GdmCl. These inverse behaviors are rationalized by a salting-out of Gdm + to the hydrophobic DNA base stack. The effect of cation-induced DNA origami denaturation by GdmCl deserves consideration in the design of single-molecule studies and may potentially be exploited in future applications such as selective denaturation for purification purposes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alcohol-induced structural transitions in the acid-denatured Bacillus licheniformis α-amylase

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Adyani Azizah Abd Halim

2017-01-01

Full Text Available Alcohol-induced structural changes in the acid-denatured Bacillus licheniformis α-amylase (BLA at pH 2.0 were studied by far-ultra violet circular dichroism, intrinsic, three-dimensional and 8-anilino-1-naphthalene sulfonic acid (ANS fluorescence, acrylamide quenching and thermal denaturation. All the alcohols used in this study produced partial refolding in the acid-denatured BLA as evident from the increased mean residue ellipticity at 222 nm, increased intrinsic fluorescence and decreased ANS fluorescence. The order of effectiveness of these alcohols to induce a partially folded state of BLA was found to be: 2,2,2-trifluoroethanol/tert-butanol > 1-propanol/2-propanol > 2-chloroethanol > ethanol > methanol. Three-dimensional fluorescence and acrylamide quenching results obtained in the presence of 5.5 M tert-butanol also suggested formation of a partially folded state in the acid-denatured BLA. However, 5.5 M tert-butanol-induced state of BLA showed a non-cooperative thermal transition. All these results suggested formation of a partially folded state of the acid-denatured BLA in the presence of these alcohols. Furthermore, their effectiveness was found to be guided by their chain length, position of methyl groups and presence of the substituents.

Visualization of early events in acetic acid denaturation of HIV-1 protease: a molecular dynamics study.

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Aditi Narendra Borkar

Full Text Available Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.

Patterns of parasite distribution in the hybrids of non-congeneric cyprinid fish species: is asymmetry in parasite infection the result of limited coadaptation?

Czech Academy of Sciences Publication Activity Database

Krasnovyd, V.; Vetešník, Lukáš; Gettová, L.; Civáňová, K.; Šimková, A.

2017-01-01

Roč. 47, č. 8 (2017), s. 471-483 ISSN 0020-7519 R&D Projects: GA ČR GAP505/12/0375 Institutional support: RVO:68081766 Keywords : Interspecific hybrids * Cyprinid fish * Parasite communities * Host specificity * Maternal ancestry Subject RIV: EG - Zoology OBOR OECD: Parasitology Impact factor: 3.730, year: 2016

Neutronics calculations for denatured molten salt reactors: Assessing resource requirements and proliferation-risk attributes

International Nuclear Information System (INIS)

Ahmad, Ali; McClamrock, Edward B.; Glaser, Alexander

2015-01-01

Highlights: • We study the proliferation-risk and resource attributes of denatured MSRs. • MSRs offer significantly better resource efficiency compared to light-water reactors. • Denatured single-fluid MSRs reactors offer promising non-proliferation attributes. - Abstract: Molten salt reactors (MSRs) are often advocated as a radical but worthwhile alternative to traditional reactor concepts based on solid fuels. This article builds upon the existing research into MSRs to model and simulate the operation of thorium-fueled single-fluid and two-fluid reactors. The analysis is based on neutronics calculations and focuses on denatured MSR systems. Resource utilization and basic proliferation-risk attributes are compared to those of standard light-water reactors. Depending on specific design choices, even fully denatured reactors could reduce uranium and enrichment requirements by a factor of 3–4. Overall, denatured single-fluid designs appear as the most promising candidate technology minimizing both design complexity and overall proliferation risks despite being somewhat less attractive from the perspective of resource utilization

Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri.

Science.gov (United States)

Yang, Huiping; Hazlewood, Leona; Heater, Sheila J; Guerrero, Paula A; Walter, Ronald B; Tiersch, Terrence R

2007-03-01

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.

A Hybrid Method for Image Segmentation Based on Artificial Fish Swarm Algorithm and Fuzzy c-Means Clustering

Directory of Open Access Journals (Sweden)

Li Ma

2015-01-01

Full Text Available Image segmentation plays an important role in medical image processing. Fuzzy c-means (FCM clustering is one of the popular clustering algorithms for medical image segmentation. However, FCM has the problems of depending on initial clustering centers, falling into local optimal solution easily, and sensitivity to noise disturbance. To solve these problems, this paper proposes a hybrid artificial fish swarm algorithm (HAFSA. The proposed algorithm combines artificial fish swarm algorithm (AFSA with FCM whose advantages of global optimization searching and parallel computing ability of AFSA are utilized to find a superior result. Meanwhile, Metropolis criterion and noise reduction mechanism are introduced to AFSA for enhancing the convergence rate and antinoise ability. The artificial grid graph and Magnetic Resonance Imaging (MRI are used in the experiments, and the experimental results show that the proposed algorithm has stronger antinoise ability and higher precision. A number of evaluation indicators also demonstrate that the effect of HAFSA is more excellent than FCM and suppressed FCM (SFCM.

Interim assessment of the denatured /sup 233/U fuel cycle: feasibility and nonproliferation characteristics

Energy Technology Data Exchange (ETDEWEB)

Abbott, L.S.; Bartine, D.E.; Burns, T.J. (eds.)

1978-12-01

A fuel cycle that employs /sup 233/U denatured with /sup 238/U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured /sup 233/U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured /sup 233/U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured /sup 233/U fuel and are based on the energy center concept are evaluated. Under this concept, dispersed power reactors fueled with denatured or low-enriched uranium fuel are supported by secure energy centers in which sensitive activities of the nuclear cycle are performed. These activities include /sup 233/U production by Pu-fueled transmuters (thermal or fast reactors) and reprocessing. A summary chapter presents the most significant conclusions from the study and recommends areas for future work.

5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH

Directory of Open Access Journals (Sweden)

Jaime Gosálvez

2013-02-01

Full Text Available We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH. A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL, 10 with high-grade SIL (HG-SIL, and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH).

Science.gov (United States)

Cortés-Gutiérrez, Elva I; Ortíz-Hernández, Brenda L; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime

2013-02-19

We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins.

Science.gov (United States)

Sherman, Eilon; Itkin, Anna; Kuttner, Yosef Yehuda; Rhoades, Elizabeth; Amir, Dan; Haas, Elisha; Haran, Gilad

2008-06-01

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.

Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions.

Science.gov (United States)

Valle, Edith R; Henderson, Gemma; Janssen, Peter H; Cox, Faith; Alexander, Trevor W; McAllister, Tim A

2015-06-01

In this study, methanogen-specific coenzyme F420 autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis.

Fluctuating partially native-like topologies in the acid denatured ensemble of autolysis resistant HIV-1 protease.

Science.gov (United States)

Rout, Manoj Kumar; Hosur, Ramakrishna V

2009-02-01

Folding, in-vivo, starts from a denatured state and thus the nature of the denatured state would play an important role in directing the folding of a protein. We report here NMR characterization of the acid-denatured state of a mutant of HIV-1 protease, designed to prevent autolysis (Q7K, L33I, L63I) and to prevent cysteine oxidation (C67A and C95A). Secondary chemical shifts, TALOS analysis of chemical shifts and (15)N relaxation data (R(1), R(2), NOE) coupled with AABUF and hydrophobicity calculations, suggest formation of hydrophobic clusters and possibility of some partially native-like topologies in the acid denatured state of the protease. The structural and dynamics characteristics of the acid denatured PR seem to be considerably different from those of the guanidine or urea denatured states of some variants of PR. These would have implications for the folding and auto-processing of the enzyme in-vivo.

9 CFR 325.13 - Denaturing procedures.

Science.gov (United States)

2010-01-01

... the appropriate agent shall be used to give the material a distinctive color, odor, or taste so that... thoroughly mixing therein denaturing oil, No. 2 fuel oil, brucine dissolved in a mixture of alcohol and pine... distinctive a color, odor, or taste that it cannot be confused with an article of human food. [35 FR 15605...

Common Fluorescence In Situ Hybridization Applications in Cytology.

Science.gov (United States)

Savic, Spasenija; Bubendorf, Lukas

2016-12-01

- Fluorescence in situ hybridization (FISH) is a well-established method for detection of genomic aberrations in diagnostic, prognostic, and predictive marker testing. - To review common applications of FISH in cytology. - The published literature was reviewed. - Cytology is particularly well suited for all kinds of FISH applications, which is highlighted in respiratory tract cytology with an increasing demand for predictive FISH testing in lung cancer. Fluorescence in situ hybridization is the gold standard for detection of predictive anaplastic lymphoma kinase gene (ALK) rearrangements, and the same evaluation criteria as in histology apply to cytology. Several other gene rearrangements, including ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), are becoming clinically important and share the same underlining cytogenetic mechanisms with ALK. MET amplification is one of the most common mechanisms of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and can be targeted by crizotinib. As genomic aberrations are a hallmark of malignant cells, FISH is a valuable objective ancillary diagnostic tool. In urinary tract cytology, atypical urothelial cells equivocal for malignancy are a common diagnostic dilemma and multitarget FISH can help clarify such cells. Diagnosis of malignant mesothelioma remains one of the most challenging fields in effusion cytology, and ancillary FISH is useful in establishing the diagnosis. Fluorescence in situ hybridization is a morphology-based technique, and the prerequisite for reliable FISH results is a targeted evaluation of the cells in question (eg, cancer or atypical cells). Cytopathologists and cytotechnicians should therefore be involved in molecular testing in order to select the best material and to provide their morphologic expertise.

The campaign to DNA barcode all fishes, FISH-BOL.

Science.gov (United States)

Ward, R D; Hanner, R; Hebert, P D N

2009-02-01

FISH-BOL, the Fish Barcode of Life campaign, is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes. Analysis is targeting a 648 base pair region of the mitochondrial cytochrome c oxidase I (COI) gene. More than 5000 species have already been DNA barcoded, with an average of five specimens per species, typically vouchers with authoritative identifications. The barcode sequence from any fish, fillet, fin, egg or larva can be matched against these reference sequences using BOLD; the Barcode of Life Data System (http://www.barcodinglife.org). The benefits of barcoding fishes include facilitating species identification, highlighting cases of range expansion for known species, flagging previously overlooked species and enabling identifications where traditional methods cannot be applied. Results thus far indicate that barcodes separate c. 98 and 93% of already described marine and freshwater fish species, respectively. Several specimens with divergent barcode sequences have been confirmed by integrative taxonomic analysis as new species. Past concerns in relation to the use of fish barcoding for species discrimination are discussed. These include hybridization, recent radiations, regional differentiation in barcode sequences and nuclear copies of the barcode region. However, current results indicate these issues are of little concern for the great majority of specimens.

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).

Science.gov (United States)

Lecrenier, M C; Ledoux, Q; Berben, G; Fumière, O; Saegerman, C; Baeten, V; Veys, P

2014-07-17

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as "mad cow disease"), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application.

On the radioimmunological determination of native and heat denaturated protein

International Nuclear Information System (INIS)

Menzel, E.J.; Glatz, F.; Technische Univ., Vienna

1981-01-01

Precipitation radioimmunoassay, solid phase radioimmunoassay and passive hemagglutination were examined for their efficiency in the determination of native or denaturated soy proteins. Native as well as autoclaved soy protein could be determined quantitatively in the precipitation radioimmunoassay, using antisera directed against the native product. In the solid phase technique only the autoclaved soy protein could be detected with high sensitivity. In the passive hemagglutination reaction, no agglutination could be observed with erythrocytes coated with autoclaved soy protein. Only antisera against the denaturated (autoclaved) soy protein agglutinated these erythrocytes. (orig.) [de

Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

Directory of Open Access Journals (Sweden)

M.R. Hespanhol

2002-03-01

Full Text Available Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18 and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA by mouse peritoneal macrophages. We observed that a macrophages are able to recognize (bind to these red cells, b this interaction can be inhibited by denatured BSA in the fluid phase, c there is no phagocytosis of these particles by normal macrophages, d phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A.

[Inactivating Effect of Heat-Denatured Lysozyme on Murine Norovirus in Bread Fillings].

Science.gov (United States)

Takahashi, Michiko; Yasuda, Yuka; Takahashi, Hajime; Takeuchi, Akira; Kuda, Takashi; Kimura, Bon

2018-01-01

In this study, we investigated the viability of murine norovirus strain 1 (MNV-1), a surrogate for human norovirus, in bread fillings used for making stuffed buns and pastries. The inactivating effect of heat-denatured lysozyme, which was recently reported to have an antiviral effect, on MNV-1 contaminating the bread fillings was also examined. MNV-1 was inoculated into two types of fillings (chocolate cream, marmalade jam) at 4.5 log PFU/g, and the bread fillings were stored at 4℃ for 5 days. MNV-1 remained viable in the bread fillings during storage. However, addition of 1% heat-denatured lysozyme to the fillings resulted in a decrease of MNV-1 infectivity immediately after inoculation, in both fillings. On the fifth day of storage, MNV-1 infectivity was decreased by 1.2 log PFU/g in chocolate cream and by 0.9 log PFU/g in marmalade jam. Although the mechanism underlying the anti-norovirus effect of heat-denatured lysozyme has not been clarified, our results suggest that heat-denatured lysozyme can be used as an inactivating agent against norovirus in bread fillings.

Parasites modify sub-cellular partitioning of metals in the gut of fish

Energy Technology Data Exchange (ETDEWEB)

Oyoo-Okoth, Elijah, E-mail: elijaoyoo2009@gmail.com [Division of Environmental Health, School of Environmental Studies, Moi University, P.O. Box 3900, Eldoret (Kenya); Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Admiraal, Wim [Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Osano, Odipo [Division of Environmental Health, School of Environmental Studies, Moi University, P.O. Box 3900, Eldoret (Kenya); Kraak, Michiel H.S. [Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Gichuki, John; Ogwai, Caleb [Kenya Marine and Fisheries Research Institute, P.O. Box 1881, Kisumu (Kenya)

2012-01-15

Infestation of fish by parasites may influence metal accumulation patterns in the host. However, the subcellular mechanisms of these processes have rarely been studied. Therefore, this study determined how a cyprinid fish (Rastrineobola argentea) partitioned four metals (Cd, Cr, Zn and Cu) in the subcellular fractions of the gut in presence of an endoparasite (Ligula intestinalis). The fish were sampled along four sites in Lake Victoria, Kenya differing in metal contamination. Accumulation of Cd, Cr and Zn was higher in the whole body and in the gut of parasitized fish compared to non-parasitized fish, while Cu was depleted in parasitized fish. Generally, for both non-parasitized and parasitized fish, Cd, Cr and Zn partitioned in the cytosolic fractions and Cu in the particulate fraction. Metal concentrations in organelles within the particulate fractions of the non-parasitized fish were statistically similar except for Cd in the lysosome, while in the parasitized fish, Cd, Cr and Zn were accumulated more by the lysosome and microsomes. In the cytosolic fractions, the non-parasitized fish accumulated Cd, Cr and Zn in the heat stable proteins (HSP), while in the parasitized fish the metals were accumulated in the heat denatured proteins (HDP). On the contrary, Cu accumulated in the HSP in parasitized fish. The present study revealed specific binding of metals to potentially sensitive sub-cellular fractions in fish in the presence of parasites, suggesting interference with metal detoxification, and potentially affecting the health status of fish hosts in Lake Victoria.

Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

Energy Technology Data Exchange (ETDEWEB)

Worley, K.C.; Lindsay, E.A.; McCabe, E.R.B. [Baylor College of Medicine, Houston, TX (United States)] [and others

1995-07-17

Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring time-consuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosome deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. 17 refs., 2 figs.

Asexual Reproduction Does Not Apparently Increase the Rate of Chromosomal Evolution: Karyotype Stability in Diploid and Triploid Clonal Hybrid Fish (Cobitis, Cypriniformes, Teleostei)

Czech Academy of Sciences Publication Activity Database

Majtánová, Zuzana; Choleva, Lukáš; Symonová, Radka; Ráb, Petr; Kotusz, J.; Pekárik, L.; Janko, Karel

2016-01-01

Roč. 11, č. 1 (2016), e0146872-e0146872 E-ISSN 1932-6203 R&D Projects: GA ČR GAP506/10/1155; GA ČR GPP506/12/P857; GA ČR GA13-12580S Institutional support: RVO:67985904 Keywords : in-situ hybridization * fresh water fish * unisexual salamanders Subject RIV: EG - Zoology Impact factor: 2.806, year: 2016

Validation of interphase fluorescence in situ hybridization (iFISH for multiple myeloma using CD138 positive cells

Directory of Open Access Journals (Sweden)

Renata Kiyomi Kishimoto

2016-06-01

Full Text Available ABSTRACT BACKGROUND: Multiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance. Multiple myeloma differs from other hematologic malignancies due to a high fraction of low proliferating malignant plasma cells and the paucity of plasma cells in bone marrow aspiration samples, making cytogenetic analysis a challenge. An abnormal karyotype is found in only one-third of patients with multiple myeloma and interphase fluorescence in situ hybridization is the most useful test for studying the chromosomal abnormalities present in almost 90% of cases. However, it is necessary to study the genetic abnormalities in plasma cells after their identification or selection by morphology, immunophenotyping or sorting. Other challenges are the selection of the most informative FISH panel and determining cut-off levels for FISH probes. This study reports the validation of interphase fluorescence in situ hybridization using CD138 positive cells, according to proposed guidelines published by the European Myeloma Network (EMN in 2012. METHOD: Bone marrow samples from patients with multiple myeloma were used to standardize a panel of five probes [1q amplification, 13q14 deletion, 17p deletion, t(4;14, and t(14;16] in CD138+ cells purified by magnetic cell sorting. RESULTS: This test was validated with a low turnaround time and good reproducibility. Five of six samples showed genetic abnormalities. Monosomy/deletion 13 plus t(4;14 were found in two cases. CONCLUSION: This technique together with magnetic cell sorting is effective and can be used in the routine laboratory practice. In addition, magnetic cell sorting provides a pure plasma cell population that allows other molecular and genomic studies.

27 CFR 19.41 - Claims on spirits, denatured spirits, articles, or wines lost or destroyed in bond.

Science.gov (United States)

2010-04-01

..., denatured spirits, articles, or wines lost or destroyed in bond. 19.41 Section 19.41 Alcohol, Tobacco... DISTILLED SPIRITS PLANTS Taxes Claims § 19.41 Claims on spirits, denatured spirits, articles, or wines lost..., relating to the destruction or loss of spirits, denatured spirits, articles, or wines in bond, shall be...

Drying and denaturation characteristics of whey protein isolate in the presence of lactose and trehalose.

Science.gov (United States)

Haque, M Amdadul; Chen, Jie; Aldred, Peter; Adhikari, Benu

2015-06-15

The denaturation kinetics of whey protein isolate (WPI), in the presence and absence of lactose and trehalose, was quantified in a convective air-drying environment. Single droplets of WPI, WPI-lactose and WPI-trehalose were dried in conditioned air (2.5% RH, 0.5m/s air velocity) at two temperatures (65°C and 80°C) for 500s. The initial solid concentration of these solutions was 10% (w/v) in all the samples. Approximately 68% of WPI was denatured when it was dried in the absence of sugars. Addition of 20% trehalose prevented the irreversible denaturation of WPI at both temperatures. Thirty percent lactose was required to prevent denaturation of WPI at 65°C and the same amount of lactose protected only 70% of WPI from denaturation at 80°C. The secondary structures of WPI were found to be altered by the drying-induced stresses, even in the presence of 20% trehalose and 30% lactose. Copyright © 2014 Elsevier Ltd. All rights reserved.

Summer yield of fish in polyculture in Transkei, South Africa, using ...

African Journals Online (AJOL)

Keywords Aquaculture, fish prOduction, polyculture, integrated farming ... *To whom all correspondence should be addressed. Introduction. The use of animal wastes in fish production systems, has been widely practised for centuries in the Far Eastern countries ..... water without problems in fish-pond management. Hybrids.

The efficacy of denaturing actinide elements as a means of decreasing materials attractiveness

Energy Technology Data Exchange (ETDEWEB)

Hase, K.R.; Bathke, C.G. [Los Alamos National Laboratory: P.O. Box 1663, Los Alamos, NM 87545 (United States); Ebbinghaus, B.B.; Sleaford, B.W.; Robel, M. [Lawrence Livermore National Laboratory: P.O. Box 808, Livermore, CA 94551 (United States); Collins, B.A.; Prichard, A.W. [Pacific Northwest National Laboratory: P.O. Box 999, Richland, WA 99352 (United States)

2013-07-01

This study considers the concept of denaturing as applied to the actinide elements present in spent fuel as a means to reduce materials attractiveness. Highly attractive materials generally have low values of bare critical mass, heat content, and dose. To denature an attractive element, its spent-fuel isotopic composition (isotopic vector) is intentionally modified by introducing sufficient quantities of a significantly less attractive isotope to dilute the concentration of a highly attractive isotope so that the overall attractiveness of the element is reduced. The authors used FOM (Figure of Merit) formula as the material attractiveness metric for their parametric determination of the attractiveness of the Pu and U. Materials attractiveness needs to be considered in three distinct phases in the process to construct a nuclear explosive device (NED): the acquisition phase, processing phase, and utilization phase. The results show that denaturing uranium with {sup 238}U is actually an effective means of reducing the attractiveness. For uranium with a large minority of {sup 235}U, a mixture of 80% {sup 238}U to 20% {sup 235}U is required to reduce the attractiveness to low. For uranium with a large concentration of {sup 233}U, a mixture of 88% {sup 238}U to 12% {sup 233}U is required to reduce the attractiveness to low. The results also show that denaturing plutonium with {sup 238}Pu is less effective than denaturing uranium with {sup 238}U. Using {sup 238}Pu as the denaturing agent would require 80% or more by mass in order to reduce the attractiveness to low. No amount of {sup 240}Pu is enough to reduce the plutonium attractiveness below medium. The combination of {sup 238}Pu and {sup 240}Pu would require approximately 70% {sup 238}Pu and 25% {sup 240}Pu by mass to reduce the plutonium attractiveness to low.

The efficacy of denaturing actinide elements as a means of decreasing materials attractiveness

International Nuclear Information System (INIS)

Hase, K.R.; Bathke, C.G.; Ebbinghaus, B.B.; Sleaford, B.W.; Robel, M.; Collins, B.A.; Prichard, A.W.

2013-01-01

This study considers the concept of denaturing as applied to the actinide elements present in spent fuel as a means to reduce materials attractiveness. Highly attractive materials generally have low values of bare critical mass, heat content, and dose. To denature an attractive element, its spent-fuel isotopic composition (isotopic vector) is intentionally modified by introducing sufficient quantities of a significantly less attractive isotope to dilute the concentration of a highly attractive isotope so that the overall attractiveness of the element is reduced. The authors used FOM (Figure of Merit) formula as the material attractiveness metric for their parametric determination of the attractiveness of the Pu and U. Materials attractiveness needs to be considered in three distinct phases in the process to construct a nuclear explosive device (NED): the acquisition phase, processing phase, and utilization phase. The results show that denaturing uranium with 238 U is actually an effective means of reducing the attractiveness. For uranium with a large minority of 235 U, a mixture of 80% 238 U to 20% 235 U is required to reduce the attractiveness to low. For uranium with a large concentration of 233 U, a mixture of 88% 238 U to 12% 233 U is required to reduce the attractiveness to low. The results also show that denaturing plutonium with 238 Pu is less effective than denaturing uranium with 238 U. Using 238 Pu as the denaturing agent would require 80% or more by mass in order to reduce the attractiveness to low. No amount of 240 Pu is enough to reduce the plutonium attractiveness below medium. The combination of 238 Pu and 240 Pu would require approximately 70% 238 Pu and 25% 240 Pu by mass to reduce the plutonium attractiveness to low

27 CFR 19.32 - Assessment of tax on spirits, denatured spirits, or wines in bond which are lost, destroyed or...

Science.gov (United States)

2010-04-01

... spirits, denatured spirits, or wines in bond which are lost, destroyed or removed without authorization... spirits, denatured spirits, or wines in bond which are lost, destroyed or removed without authorization. When spirits, denatured spirits, or wines in bond are lost or destroyed (except spirits, denatured...

Nonsurgical Transurethral Radiofrequency Collagen Denaturation: Results at Three Years after Treatment

Directory of Open Access Journals (Sweden)

Denise M. Elser

2011-01-01

Full Text Available Objective. To assess treatment efficacy and quality of life in women with stress urinary incontinence 3 years after treatment with nonsurgical transurethral radiofrequency collagen denaturation. Methods. This prospective study included 139 women with stress urinary incontinence due to bladder outlet hypermobility. Radiofrequency collagen denaturation was performed using local anesthesia in an office setting. Assessments included incontinence quality of life (I-QOL and urogenital distress inventory (UDI-6 instruments. Results. In total, 139 women were enrolled and 136 women were treated (mean age, 47 years. At 36 months, intent-to-treat analysis (n=139 revealed significant improvements in quality of life. Mean I-QOL score improved 17 points from baseline (P=.0004, while mean UDI-6 score improved (decreased 19 points (P=.0005. Conclusions. Transurethral collagen denaturation is a low-risk, office-based procedure that results in durable quality-of-life improvements in a significant proportion of women for as long as 3 years.

The expression of miR-125b regulates angiogenesis during the recovery of heat-denatured HUVECs.

Science.gov (United States)

Zhou, Situo; Zhang, Pihong; Liang, Pengfei; Huang, Xiaoyuan

2015-06-01

In previous studies we found that miR-125b was down-regulated in denatured dermis of deep partial thickness burn patients. Moreover, miR-125b inhibited tumor-angiogenesis associated with the decrease of ERBB2 and VEGF expression in ovarian cancer cells and breast cancer cells, etc. In this study, we investigated the expression patterns and roles of miR-125b during the recovery of denatured dermis and heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burns in Sprague-Dawley rats and the heat-denatured cells (52°C, 35 s) were used for analysis. Western blot analysis and real-time PCR were applied to evaluate the expression of miR-125b and ERBB2 and VEGF. The ability of angiogenesis in heat-denatured HUVECs was analyzed by scratch wound healing and tube formation assay after pri-miR-125b or anti-miR-125b transfection. miR-125b expression was time-dependent during the recovery of heat-denatured dermis and HUVECs. Moreover, miR-125b regulated ERBB2 mRNA and Protein Expression and regulated angiogenesis association with regulating the expression of VEGF in heat-denatured HUVECs. Taken together our results show that the expression of miR-125b is time-dependent and miR-125b plays a regulatory role of angiogenesis during wound healing after burns. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

Science.gov (United States)

Cai, Junchao; Terasaki, Paul I; Zhu, Dong; Lachmann, Nils; Schönemann, Constanze; Everly, Matthew J; Qing, Xin

2016-02-01

We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, pantibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein denaturation and functional properties of Lenient Steam Injection heat treated whey protein concentrate

DEFF Research Database (Denmark)

Dickow, Jonatan Ahrens; Kaufmann, Niels; Wiking, Lars

2012-01-01

Whey protein concentrate (WPC) was heat treated by use of the novel heat treatment method of Lenient Steam Injection (LSI) to elucidate new functional properties in relation to heat-induced gelation of heat treated WPC. Denaturation was measured by both DSC and FPLC, and the results of the two...... methods were highly correlated. Temperatures of up to 90 °C were applicable using LSI, whereas only 68 °C could be reached by plate heat exchange before coagulation/fouling. Denaturation of whey proteins increased with increasing heat treatment temperature up to a degree of 30–35% denaturation at 90 °C...

How Chain Length and Charge Affect Surfactant Denaturation of Acyl Coenzyme A Binding Protein (ACBP)

DEFF Research Database (Denmark)

Andersen, Kell Kleiner; Otzen, Daniel

2009-01-01

maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the denaturation mechanism. ACBP denatures in two steps irrespective of surfactant chain length, but with increasing chain length, the potency of the denaturant rises more rapidly than the critical micelle......Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl...... constants increases linearly with denaturant concentration below the cmc but declines at higher concentrations. Both shortening chain length and decreasing micellar charge reduce the overall kinetics of unfolding and makes the dependence of unfolding rate constants on surfactant concentration more complex...

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length.

Science.gov (United States)

Kobayashi, Shinya; Fujii, Sotaro; Koga, Aya; Wakai, Satoshi; Matubayasi, Nobuyuki; Sambongi, Yoshihiro

2017-07-01

Reversible denaturation of Pseudomonas aeruginosa cytochrome c 551 (PAc 551 ) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc 551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc 551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc 551 , for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.

Detection of denitrification genes by in situ rolling circle amplification - fluorescence in situ hybridization (in situ RCA-FISH) to link metabolic potential with identity inside bacterial cells

DEFF Research Database (Denmark)

Hoshino, Tatsuhiko; Schramm, Andreas

2010-01-01

target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal-to-noise ratio but low detection frequency (up to 15% for single-copy genes and up to 43% for the multi-copy 16S rRNA gene...... as Candidatus Accumulibacter phosphatis by combining in situ RCA-FISH with 16S rRNA-targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA-FISH will allow to link metabolic potential with 16S rRNA (gene)-based identification of single microbial cells.......). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was demonstrated on activated sludge by the differential detection of two types of nirS-defined denitrifiers; one of them was identified...

On the Use of Potential Denaturing Agents for Ethanol in Direct Ethanol Fuel Cells

Directory of Open Access Journals (Sweden)

Domnik Bayer

2011-01-01

Full Text Available Acidic or alkaline direct ethanol fuel cells (DEFCs can be a sustainable alternative for power generation if they are fuelled with bio-ethanol. However, in order to keep the fuel cheap, ethanol has to be exempted from tax on spirits by denaturing. In this investigation the potential denaturing agents fusel oil, tert-butyl ethyl ether, and Bitrex were tested with regard to their compatibility with fuel cells. Experiments were carried out both in sulphuric acid and potassium hydroxide solution. Beside, basic electrochemical tests, differential electrochemical mass spectrometry (DEMS and fuel cell tests were conducted. It was found that fusel oil is not suitable as denaturing agent for DEFC. However, tert-butyl ethyl ether does not seem to hinder the ethanol conversion as much. Finally, a mixture of tert-butyl ethyl ether and Bitrex can be proposed as promising candidate as denaturing agent for use in acidic and alkaline DEFC.

Denaturation of membrane proteins and hyperthermic cell killing

NARCIS (Netherlands)

Burgman, Paulus Wilhelmus Johannes Jozef

1993-01-01

Summarizing: heat induced denaturation of membrane proteins is probably related to hyperthermic cell killing. Induced resistance of heat sensitive proteins seems to be involved in the development of thermotolerance. Although many questions remain still to be answered, it appears that HSP72, when

Comparison of Chromogenic In Situ Hybridization and Fluorescence In Situ Hybridization for the Evaluation of MDM2 Amplification in Adipocytic Tumors.

Science.gov (United States)

Mardekian, Stacey K; Solomides, Charalambos C; Gong, Jerald Z; Peiper, Stephen C; Wang, Zi-Xuan; Bajaj, Renu

2015-11-01

Atypical lipomatous tumor/well-differentiated liposarcoma (ALT-WDLPS) and dedifferentiated liposarcoma (DDLPS) are characterized cytogenetically by a 12q13-15 amplification involving the mouse double minute 2 (MDM2) oncogene. Fluorescence in situ hybridization (FISH) is used frequently to detect this amplification and aid with the diagnosis of these entities, which is difficult by morphology alone. Recently, bright-field in situ hybridization techniques such as chromogenic in situ hybridization (CISH) have been introduced for the determination of MDM2 amplification status. The present study compared the results of FISH and CISH for detecting MDM2 amplification in 41 cases of adipocytic tumors. Amplification was defined in both techniques as a MDM2/CEN12 ratio of 2 or greater. Eleven cases showed amplification with both FISH and CISH, and 26 cases showed no amplification with both methods. Two cases had discordant results between CISH and FISH, and two cases were not interpretable by CISH. CISH is advantageous for allowing pathologists to evaluate the histologic and molecular alterations occurring simultaneously in a specimen. Moreover, CISH is found to be more cost- and time-efficient when used with automation, and the signals do not quench over time. CISH technique is a reliable alternative to FISH in the evaluation of adipocytic tumors for MDM2 amplification. © 2014 Wiley Periodicals, Inc.

Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens

Science.gov (United States)

García-Caballero, Tomás; Grabau, Dorthe; Green, Andrew R; Gregory, John; Schad, Arno; Kohlwes, Elke; Ellis, Ian O; Watts, Sarah; Mollerup, Jens

2010-01-01

García-Caballero T, Grabau D, Green A R, Gregory J, Schad A, Kohlwes E, Ellis I O, Watts S & Mollerup J (2010) Histopathology56, 472–480 Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens Aims: Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual-colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual-colour CISH and FISH and compared the results. Methods and results: We found 100% agreement between HER2 status determined by FISH and dual-colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual-colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual-colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual-colour CISH and FISH was highly significant with an overall correlation coefficient (ρ) of 0.96. Conclusions: We conclude that dual-colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer. PMID:20459554

Effects of thermally induced denaturation on technological-functional properties of whey protein isolate-based films.

Science.gov (United States)

Schmid, M; Krimmel, B; Grupa, U; Noller, K

2014-09-01

This study examined how and to what extent the degree of denaturation affected the technological-functional properties of whey protein isolate (WPI)-based coatings. It was observed that denaturation affected the material properties of WPI-coated films significantly. Surface energy decreased by approximately 20% compared with native coatings. Because the surface energy of a coating should be lower than that of the substrate, this might result in enhanced wettability characteristics between WPI-based solution and substrate surface. Water vapor barrier properties increased by about 35% and oxygen barrier properties increased by approximately 33%. However, significant differences were mainly observed between coatings made of fully native WPI and ones with a degree of denaturation of 25%. Higher degrees of denaturation did not lead to further improvement of material properties. This observation offers cost-saving potential: a major share of denatured whey proteins may be replaced by fully native ones that are not exposed to energy-intensive heat treatment. Furthermore, native WPI solutions can be produced with higher dry matter content without gelatinizing. Hence, less moisture has to be removed through drying, resulting in reduced energy consumption. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Fish Vaccine Development and Use to Prevent Streptococcal Diseases

Science.gov (United States)

An important pathogen of tilapia, hybrid striped bass and trout raised in intensive aquaculture is Streptococcus sp., a cause of severe economic losses in the fish farming industry. Infected fish experience severe to moderate mortality due to Streptococcus iniae and/or S. agalactiae. The diseased ...

Comparison of Fluorescence In Situ Hybridization and Chromogenic In Situ Hybridization for Low and High Throughput HER2 Genetic Testing

DEFF Research Database (Denmark)

Poulsen, Tim S; Espersen, Maiken Lise Marcker; Kofoed, Vibeke

2013-01-01

cancer patients with HER2 immunohistochemistry (IHC) results scored as 0/1+, 2+, and 3+. HER2 genetic status was analysed using chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH). Scoring results were documented through digital image analysis. The cancer region...

Species integrity enhanced by a predation cost to hybrids in the wild

DEFF Research Database (Denmark)

Nilsson, P. A.; Hulthén, Kaj; Chapman, Ben B.

2017-01-01

Species integrity can be challenged, and even eroded, if closely related species can hybridize and produce fertile offspring of comparable fitness to that of parental species. The maintenance of newly diverged or closely related species therefore hinges on the establishment and effectiveness of pre...... barrier to gene flow in the wild. Cyprinid fishes commonly produce fertile, viable hybrid offspring and therefore make excellent study organisms to investigate ecological costs to hybrids. We electronically tagged two freshwater cyprinid fish species (roach Rutilus rutilus and bream Abramis brama...... to directly test for a predation cost to hybrids in the wild. Hybrid individuals were found significantly more susceptible to cormorant predation than individuals from either parental species. Such ecological selection against hybrids contributes to species integrity, and can enhance species diversification....

Spectral shift controlled reactors, denatured U-233/thorium cycle

International Nuclear Information System (INIS)

1978-05-01

This paper presents technical and economic data on the SSCR which may be of use in the International Fuel Cycle Evaluation Program to intercompare alternative nuclear systems. Included in this paper are data on the denatured U-233/thorium cycle. This cycle shows a proliferation advantage over more classical thorium fuel cycle (e.g., highly-enriched U-235/thorium or plutonium/thorium) due to the elimination of chemically-separable, concentrated fissile material from unirradiated nuclear fuel. The U-233 is denatured by mixing with depleted uranium to a concentration no greater than 12 w/o. An exogenous source of U-233 is assumed in this paper, since U-233 does not occur in nature and only a limited supply has been produced to date for research and development work

Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water

Science.gov (United States)

Siepi, Marialuisa; Politi, Jane; Dardano, Principia; Amoresano, Angela; De Stefano, Luca; Monti, Daria Maria; Notomista, Eugenio

2017-08-01

Fullerenes, allotropic forms of carbon, have very interesting pharmacological effects and engineering applications. However, a very low solubility both in organic solvents and water hinders their use. Fullerene C60, the most studied among fullerenes, can be dissolved in water only in the form of nanoparticles of variable dimensions and limited stability. Here the effect on the production of C60 nanoparticles by a native and denatured hen egg white lysozyme, a highly basic protein, has been systematically studied. In order to obtain a denatured, yet soluble, lysozyme derivative, the four disulfides of the native protein were reduced and exposed cysteines were alkylated by 3-bromopropylamine, thus introducing eight additional positive charges. The C60 solubilizing properties of the modified denatured lysozyme proved to be superior to those of the native protein, allowing the preparation of biocompatible highly homogeneous and stable C60 nanoparticles using lower amounts of protein, as demonstrated by dynamic light scattering, transmission electron microscopy and atomic force microscopy studies. This lysozyme derivative could represent an effective tool for the solubilization of other carbon allotropes.

Expanding probe repertoire and improving reproducibility in human genomic hybridization

Science.gov (United States)

Dorman, Stephanie N.; Shirley, Ben C.; Knoll, Joan H. M.; Rogan, Peter K.

2013-01-01

Diagnostic DNA hybridization relies on probes composed of single copy (sc) genomic sequences. Sc sequences in probe design ensure high specificity and avoid cross-hybridization to other regions of the genome, which could lead to ambiguous results that are difficult to interpret. We examine how the distribution and composition of repetitive sequences in the genome affects sc probe performance. A divide and conquer algorithm was implemented to design sc probes. With this approach, sc probes can include divergent repetitive elements, which hybridize to unique genomic targets under higher stringency experimental conditions. Genome-wide custom probe sets were created for fluorescent in situ hybridization (FISH) and microarray genomic hybridization. The scFISH probes were developed for detection of copy number changes within small tumour suppressor genes and oncogenes. The microarrays demonstrated increased reproducibility by eliminating cross-hybridization to repetitive sequences adjacent to probe targets. The genome-wide microarrays exhibited lower median coefficients of variation (17.8%) for two HapMap family trios. The coefficients of variations of commercial probes within 300 nt of a repetitive element were 48.3% higher than the nearest custom probe. Furthermore, the custom microarray called a chromosome 15q11.2q13 deletion more consistently. This method for sc probe design increases probe coverage for FISH and lowers variability in genomic microarrays. PMID:23376933

Isolation and characterization of acid-soluble collagen from the scales of marine fishes from Japan and Vietnam.

Science.gov (United States)

Minh Thuy, Le Thi; Okazaki, Emiko; Osako, Kazufumi

2014-04-15

Acid-soluble collagen (ASC) was successfully extracted from the scales of lizard fish (Saurida spp.) and horse mackerel (Trachurus japonicus) from Japan and Vietnam and grey mullet (Mugil cephalis), flying fish (Cypselurus melanurus) and yellowback seabream (Dentex tumifrons) from Japan. ASC yields were about 0.43-1.5% (on a dry weight basis), depending on the species. The SDS-PAGE profile showed that the ASCs were type I collagens, and consisted of two different α chains, α1 and α2, as well as a β component. ASC of horse mackerel from Vietnam contained a higher imino acid level than that from Japan. ASC denaturation temperature (Td) ranged from 26 to 29 °C, depending on fish species and imino acid content (p0.4M, regardless of fish type. Copyright © 2013 Elsevier Ltd. All rights reserved.

Role of chromogenic in situ hybridization (CISH) in the evaluation of HER2 status in breast carcinoma: comparison with immunohistochemistry and FISH.

Science.gov (United States)

Li-Ning-T, Elsa; Ronchetti, Ruben; Torres-Cabala, Carlos; Merino, Maria J

2005-10-01

We report our experience with Chromogenic in Situ Hybridization (CISH) for the evaluation of HER2 amplification on 55 cases of formalin-fixed, paraffin-embedded invasive breast carcinomas of different histology. All the results were corrected for chromosome 17 aneusomy and compared with immunohistochemistry (IHC); a subset of cases was compared to FISH. Thirty-one of 32 cases in which FISH and CISH were performed yielded the same results. CISH and IHC showed a good concordance in the 0/1+ and 3+ category, while a poor agreement with weakly protein overexpression was confirmed. Chromosome 17 analysis was necessary in cases with a low number of HER2 gene copies. CISH is a useful tool to evaluate breast cancer HER2 status that can be easily implemented in a laboratory of surgical pathology.

Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

Science.gov (United States)

Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

2015-01-01

In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the

Evaluation of denaturing gradient gel electrophoresis (DGGE) used ...

African Journals Online (AJOL)

Denaturing gradient gel electrophoresis (DGGE) is a powerful method used to study structure of bacterial communities, without cultivation, based on the diversity of the genes coding for ribosomal RNA. However, the results are strongly dependent on the respective target region of the used primer systems. Therefore, three ...

Genetic population structure of turbot ( Scophthalmus maximus L.) supports the presence of multiple hybrid zones for marine fishes in the transition zone between the Baltic Sea and the North Sea

DEFF Research Database (Denmark)

Eg Nielsen, Einar; Nielsen, P.H.; Meldrup, Dorte

2004-01-01

Sea, suggesting high gene flow among populations in these areas. In contrast, there was a sharp cline in genetic differentiation going from the low saline Baltic Sea to the high saline North Sea. The data were explained best by two divergent populations connected by a hybrid zone; however......, a mechanical mixing model could not be ruled out. A significant part of the genetic variance could be ascribed to variation among years within locality. Nevertheless, the population structure was relatively stable over time, suggesting that the observed pattern of genetic differentiation is biologically...... significant. This study suggests that hybrid zones are a common phenomenon for marine fishes in the transition area between the North Sea and the Baltic Sea and highlights the importance of using interspecific comparisons for inferring population structure in high gene flow species such as most marine fishes....

Introgressive hybridization as a promoter of genome reshuffling in natural homoploid fish hybrids (Cyprinidae, Leuciscinae)

Czech Academy of Sciences Publication Activity Database

Pereira, C. S.; Aboim, M. A.; Ráb, Petr; Collares-Pereira, M. J.

2014-01-01

Roč. 112, č. 3 (2014), s. 343-350 ISSN 0018-067X Institutional support: RVO:67985904 Keywords : comparative genome hybridization * hybrid zones * introgression Subject RIV: EG - Zoology Impact factor: 3.805, year: 2014

Isothermal chemical denaturation of large proteins: Path-dependence and irreversibility.

Science.gov (United States)

Wafer, Lucas; Kloczewiak, Marek; Polleck, Sharon M; Luo, Yin

2017-12-15

State functions (e.g., ΔG) are path independent and quantitatively describe the equilibrium states of a thermodynamic system. Isothermal chemical denaturation (ICD) is often used to extrapolate state function parameters for protein unfolding in native buffer conditions. The approach is prudent when the unfolding/refolding processes are path independent and reversible, but may lead to erroneous results if the processes are not reversible. The reversibility was demonstrated in several early studies for smaller proteins, but was assumed in some reports for large proteins with complex structures. In this work, the unfolding/refolding of several proteins were systematically studied using an automated ICD instrument. It is shown that: (i) the apparent unfolding mechanism and conformational stability of large proteins can be denaturant-dependent, (ii) equilibration times for large proteins are non-trivial and may introduce significant error into calculations of ΔG, (iii) fluorescence emission spectroscopy may not correspond to other methods, such as circular dichroism, when used to measure protein unfolding, and (iv) irreversible unfolding and hysteresis can occur in the absence of aggregation. These results suggest that thorough confirmation of the state functions by, for example, performing refolding experiments or using additional denaturants, is needed when quantitatively studying the thermodynamics of protein unfolding using ICD. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

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Jeangkhwoa, Pattraporn; Bandhaya, Achirapa; Umpunjun, Puangpaka; Chuenboonngarm, Ngarmnij; Panvisavas, Nathinee

2017-03-01

This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace. Copyright © 2016. Published by Elsevier B.V.

Chromosomal imbalances detected in primary bone tumors by comparative genomic hybridization and interphase fluorescence in situ hybridization

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Marcelo Razera Baruffi

2003-01-01

Full Text Available We applied a combination of comparative genomic hybridization (CGH and fluorescence in situ hybridization (FISH, to characterize the genetic aberrations in three osteosarcomas (OS and one Ewing's sarcoma. CGH identified recurrent chromosomal losses at 10p14-pter and gains at 8q22.3-24.1 in OS. Interphase FISH allowed to confirm 8q gain in two cases. A high amplification level of 11q12-qter was detected in one OS. The Ewing's sarcoma showed gain at 1p32-36.1 as the sole chromosome alteration. These studies demonstrate the value of molecular cytogenetic methods in the characterization of recurrent genomic alterations in bone tumor tissue.

Chromogenic in situ hybridization for Her-2/neu-oncogene in breast cancer: comparison of a new dual-colour chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization.

Science.gov (United States)

Mayr, Doris; Heim, Sibylle; Weyrauch, Kerstin; Zeindl-Eberhart, Evelyn; Kunz, Anne; Engel, Jutta; Kirchner, Thomas

2009-12-01

Her-2/neu testing is used as a marker for Herceptin therapy. The aim was to investigate new dual-colour chromogenic in situ hybridization (CISH), in a large number of breast carcinomas (n = 205) with DNA-specific dual-colour probes (ZytoVision, Bremerhaven, Germany) and to compare the results with immunohistochemistry (n = 205) and fluorescence in situ hybridization (FISH) (n = 129). Paraffin-embedded tissue of 205 patients was used. After immunohistochemistry with a focus on immunohistochemically uncertain cases, Her-2/neu amplification using dual-colour CISH (ZytoVision) was analysed. Validation by FISH was performed. The results were: immunohistochemistry, 27.8% with strong expression, 53.7% with uncertain overexpression and 18.5% with no expression; FISH, 25.6% amplified and 74.4% negative; CISH, 35.6% amplified, 62.9% negative and 1.5% not evaluable. Comparison of immunohistochemistry with CISH: CISH negative in 100% with immunohistochemistry 0/1+, amplified in 82.5% with immunohistochemistry 3+; 5.9% contradictory results: 4.4% immunohistochemistry 3+ and negative by CISH, 1.5% negative in immunohistochemistry but amplified by CISH; FISH (129 cases), 8.5% contradictory results to immunohistochemistry, 6.2% immunohistochemistry 3+ and negative by FISH, 2.3% negative by immunohistochemistry and amplified by FISH; comparison of CISH and FISH, 94.6% same results, 3.9% different ones, 1.6% CISH not analysable. CISH, using dual-colour probes (ZytoVision) is as good as FISH for Her-2/neu analysis. The few discrepant results are likely to be caused by polysomy or tumour heterogeneity.

U.S. leans toward denatured thorium cycle

International Nuclear Information System (INIS)

Smock, R.

1977-01-01

Denatured thorium appears to be the most promising among the nonproliferating alternatives to the plutonium cycle, which the Carter Administration is trying to cancel. Criteria for a better system include uranium utilization comparable to current light water reactors and minimal separation of fissile material into the waste stream. Comparisons with other systems conclude that thorium is preferable because it can lead to an acceptable fast breeder. The thorium cycle can be placed in energy centers for sensitive facilities and can also be introduced into ongoing light water systems. Reprocessing can be handled in the centers, where thorium can be mixed with plutonium for use in reactors within the center, while light water reactors operate on the outside. Any fuel leaving the center would be unsuitable for weapons. Later adaptation to in-center fast breeders will extend energy supplies, although a thorium breeder will be less efficient than a plutonium fast breeder. Denatured thorium is a technical answer to a complex political problem, but those in the nuclear industry see the U.S. goal of a nonproliferating fuel as futile in the light of world politics and breeder efforts in other countries

Partially folded intermediates during trypsinogen denaturation

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Martins N.F.

1999-01-01

Full Text Available The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

International Nuclear Information System (INIS)

Ahluwalia, Unnati; Deep, Shashank

2012-01-01

Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

Cold denaturation of the HIV-1 protease monomer

DEFF Research Database (Denmark)

Rösner, Heike Ilona; Caldarini, Martina; Prestel, Andreas

2017-01-01

The HIV-1-protease is a complex protein which in its active form adopts a homodimer dominated by -sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1-protease which is populated above 0ºC and therefore directly accessible to various spectroscopic approac...

Annual trends in catchability and fish stock assessments

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Marchal Paul

2003-04-01

Full Text Available A key assumption of many fish stock assessment models is that catchability is constant over time. We assume here that trends in catchability may occur through fishing power creeping. The tuning fleets, which are prone to fishing power development, may be identified using the Hybrid method. A range of catchability trends, including values derived from the Hybrid method, is then implemented to standardise the fishing effort of some tuning fleets used in the stock assessments performed by XSA (eXtended Survivors Analysis. Stocks being assessed are the North Sea cod, saithe, plaice and sole. The performances of the new and traditional XSA assessments are compared using criteria based on the precision of catchability estimates, stationarity of Log-catchability residuals and retrospective patterns relative to fishing mortality, spawning stock biomass and recruitment estimates. The performances of the North Sea cod, plaice and sole assessments could be enhanced by accounting for an overall annual increase in the catchability of some of the tuning fleets. No significant trends could be detected in the catchability of the tuning fleets relative to the assessment of the North Sea saithe. By contrast with the traditional assessment, the spawning biomass of cod is expected not to have increased between 1997 and 1998, while the fishing mortality of sole is expected to have increased over the same period.

Asexual Reproduction Does Not Apparently Increase the Rate of Chromosomal Evolution: Karyotype Stability in Diploid and Triploid Clonal Hybrid Fish (Cobitis, Cypriniformes, Teleostei).

Science.gov (United States)

Majtánová, Zuzana; Choleva, Lukáš; Symonová, Radka; Ráb, Petr; Kotusz, Jan; Pekárik, Ladislav; Janko, Karel

2016-01-01

Interspecific hybridization, polyploidization and transitions from sexuality to asexuality considerably affect organismal genomes. Especially the last mentioned process has been assumed to play a significant role in the initiation of chromosomal rearrangements, causing increased rates of karyotype evolution. We used cytogenetic analysis and molecular dating of cladogenetic events to compare the rate of changes of chromosome morphology and karyotype in asexually and sexually reproducing counterparts in European spined loach fish (Cobitis). We studied metaphases of three sexually reproducing species and their diploid and polyploid hybrid clones of different age of origin. The material includes artificial F1 hybrid strains, representatives of lineage originated in Holocene epoch, and also individuals of an oldest known age to date (roughly 0.37 MYA). Thereafter we applied GISH technique as a marker to differentiate parental chromosomal sets in hybrids. Although the sexual species accumulated remarkable chromosomal rearrangements after their speciation, we observed no differences in chromosome numbers and/or morphology among karyotypes of asexual hybrids. These hybrids possess chromosome sets originating from respective parental species with no cytogenetically detectable recombinations, suggesting their integrity even in a long term. The switch to asexual reproduction thus did not provoke any significant acceleration of the rate of chromosomal evolution in Cobitis. Asexual animals described in other case studies reproduce ameiotically, while Cobitis hybrids described here produce eggs likely through modified meiosis. Therefore, our findings indicate that the effect of asexuality on the rate of chromosomal change may be context-dependent rather than universal and related to particular type of asexual reproduction.

Asexual Reproduction Does Not Apparently Increase the Rate of Chromosomal Evolution: Karyotype Stability in Diploid and Triploid Clonal Hybrid Fish (Cobitis, Cypriniformes, Teleostei.

Directory of Open Access Journals (Sweden)

Zuzana Majtánová

Full Text Available Interspecific hybridization, polyploidization and transitions from sexuality to asexuality considerably affect organismal genomes. Especially the last mentioned process has been assumed to play a significant role in the initiation of chromosomal rearrangements, causing increased rates of karyotype evolution. We used cytogenetic analysis and molecular dating of cladogenetic events to compare the rate of changes of chromosome morphology and karyotype in asexually and sexually reproducing counterparts in European spined loach fish (Cobitis. We studied metaphases of three sexually reproducing species and their diploid and polyploid hybrid clones of different age of origin. The material includes artificial F1 hybrid strains, representatives of lineage originated in Holocene epoch, and also individuals of an oldest known age to date (roughly 0.37 MYA. Thereafter we applied GISH technique as a marker to differentiate parental chromosomal sets in hybrids. Although the sexual species accumulated remarkable chromosomal rearrangements after their speciation, we observed no differences in chromosome numbers and/or morphology among karyotypes of asexual hybrids. These hybrids possess chromosome sets originating from respective parental species with no cytogenetically detectable recombinations, suggesting their integrity even in a long term. The switch to asexual reproduction thus did not provoke any significant acceleration of the rate of chromosomal evolution in Cobitis. Asexual animals described in other case studies reproduce ameiotically, while Cobitis hybrids described here produce eggs likely through modified meiosis. Therefore, our findings indicate that the effect of asexuality on the rate of chromosomal change may be context-dependent rather than universal and related to particular type of asexual reproduction.

Apparent digestibility of Asian carp and common carp-derived fish meals in feeds for hybrid striped bass Morone saxatilis female x M. chrysops male and rainbow trout Oncorhynchus mykiss

Science.gov (United States)

Apparent digestibility coefficients (ADCs) of nutrients (crude protein, amino acids, crude lipid, fatty acids, and minerals) were determined for fish meals derived from menhaden, Asian carp (combination of silver and bighead carps), and common carp in feeds for hybrid striped bass and rainbow trout....

Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

Science.gov (United States)

Bian, Liujiao; Ji, Xu

2014-01-01

Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

KAUST Repository

Harrison, H.B.

2014-04-24

Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo-Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co-amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent-offspring relationships in natural populations, with over 99.6% accuracy in parent-offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28-96% of loci. The multiplex PCRs developed here provide a reliable and cost-effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species. 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

KAUST Repository

Harrison, H.B.; Feldheim, K.A.; Jones, G.P.; Ma, K.; Mansour, H.; Perumal, S.; Williamson, D.H.; Berumen, Michael L.

2014-01-01

Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo-Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co-amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent-offspring relationships in natural populations, with over 99.6% accuracy in parent-offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28-96% of loci. The multiplex PCRs developed here provide a reliable and cost-effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species. 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

HER2 Gene Amplification Testing by Fluorescent In Situ Hybridization (FISH): Comparison of the ASCO-College of American Pathologists Guidelines With FISH Scores Used for Enrollment in Breast Cancer International Research Group Clinical Trials.

Science.gov (United States)

Press, Michael F; Sauter, Guido; Buyse, Marc; Fourmanoir, Hélène; Quinaux, Emmanuel; Tsao-Wei, Denice D; Eiermann, Wolfgang; Robert, Nicholas; Pienkowski, Tadeusz; Crown, John; Martin, Miguel; Valero, Vicente; Mackey, John R; Bee, Valerie; Ma, Yanling; Villalobos, Ivonne; Campeau, Anaamika; Mirlacher, Martina; Lindsay, Mary-Ann; Slamon, Dennis J

2016-10-10

Purpose ASCO and the College of American Pathologists (ASCO-CAP) recently recommended further changes to the evaluation of human epidermal growth factor receptor 2 gene (HER2) amplification by fluorescent in situ hybridization (FISH). We retrospectively assessed the impact of these new guidelines by using annotated Breast Cancer International Research Group (BCIRG) -005, BCIRG-006, and BCIRG-007 clinical trials data for which we have detailed outcomes. Patients and Methods The HER2 FISH status of BCIRG-005/006/007 patients with breast cancers was re-evaluated according to current ASCO-CAP guidelines, which designates five different groups according to HER2 FISH ratio and average HER2 gene copy number per tumor cell: group 1 (in situ hybridization [ISH]-positive): HER2-to-chromosome 17 centromere ratio ≥ 2.0, average HER2 copies ≥ 4.0; group 2 (ISH-positive): ratio ≥ 2.0, copies < 4.0; group 3 (ISH-positive): ratio < 2.0, copies ≥ 6.0; group 4 (ISH-equivocal): ratio < 2.0, copies ≥ 4.0 and < 6.0; and group 5 (ISH-negative): ratio < 2.0, copies < 4.0. We assessed correlations with HER2 protein, clinical outcomes by disease-free survival (DFS) and overall survival (OS) and benefit from trastuzumab therapy (hazard ratio [HR]). Results Among 10,468 patients with breast cancers who were successfully screened for trial entry, 40.8% were in ASCO-CAP ISH group 1, 0.7% in group 2; 0.5% in group 3, 4.1% in group 4, and 53.9% in group 5. Distributions were similar in screened compared with accrued subpopulations. Among accrued patients, FISH group 1 breast cancers were strongly correlated with immunohistochemistry 3+ status (P < .0001), whereas groups 2, 3, 4, and 5 were not; however, groups 2, 4 and, 5 were strongly correlated with immunohistochemistry 0/1+ status (all P < .0001), whereas group 3 was not. Among patients accrued to BCIRG-005, group 4 was not associated with significantly worse DFS or OS compared with group 5. Among patients accrued to BCIRG-006, only

Membrane bridging and hemifusion by denaturated Munc18.

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Yi Xu

Full Text Available Neuronal Munc18-1 and members of the Sec1/Munc18 (SM protein family play a critical function(s in intracellular membrane fusion together with SNARE proteins, but the mechanism of action of SM proteins remains highly enigmatic. During experiments designed to address this question employing a 7-nitrobenz-2-oxa-1,3-diazole (NBD fluorescence de-quenching assay that is widely used to study lipid mixing between reconstituted proteoliposomes, we observed that Munc18-1 from squid (sMunc18-1 was able to increase the apparent NBD fluorescence emission intensity even in the absence of SNARE proteins. Fluorescence emission scans and dynamic light scattering experiments show that this phenomenon arises at least in part from increased light scattering due to sMunc18-1-induced liposome clustering. Nuclear magnetic resonance and circular dichroism data suggest that, although native sMunc18-1 does not bind significantly to lipids, sMunc18-1 denaturation at 37 °C leads to insertion into membranes. The liposome clustering activity of sMunc18-1 can thus be attributed to its ability to bridge two membranes upon (perhaps partial denaturation; correspondingly, this activity is hindered by addition of glycerol. Cryo-electron microscopy shows that liposome clusters induced by sMunc18-1 include extended interfaces where the bilayers of two liposomes come into very close proximity, and clear hemifusion diaphragms. Although the physiological relevance of our results is uncertain, they emphasize the necessity of complementing fluorescence de-quenching assays with alternative experiments in studies of membrane fusion, as well as the importance of considering the potential effects of protein denaturation. In addition, our data suggest a novel mechanism of membrane hemifusion induced by amphipathic macromolecules that does not involve formation of a stalk intermediate.

Dual-colour chromogenic in-situ hybridization is a potential alternative to fluorescence in-situ hybridization in HER2 testing.

Science.gov (United States)

Hwang, Cheng-Cheng; Pintye, Mariann; Chang, Liang-Che; Chen, Huang-Yang; Yeh, Kun-Yan; Chein, Hui-Ping; Lee, Nin; Chen, Jim-Ray

2011-11-01

Dual-colour chromogenic in-situ hybridization (dc-CISH) is an emerging methodology for characterizing genomic alterations. This study was aimed at evaluating the performance of a dc-CISH kit (ZytoVision) in determining human epidermal growth factor receptor 2 (HER2) status in breast cancer. Two hundred and twenty-eight invasive breast carcinomas arranged in tissue microarrays were analysed in parallel with dc-CISH, fluorescence in-situ hybridization (FISH), and immunohistochemistry. Of 227 tumours with available FISH and dc-CISH results, HER2 amplification and non-amplification were detected in 49 (21.6%) and 178 (78.4%) tumours, respectively, by both assays. The concordance between dc-CISH and FISH results showed 100% agreement (κ-coefficient=1.00). Immunohistochemically, 162 (71%), 25 (11.0%) and 41 (18%) tumours were scored 0/1+, 2+, and 3+, respectively. The corresponding results with both FISH and dc-CISH demonstrated HER2 amplification in two (3.2%), nine (36%) and 38 (93%) tumours, respectively. Complete consensus among these three methods was observed in 197 cases, representing 98% of all 3+ and 0/1+ tumours (κ-coefficient=0.92). Confirmatory testing of 25 2+ tumours showed complete consensus between FISH and dc-CISH. dc-CISH is a promising alternative to FISH in HER2 testing, and the single-institute incidence of HER2 amplification in breast cancer in Taiwan is 21.2%. © 2011 Blackwell Publishing Limited.

Perulernaea gamitanae (Copepoda: Lernaeidae parasitizing tambaqui (Colossoma macropomum (Characidae and the hybrids tambacu and tambatinga, cultured in northern Brazil

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M. Tavares-Dias

2011-08-01

Full Text Available The infestation rate in Colossoma macropomum, hybrid tambacu (C. macropomum x Piaractus mesopotamicus and hybrid tambatinga (C. macropomum x Piaractus brachypomum with Perulernaea gamitanae Thatcher and Paredes, 1985 from two fish farms in Amapá State, Brazil was studied. Lernaeid parasites (n=2887 were collected mainly on the tongue and the mouth cavity and also on cartilage of gill arches and filaments. Inflammation and fibrous nodules were observed on the attachment sites of the parasites. The infestation rate varied according to the fish farm and host. The prevalence of P. gamitanae was of 100% in hosts from one fish farm and was lower in the other fish farm. Higher intensity of P. gamitanae occurred in hybrids tambacu and tambatinga, but despite the high prevalence its intensity was moderate. This is the first report on epidemiology of P. gamitanae in cultured fishes from Brazilian Amazonia, and the occurrence of this crustacean parasite in two new hosts, the hybrids tambacu and tambatinga.

Insight into the effect mechanism of urea-induced protein denaturation by dielectric spectroscopy.

Science.gov (United States)

Zhang, Cancan; Yang, Man; Zhao, Kongshuang

2017-12-06

Dielectric relaxation spectroscopy was applied to study how urea affects the phase transition of a thermosensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), which has been widely used as a protein model. It was found that there is a pronounced relaxation near 10 GHz for the ternary system of PNIPAM in urea aqueous solution. The temperature dependence of dielectric parameters indicates that urea can reduce the lower critical solution temperature (LCST) of PNIPAM, i.e., stabilize the globule state of PNIPAM and collapse the PNIPAM chains. Based on our results, the interaction mechanism of urea on the conformational transition of PNIPAM was presented: urea replaces water molecules directly bonding with PNIPAM and acts as the bridging agent for the adjacent side chains of PNIPAM. Accordingly, the mechanism with which urea denatures protein was deduced. In addition, it is worth mentioning that, from the temperature dependence of the dielectric parameters obtained in the presence of urea, an interesting phenomenon was found in which the effect of urea on PNIPAM seems to take 2 M as a unit. This result may be the reason why urea and TMAO exit marine fishes at a specific ratio of 2 : 1.

Dynamics of Ionic Liquid-Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions toward Renaturation.

Science.gov (United States)

Singh, Upendra Kumar; Patel, Rajan

2018-05-25

In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C 8 mim][Cl], and 1-decyl-3-methylimidazolium chloride [C 10 mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was correlated to understand the mechanism of the conformational stability of proteins in aqueous solutions of ILs. The results showed that the long chain ILs having the [C 8 mim] + and [C 10 mim] + cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C 10 mim] + cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C 8 mim] + cation, which is known to be compatible for protein stability, shows slight refolding and forms a different transition state. The lifetime results show successful refolding of alkaline-denatured h-cyt c by both of the ILs, however, more refolding was observed in the case of [C 10 mim][Cl], and this was correlated with the fast and medium lifetimes (τ 1 and τ 2 ) obtained, which show an increase accompanied by an increase in secondary structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of the MG state by [C 10 mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C 10 mim] + cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.

Heat denaturation of soy glycinin : Influence of pH and ionic strength on molecular structure

NARCIS (Netherlands)

Lakemond, C.M.M.; Jongh, de H.H.J.; Hessing, M.; Gruppen, H.; Voragen, A.G.J.

2000-01-01

The 7S/11S glycinin equilibrium as found in Lakemond et al. (J. Agric. Food Chem. 2000, 48, xxxx-xxxx) at ambient temperatures influences heat denaturation. It is found that the 7S form of glycinin denatures at a lower temperature than the 11S form, as demonstrated by a combination of calorimetric

Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride.

Science.gov (United States)

Jha, Santosh Kumar; Marqusee, Susan

2014-04-01

Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).

Determination of 24 personal care products in fish bile using hybrid solvent precipitation and dispersive solid phase extraction cleanup with ultrahigh performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry.

Science.gov (United States)

Yao, Li; Lv, Yin-Zhi; Zhang, Li-Juan; Liu, Wang-Rong; Zhao, Jian-Liang; Liu, You-Sheng; Zhang, Qian-Qian; Ying, Guang-Guo

2018-05-25

Personal care products (PCPs) are ubiquitous in aquatic environments owing to the continuous discharge of domestic wastewater from highly urbanized regions. These PCPs can be adsorbed by fish and thereafter usually enter the bile of the fish through biliary excretion. In this study, a sensitive method based on a combination of hybrid solvent precipitation and dispersive solid phase extraction (d-SPE) purification was developed to simultaneously extract and detect 24 PCPs, namely, 16 biocides, 4 synthetic musks, and 4 benzotriazoles, from fish bile. Hybrid precipitation on solid phase extraction (SPE) tubes was applied to remove phospholipids and proteins, and a d-SPE procedure was used for further purification. The extraction solvents for the hybrid precipitation/SPE tubes and d-SPE materials were optimized. The method performance for bile samples both with and without enzyme hydrolysis using β-glucuronidase/aryl-sulfatase were validated. The 24 PCPs in fish bile were spiked with standard concentrations of 10 ng/mL, 20 ng/mL, 100 ng/mL, and 200 ng/mL to evaluate recoveries, which ranged from 70 to 120% for 16, 16, 22, and 21 analytes with hydrolysis, respectively, and 70-120% for 14, 15, 23, and 23 analytes without hydrolysis, respectively. The quantification limits for target PCPs were in the range 0.26-7.38 ng/mL [excluding musk xylene (MX) and musk ketone (MK)] and 0.20-9.48 ng/mL (excluding MX and MK) for bile samples with and without enzyme hydrolysis, respectively. After enzyme hydrolysis, 12 PCPs were detected in bile from fish collected from the Yangtze River, with a maximum detected concentration of 460 ng/mL, for triclosan (TCS). The hydrolysis reaction indicated that high percentages of glucuronide and sulfate metabolites for some PCPs, i.e. four parabens and TCS, existed in the bile. Copyright © 2018 Elsevier B.V. All rights reserved.

RNA detection in situ with FISH-STICs.

Science.gov (United States)

Sinnamon, John R; Czaplinski, Kevin

2014-02-01

The ability to detect RNA molecules in situ has long had important applications for molecular biological studies. Enzyme or dye-labeled antisense in vitro runoff transcripts and synthetic oligodeoxynucleotides (ODN) both have a proven track record of success, but each of these also has scientific and practical drawbacks and limitations to its use. We devised a means to use commercially synthesized oligonucleotides as RNA-FISH probes without further modification and show that such probes work well for detection of RNA in cultured cells. This approach can bind a high concentration of fluorescent ODN to a short stretch of an RNA using commercial DNA synthesis outlets available to any laboratory. We call this approach for creating in situ hybridization probes Fluorescence In Situ Hybridization with Sequential Tethered and Intertwined ODN Complexes (FISH-STICs). We demonstrate that one FISH-STIC probe can detect mRNA molecules in culture, and that probe detection can be improved by the addition of multiple probes that can be easily adapted for robust mRNA quantification. Using FISH-STICs, we demonstrate a nonoverlapping distribution for β-actin and γ-actin mRNA in cultured fibroblasts, and the detection of neuron-specific transcripts within cultured primary hippocampal neurons.

Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia

Directory of Open Access Journals (Sweden)

KAVEH RAHMANIFARAH

2014-05-01

Full Text Available Rahmanifarah K, Shabanpour B, Shaviklo AR, Aalami M. 2014. Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia. Nusantara Bioscience 6: 1-6. The effect of protein extraction procedures (leached mince and heated suspension on selected properties of fish protein powder (proximate composition, pH, color, density, viscosity, fat adsorption, emulsifying capacity, emulsifying stability, foaming capacity, foaming stability, WBC, protein solubility in water, hygroscopicity, Trichloroacetic acid (TCA-soluble peptides and free sulfhydryl groups was investigated. Results showed that Fish protein powder (FPP produced by leaching mince (LM have higher protein, moisture, ash, pH, L*, viscosity, emulsion capacity, emulsion stability, foam capacity, foam stability, water binding capacity (WBC, protein solubility, hygroscopicity, TCA soluble peptides and free sulfhydryl group content than heated suspension (HS (P0.05. Overall, it was observed that high temperature during heating of suspension in HS method makes possible protein denaturation and aggregation. Consequently, based on functional, chemical and physical properties, extraction of fish protein by leaching process was found to be suitable for the production of fish protein powder.

The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

Science.gov (United States)

Fischer, T H

1983-01-01

To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy. PMID:6309139

Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications.

Science.gov (United States)

Cui, Chenghua; Shu, Wei; Li, Peining

2016-01-01

Fluorescence in situ hybridization (FISH) is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes, and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains, and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbias and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH) allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH) using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells DNA and RNA FISH

Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications

Directory of Open Access Journals (Sweden)

Chenghua Cui

2016-09-01

Full Text Available Fluorescence in situ hybridization (FISH is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbials and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells

Comparative analysis of the aquaculture potential of hybrid Tilapia ...

African Journals Online (AJOL)

, on the growth rate, feeding efficiency and mortality rates of hybrid tilapia — Tilapia zillii (male) x T. guineensis (female) — was evaluated for 233 days. Fish of average weight 12.59g were stocked at a density of 20 fish m–³ and were fed a 30% ...

In Situ Hybridization Pada Kanker Payudara

OpenAIRE

Diah Witari, Ni Putu

2014-01-01

Kesulitan yang dijumpai pada penanganan kanker payudara adalah terjadinya kekambuhan atau relaps. Deteksi status HER2 pada pasien merupakan salah satu upaya untuk mendeteksi terjadinya relaps dan juga untuk menentukan jenis terapi yang ada diberikan. Ekspresi protein HER2 dapat dideteksi dengan immunohistochemistry (IHC), sedangkan mutasi gen HER2 dapat dideteksi dengan teknik in situ hybridization baik berupa fluorescence in situ hybridization (FISH) ataupun chromogenic in situ hy...

Denaturing of plutonium by transmutation of minor-actinides for enhancement of proliferation resistance

International Nuclear Information System (INIS)

Sagara, Hiroshi; Saito, Masaki; Peryoga, Yoga; Ezoubtchenko, Alexey; Takivayev, Alan

2005-01-01

Feasibility study for the plutonium denaturing by utilizing minor-actinide transmutation in light water reactors has been performed. And the intrinsic feature of proliferation resistance of plutonium has been discussed based on IAEA's publication and Kessler's proposal. The analytical results show that not only 238 Pu but also other plutonium isotopes with even-mass-number have very important role for denaturing of plutonium due to their relatively large critical mass and noticeably high spontaneous fission neutron generation. With the change of the minor-actinide doping ratio in U-Pu mix oxide fuel and moderator to fuel ratio, it is found that the reactor-grade plutonium from conventional light water reactors can be denatured to satisfy the proliferation resistance criterion based on the Kessler's proposal but not to be sufficient for the criterion based on IAEA's publication. It has been also confirmed that all the safety coefficients take negative value throughout the irradiation. (author)

Thermal denaturation of sunflower globulins in low moisture conditions

International Nuclear Information System (INIS)

Rouilly, A.; Orliac, O.; Silvestre, F.; Rigal, L.

2003-01-01

DSC analysis in pressure resisting pans of sunflower oil cake makes appear the endothermic peak of sunflower globulins denaturation. Its temperature decreases from 189.5 to 119.9 deg. C while the corresponding enthalpy increases from 2.6 to 3.3 J/g of sample, or from 6.7 to 12.2 J/g of dry protein, when the samples moisture content varies from 0 to 30.0% of the total weight. The plot of the denaturation temperature versus the moisture content is not linear but has a rounded global shape and seems to follow the hydration behavior of the proteins, modeled with the sorption isotherm. As it can be seen on scanning electron microscopy (SEM) micrographs, protein corpuscles 'melt' after such a thermal treatment and large aggregates form by coagulation. Moisture dependence of the 'fusion' temperature of native proteic organization, in low moisture conditions, offers so a new characterization method for the use of vegetable proteins in agro-materials

Thermal denaturation of sunflower globulins in low moisture conditions

Energy Technology Data Exchange (ETDEWEB)

Rouilly, A.; Orliac, O.; Silvestre, F.; Rigal, L

2003-03-05

DSC analysis in pressure resisting pans of sunflower oil cake makes appear the endothermic peak of sunflower globulins denaturation. Its temperature decreases from 189.5 to 119.9 deg. C while the corresponding enthalpy increases from 2.6 to 3.3 J/g of sample, or from 6.7 to 12.2 J/g of dry protein, when the samples moisture content varies from 0 to 30.0% of the total weight. The plot of the denaturation temperature versus the moisture content is not linear but has a rounded global shape and seems to follow the hydration behavior of the proteins, modeled with the sorption isotherm. As it can be seen on scanning electron microscopy (SEM) micrographs, protein corpuscles 'melt' after such a thermal treatment and large aggregates form by coagulation. Moisture dependence of the 'fusion' temperature of native proteic organization, in low moisture conditions, offers so a new characterization method for the use of vegetable proteins in agro-materials.

FISH and PNA FISH for the diagnosis of Q fever endocarditis and vascular infections.

Science.gov (United States)

Prudent, Elsa; Lepidi, Hubert; Angelakis, Emmanouil; Raoult, Didier

2018-06-13

Purpose. Endocarditis and vascular infections are common manifestations of persistent localized infection due to Coxiella burnetii and recently, fluorescence in situ hybridization (FISH) was proposed as an alternative tool for their diagnosis. In this study, we evaluated the efficiency of FISH in a series of valve and vascular samples infected by C. burnetii. Methods. We tested 23 C. burnetii -positive valves and thrombus samples obtained from patients with Q fever endocarditis. Seven aneurysms and thrombus specimens were retrieved from patients with Q fever vascular infection. Samples were analyzed by culture, immunochemistry and FISH with oligonucleotide and PNA probes targeting C. burnetii -specific 16S ribosomal RNA sequences. Results. Immunohistochemical analysis was positive for five (17%) samples with significantly more copies of C. burnetii DNA than the negative ones ( p= 0.02). FISH was positive for 13 (43%) samples and presented 43% and 40% sensitivity compared to qPCR and culture, respectively. PNA FISH detected C. burnetii in 18 (60%) samples and presented 60% and 55% sensitivity compared to qPCR and culture, respectively. Immunohistochemistry had 38% and 28% sensitivity compared to FISH and PNA FISH, respectively. Samples found positive by both immunohistochemistry and PNA FISH contained significantly more copies of C. burnetii DNA than the negative ones ( p= 0.03). Finally, PNA FISH was more sensitive than FISH (60% versus 43%) for the detection of C. burnetii Conclusion. We provide evidence that PNA FISH and FISH are important assays for the diagnosis of C. burnetii endocarditis and vascular infections. Copyright © 2018 American Society for Microbiology.

Evaluation of gasoline-denatured ethanol as a carbon source for denitrification.

Science.gov (United States)

Kazasi, Anna; Boardman, Gregory D; Bott, Charles B

2013-06-01

In this study concerning denitrification, the performance of three carbon sources, methanol (MeOH), ethanol (EtOH) and gasoline-denatured ethanol (dEtOH), was compared and evaluated on the basis of treatment efficiency, inhibition potential and cost. The gasoline denaturant considered here contained mostly aliphatic compounds and little of the components that typically boost the octane rating, such as benzene, toluene, ethylbenzene and xylenes. Results were obtained using three lab-scale SBRs operated at SRT of 12.0 +/- 0.9 days. After biomass was acclimated, denitrification rates with dEtOH were similar to those of EtOH (201 +/- 50 and 197 +/- 28 NO3-N/g MLVSS x d, respectively), and higher than those of MeOH (165 +/- 49 mg NO3-N/g MLVSS x d). The denaturant did not affect biomass production, nitrification or denitrification. Effluent soluble COD concentrations were always less than the analytical detection limit. Although the cost of dEtOH ($2.00/kg nitrate removed) was somewhat higher than that of methanol ($1.63/kg nitrate removed), the use of dEtOH is very promising and utilities will have to decide if it is worth paying a little extra to take advantage of its benefits.

A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization (FISH

Directory of Open Access Journals (Sweden)

Jinzhong FU

2009-04-01

Full Text Available The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA. Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution.

Accuracy of HER2 status determination on breast core-needle biopsies (immunohistochemistry, FISH, CISH and SISH vs FISH).

Science.gov (United States)

Arnould, Laurent; Roger, Pascal; Macgrogan, Gaëtan; Chenard, Marie-Pierre; Balaton, André; Beauclair, Sophie; Penault-Llorca, Frederique

2012-05-01

Preoperative breast cancer diagnosis on core biopsies has become a standard of care in many countries. Controversies exist concerning the accuracy of HER2 testing on biopsies as compared with surgical specimens, and few data exist concerning the use of emerging technologies such as bright-field in-situ hybridization in such a setting. A French multicenter, cross-sectional, histopathological study assessed the concordance of HER2 status determined by immunohistochemistry and silver (SISH) or chromogenic in-situ hybridization (CISH) on core-needle biopsies with HER2 status determined by fluorescence in-situ hybridization (FISH) on surgical specimens. The concordance between biopsy and operative results was also assessed for each method. We studied 260 breast tumors from 24 centers between April 2003 and August 2009. Excellent concordance (κ: 0.92-0.97) was shown between immunohistochemistry and FISH with low discordance rates (2-4%), high specificity (97-98%) and sensitivity values (95-99%), with no significant difference according to the immunohistochemistry interpretation guidelines used. The correlation between SISH and CISH on biopsies and FISH on surgical samples was strong (κ: 0.96 and 0.94, respectively), with no significant difference between false negative rates or sensitivity and specificity values (2 and 5%, 99 and 96%, 98 and 98%, respectively). Whatever the evaluation technique, excellent concordance between biopsies and surgical specimens was observed (κ ≥ 0.97; discordance rates between 1 and 2%), with high sensitivity (98-99%) and specificity (98-100%). Based on these results, when FISH cannot be used, SISH and/or CISH could be proposed as an alternative method to determine HER2 status and to confirm any ambiguous immunohistochemistry results, either for preoperative percutaneous biopsies or for surgical specimens. They could also be used for quality controls and immunohistochemistry calibration.

[HER2 gene amplification assay: is CISH an alternative to FISH?].

Science.gov (United States)

Denoux, Yves; Arnould, Laurent; Fiche, Maryse; Lannes, B; Couturier, Jérôme; Vincent-Salomon, Anne; Penault-Llorca, Frédérique; Antoine, M; Balaton, A; Baranzelli, M C; Becette, V; Bellocq, J P; Bibeau, F; Ettore, F; Fridman, V; Gnassia, J P; Jacquemier, J; MacGrogan, G; Mathieu, M C; Migeon, C; Rigaud, C; Roger, P; Sigal-Zafrani, B; Simony-Lafontaine, J; Trassard, M; Treilleux, I; Verriele, V; Voigt, J J

2003-12-01

The HER2 proto-oncogene encodes a transmembrane protein, which is considered to function as a growth factor receptor. Overexpression of this protein found by immunohistochemistry in about 20% of infiltrating breast carcinomas, has a predictive value of response to treatment by trastuzumab, an anti-HER2 humanized monoclonal antibody. Search for HER2 gene amplification is necessary to adapt the immunohistochemical technique quality and also in the cases of delicate analysis or weak overexpression. It is usually carried out by Fluorescence In Situ Hybridization (FISH). A more recent hybridization technique, named CISH because of its chromogenic revelation is an alternative method, which gives highly correlated results with FISH. We present details of this technique, which may be more familiar for the pathologists than FISH, because reading analysis is similar to that of immunohistochemical staining.

Unravelling the hydrophobicity of urea in water using thermodiffusion: implications for protein denaturation.

Science.gov (United States)

Niether, Doreen; Di Lecce, Silvia; Bresme, Fernando; Wiegand, Simone

2018-01-03

Urea is widely used as a protein denaturant in aqueous solutions. Experimental and computer simulation studies have shown that it dissolves in water almost ideally at high concentrations, introducing little disruption in the water hydrogen bonded structure. However, at concentrations of the order of 5 M or higher, urea induces denaturation in a wide range of proteins. The origin of this behaviour is not completely understood, but it is believed to stem from a balance between urea-protein and urea-water interactions, with urea becoming possibly hydrophobic at a specific concentration range. The small changes observed in the water structure make it difficult to connect the denaturation effects to the solvation properties. Here we show that the exquisite sensitivity of thermodiffusion to solute-water interactions allows the identification of the onset of hydrophobicity of urea-water mixtures. The hydrophobic behaviour is reflected in a sign reversal of the temperature dependent slope of the Soret coefficient, which is observed, both in experiments and non-equilibrium computer simulations at ∼5 M concentration of urea in water. This concentration regime corresponds to the one where abrupt changes in the denaturation of proteins are commonly observed. We show that the onset of hydrophobicity is intrinsically connected to the urea-water interactions. Our results allow us to identify correlations between the Soret coefficient and the partition coefficient, log P, hence establishing the thermodiffusion technique as a powerful approach to study hydrophobicity.

A correlated Walks' theory for DNA denaturation

International Nuclear Information System (INIS)

Mejdani, R.

1994-08-01

We have shown that by using a correlated Walks' theory for the lattice gas model on a one-dimensional lattice, we can study, beside the saturation curves obtained before for the enzyme kinetics, also the DNA denaturation process. In the limit of no interactions between sites the equation for melting curves of DNA reduces to the random model equation. Thus our leads naturally to this classical equation in the limiting case. (author). 22 refs, 3 figs

Temperature effect on gastric emptying time of hybrid grouper (Epinephelus spp.)

Energy Technology Data Exchange (ETDEWEB)

De, Moumita; Ghaffar, Mazlan Abd. [School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor (Malaysia); Das, Simon K. [School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia and Marine Ecosystem Research Centre (EKOMAR), Faculty of Science and Technology, Universiti (Malaysia)

2014-09-03

Knowledge of fish gastric emptying time is a necessary component for understanding the fish feeding rates, energy budgets and commercial production of fishes in aquaculture. The hybrid grouper Epinephelus spp. is getting popular as a culture species in Malaysia for their faster growth rate compared to commonly cultured grouper species (giant grouper Epinephelus lanceolatus and tiger grouper Epinephelus fuscoguttatus). There are data suggests that elevated sea water temperature affects gastric emptying time (GET) of fishes. Hence, this study aims to study the GET of hybrid grouper at different temperature (22, 26, 30, 34°C) in laboratory condition with commercial diet pellet. The gastric emptying times (GETs) at different temperatures were determined X-radiographically, using barium sulfate (BaSO{sub 4}) as a contrast medium food marker. The food marker and X-radiography showed that initial voidance of fecal matter began 4-6 h after feeding at all temperature. The fastest GET (13 h) was obsereved in the 30°C group, whereas the longest (17 h) GET was seen in 22°C group fed with artificial diet pellet. Not much differences in GET were recorded between the 26 and 34°C groups as 34°C groups fed lesser amount compared to 26°C groups. Nevertheless a substantial delay in GET was observed in the 22°C group. The findings of this study suggest to culture hybrid grouper between 26 to 30°C with commercial diet pellet as this temperature ranges proliferate the faster digestion process which may contribute faster growth rate of this commerical important fish species. Overall, these findings may have important consequences for optimization of commercial production of hybrid grouper.

Temperature effect on gastric emptying time of hybrid grouper (Epinephelus spp.)

Science.gov (United States)

De, Moumita; Ghaffar, Mazlan Abd.; Das, Simon K.

2014-09-01

Knowledge of fish gastric emptying time is a necessary component for understanding the fish feeding rates, energy budgets and commercial production of fishes in aquaculture. The hybrid grouper Epinephelus spp. is getting popular as a culture species in Malaysia for their faster growth rate compared to commonly cultured grouper species (giant grouper Epinephelus lanceolatus and tiger grouper Epinephelus fuscoguttatus). There are data suggests that elevated sea water temperature affects gastric emptying time (GET) of fishes. Hence, this study aims to study the GET of hybrid grouper at different temperature (22, 26, 30, 34°C) in laboratory condition with commercial diet pellet. The gastric emptying times (GETs) at different temperatures were determined X-radiographically, using barium sulfate (BaSO4) as a contrast medium food marker. The food marker and X-radiography showed that initial voidance of fecal matter began 4-6 h after feeding at all temperature. The fastest GET (13 h) was obsereved in the 30°C group, whereas the longest (17 h) GET was seen in 22°C group fed with artificial diet pellet. Not much differences in GET were recorded between the 26 and 34°C groups as 34°C groups fed lesser amount compared to 26°C groups. Nevertheless a substantial delay in GET was observed in the 22°C group. The findings of this study suggest to culture hybrid grouper between 26 to 30°C with commercial diet pellet as this temperature ranges proliferate the faster digestion process which may contribute faster growth rate of this commerical important fish species. Overall, these findings may have important consequences for optimization of commercial production of hybrid grouper.

Temperature effect on gastric emptying time of hybrid grouper (Epinephelus spp.)

International Nuclear Information System (INIS)

De, Moumita; Ghaffar, Mazlan Abd.; Das, Simon K.

2014-01-01

Knowledge of fish gastric emptying time is a necessary component for understanding the fish feeding rates, energy budgets and commercial production of fishes in aquaculture. The hybrid grouper Epinephelus spp. is getting popular as a culture species in Malaysia for their faster growth rate compared to commonly cultured grouper species (giant grouper Epinephelus lanceolatus and tiger grouper Epinephelus fuscoguttatus). There are data suggests that elevated sea water temperature affects gastric emptying time (GET) of fishes. Hence, this study aims to study the GET of hybrid grouper at different temperature (22, 26, 30, 34°C) in laboratory condition with commercial diet pellet. The gastric emptying times (GETs) at different temperatures were determined X-radiographically, using barium sulfate (BaSO 4 ) as a contrast medium food marker. The food marker and X-radiography showed that initial voidance of fecal matter began 4-6 h after feeding at all temperature. The fastest GET (13 h) was obsereved in the 30°C group, whereas the longest (17 h) GET was seen in 22°C group fed with artificial diet pellet. Not much differences in GET were recorded between the 26 and 34°C groups as 34°C groups fed lesser amount compared to 26°C groups. Nevertheless a substantial delay in GET was observed in the 22°C group. The findings of this study suggest to culture hybrid grouper between 26 to 30°C with commercial diet pellet as this temperature ranges proliferate the faster digestion process which may contribute faster growth rate of this commerical important fish species. Overall, these findings may have important consequences for optimization of commercial production of hybrid grouper

The correlation between dual-color chromogenic in situ hybridization and fluorescence in situ hybridization in assessing HER2 gene amplification in breast cancer.

Science.gov (United States)

Pedersen, Marianne; Rasmussen, Birgitte Bruun

2009-06-01

Fluorescence in situ hybridization (FISH) is regarded as the gold standard method for detecting HER2 gene amplification. Chromogenic in situ hybridization (CISH) is a promising alternative to FISH because CISH has the advantages of being a method evaluated by bright-field microscopy and the generated chromogenic signals are also stable. This study presents a dual color CISH for simultaneous detection of the HER2 gene and chromosome 17. The CISH method performs a chromogenic detection "on top" of the Food and Drug Administration (FDA)-approved HER2 FISH pharmDx method, where the fluorochrome-labeled probes are detected using enzyme-labeled antibodies and visualized by chromogenic enzymatic reactions. The HER2 status (amplified/not amplified and HER2 ratios) was evaluated by the CISH method and compared with results obtained by the FDA-approved FISH method. Of the 72 successfully investigated invasive breast carcinomas, both FISH and CISH detected HER2 amplification in 24 cases and nonamplification was detected in 47 cases. One case showed a discrepancy between FISH and CISH. The concordance between CISH and FISH was found to be almost perfect (98.6%). The correlation between the HER2 ratios obtained by the 2 methods showed excellent correlation (correlation coefficient 0.95). In conclusion, it is possible by dual-color CISH method to demonstrate HER2 genes and chromosome 17 genes, in the same tissue section and reliably assess HER2 status. The CISH method is a very promising alternative to the FISH method.

On the Use of Potential Denaturing Agents for Ethanol in Direct Ethanol Fuel Cells

OpenAIRE

Domnik Bayer; Florina Jung; Birgit Kintzel; Martin Joos; Carsten Cremers; Dierk Martin; Jörg Bernard; Jens Tübke

2011-01-01

Acidic or alkaline direct ethanol fuel cells (DEFCs) can be a sustainable alternative for power generation if they are fuelled with bio-ethanol. However, in order to keep the fuel cheap, ethanol has to be exempted from tax on spirits by denaturing. In this investigation the potential denaturing agents fusel oil, tert-butyl ethyl ether, and Bitrex were tested with regard to their compatibility with fuel cells. Experiments were carried out both in sulphuric acid and potassium hydroxide solution...

Metaphase FISH on a Chip: Miniaturized Microfluidic Device for Fluorescence in situ Hybridization

DEFF Research Database (Denmark)

Vedarethinam, Indumathi; Shah, Pranjul Jaykumar; Dimaki, Maria

2010-01-01

-FISH, the process continues to be a manual, labour intensive, expensive and time consuming technique, often taking over 3-5 days, even in dedicated labs. We have developed a novel microFISH device to perform metaphase FISH on a chip which overcomes many shortcomings of the current laboratory protocols. This work...... also introduces a novel splashing device for preparing metaphase spreads on a microscope glass slide, followed by a rapid adhesive tape-based bonding protocol leading to rapid fabrication of the microFISH device. The microFISH device allows for an optimized metaphase FISH protocol on a chip with over...... a 20-fold reduction in the reagent volume. This is the first demonstration of metaphase FISH on a microfluidic device and offers a possibility of automation and significant cost reduction of many routine diagnostic tests of genetic anomalies....

Denaturation of proteins by surfactants studied by the Taylor dispersion analysis.

Directory of Open Access Journals (Sweden)

Aldona Jelińska

Full Text Available We showed that the Taylor Dispersion Analysis (TDA is a fast and easy to use method for the study of denaturation proteins. We applied TDA to study denaturation of β-lactoglobulin, transferrin, and human insulin by anionic surfactant sodium dodecyl sulfate (SDS. A series of measurements at constant protein concentration (for transferrin was 1.9 x 10-5 M, for β- lactoglobulin was 7.6 x 10-5 M, and for insulin was 1.2 x 10-4 M and varying SDS concentrations were carried out in the phosphate-buffered saline (PBS. The structural changes were analyzed based on the diffusion coefficients of the complexes formed at various surfactant concentrations. The concentration of surfactant was varied in the range from 1.2 x 10-4 M to 8.7 x 10-2 M. We determined the minimum concentration of the surfactant necessary to change the native conformation of the proteins. The minimal concentration of SDS for β-lactoglobulin and transferrin was 4.3 x 10-4 M and for insulin 2.3 x 10-4 M. To evaluate the TDA as a novel method for studying denaturation of proteins we also applied other methods i.e. electronic circular dichroism (ECD and dynamic light scattering (DLS to study the same phenomenon. The results obtained using these methods were in agreement with the results from TDA.

Effect of heating strategies on whey protein denaturation--Revisited by liquid chromatography quadrupole time-of-flight mass spectrometry.

Science.gov (United States)

Akkerman, M; Rauh, V M; Christensen, M; Johansen, L B; Hammershøj, M; Larsen, L B

2016-01-01

Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of β-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5-soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Ion-ion interactions in the denatured state contribute to the stabilization of CutA1 proteins.

Science.gov (United States)

Yutani, Katsuhide; Matsuura, Yoshinori; Naitow, Hisashi; Joti, Yasumasa

2018-05-16

In order to elucidate features of the denatured state ensembles that exist in equilibrium with the native state under physiological conditions, we performed 1.4-μs molecular dynamics (MD) simulations at 400 K and 450 K using the monomer subunits of three CutA1 mutants from Escherichia coli: an SH-free mutant (Ec0SH) with denaturation temperature (T d ) = 85.6 °C, a hydrophobic mutant (Ec0VV) with T d  = 113.3 °C, and an ionic mutant (Ec0VV_6) with T d  = 136.8 °C. The occupancy of salt bridges by the six substituted charged residues in Ec0VV_6 was 140.1% at 300 K and 89.5% at 450 K, indicating that even in the denatured state, salt bridge occupancy was high, approximately 60% of that at 300 K. From these results, we can infer that proteins from hyperthermophiles with a high ratio of charged residues are stabilized by a decrease in conformational entropy due to ion-ion interactions in the denatured state. The mechanism must be comparable to the stabilization conferred by disulfide bonds within a protein. This suggests that introduction of charged residues, to promote formation of salt bridges in the denatured state, would be a simple way to rationally design stability-enhanced mutants.

Denaturation/Renaturation of Organophosphorus Acid Anhydrolase (OPAA) Using Guanidinium Hydrochloride and Urea

National Research Council Canada - National Science Library

Ong, K. K; Sun, Z; Cheng, T. C; Wei, Y; Yuan, J. M; Yin, R

2004-01-01

.... Using organophosphorus acid anhydrolase (OPAA) as the model protein, a guanidinium hydrochloride and urea denaturation/renaturation study was conducted and measured both optically and enzymatically...

Denaturation/Renaturation of Organophosphorus Acid Anhydrolase (OPAA) Using Guanidinium Hydrochloride and Urea

National Research Council Canada - National Science Library

Ong, K. K; Sun, Z; Cheng, T. C; Wei, Y; Yuan, J. M; Yin, R

2004-01-01

...; thereby indicating conformational changes. Similar results were obtained with circular dichroism as the peak representing the alpha-helix conformation decreased as denaturant concentration was increased...

Marker chromosome 21 identified by microdissection and FISH

Energy Technology Data Exchange (ETDEWEB)

Sun, Y.; Palmer, C.G. [Indiana Univ. School of Medicine, Indianapolis, IN (United States); Rubinstein, J. [Univ. Affiliated Cincinnati Center for Developmental Disorders, OH (United States)] [and others

1995-03-27

A child without Down`s syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with {alpha}-satellite DNA probes for acrocentric chromosomes and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21. 14 refs., 3 figs.

THE RESEARCH IN FISH GENETICS IN CROATIA AND FORMER YUGOSLAVIA

Directory of Open Access Journals (Sweden)

Tomislav Treer

1994-03-01

Full Text Available This is a review on fish genetics research in Croatia and former Yugoslavia, based on the analyses of all the articles published in four main journals (Ribarstvo Jugoslavije, Morsko ribarstvo, Ichthyologia and Acta Adriatica since 1945 till disintegration of Yugoslavia in 1991. Most of the papers cover the fields on cytogenetics and hybridization (24 and 13 respectively. Eight papers were on fish selection and five on population genetics. Apart from those, five papers were written by foreign authors. Two groups of researchers from the University of Sarajevo were specially active. One of them lead by B e r b e r o v i ć and S o f r a d ž i j a did extensive work in cytogenetics, analyzing the karyotypes of many fish species, some of them endemic. Another one lead by V u k o v i ć , investigated some natural hybrids and created many of them artificially, particulary among cyprinids. These results are presented in a special table. Contrary to the mountainous Bosnia where this type of research was of systematic and ecologic importance, in Croatia whwrw aquaculture was highly developed, the approach was quite different. The scientists from the University of Zagreb, H a b e k o v i ć and T u r k , studied the hybridization and selection of important cultured cyprinids. Apart from these scientific groups, many papers were published by A l - S a b t i , who later became world famous in fish cytogenetics. The works of many other authors who contributed with papers in different fields of fish genetics are also described.

Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

Energy Technology Data Exchange (ETDEWEB)

Nagy, Attila; Malnasi-Csizmadia, Andras; Somogyi, Bela; Lorinczy, Denes

2004-02-09

Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light ({lambda}{sub max}=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser{sup 65}-Tyr{sup 66}-Gly{sup 67}-, which buried into {beta}-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 deg. C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol{sup -1}), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol{sup -1}. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in {beta}-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.

Fluorescence In Situ Hybridization (FISH-Based Karyotyping Reveals Rapid Evolution of Centromeric and Subtelomeric Repeats in Common Bean (Phaseolus vulgaris and Relatives

Directory of Open Access Journals (Sweden)

Aiko Iwata-Otsubo

2016-04-01

Full Text Available Fluorescence in situ hybridization (FISH-based karyotyping is a powerful cytogenetics tool to study chromosome organization, behavior, and chromosome evolution. Here, we developed a FISH-based karyotyping system using a probe mixture comprised of centromeric and subtelomeric satellite repeats, 5S rDNA, and chromosome-specific BAC clones in common bean, which enables one to unambiguously distinguish all 11 chromosome pairs. Furthermore, we applied the karyotyping system to several wild relatives and landraces of common bean from two distinct gene pools, as well as other related Phaseolus species, to investigate repeat evolution in the genus Phaseolus. Comparison of karyotype maps within common bean indicates that chromosomal distribution of the centromeric and subtelomeric satellite repeats is stable, whereas the copy number of the repeats was variable, indicating rapid amplification/reduction of the repeats in specific genomic regions. In Phaseolus species that diverged approximately 2–4 million yr ago, copy numbers of centromeric repeats were largely reduced or diverged, and chromosomal distributions have changed, suggesting rapid evolution of centromeric repeats. We also detected variation in the distribution pattern of subtelomeric repeats in Phaseolus species. The FISH-based karyotyping system revealed that satellite repeats are actively and rapidly evolving, forming genomic features unique to individual common bean accessions and Phaseolus species.

Comparison of thermal properties of fish collagen and bovine collagen in the temperature range 298-670K.

Science.gov (United States)

Gauza-Włodarczyk, Marlena; Kubisz, Leszek; Mielcarek, Sławomir; Włodarczyk, Dariusz

2017-11-01

The increased interest in fish collagen is a consequence of the risk of exposure to Creutzfeld-Jacob disease (CJD) and the bovine spongiform encephalopathy (BSE), whose occurrence is associated with prions carried by bovine collagen. Collagen is the main biopolymer in living organisms and the main component of the skin and bones. Until the discovery of the BSE, bovine collagen had been widely used. The BSE epidemic increased the interest in new sources of collagen such as fish skin collagen (FSC) and its properties. Although the thermal properties of collagen originating from mammals have been well described, less attention has been paid to the thermal properties of FSC. Denaturation temperature is a particularly important parameter, depending on the collagen origin and hydration level. In the reported experiment, the free water and bound water release processes along with thermal denaturation process were studied by means of the differential scanning calorimetry (DSC). Measurements were carried out using a DSC 7 instrument (Elmer-Perkin), in the temperature range 298-670K. The study material was FSC derived by acidic hydration method. The bovine Achilles tendon (BAT) collagen type I was used as the control material. The thermograms recorded revealed both, exothermic and endothermic peaks. For both materials, the peaks in the temperature range of 330-360K were assigned to the release of free water and bound water. The denaturation temperatures of FSC and BAT collagen were determined as 420K and 493K, respectively. Thermal decomposition process was observed at about 500K for FSC and at about 510K for BAT collagen. These results show that FSC is less resistant to high temperature than BAT collagen. Copyright © 2017 Elsevier B.V. All rights reserved.

Reversible thermal denaturation of immobilized rhodanese

International Nuclear Information System (INIS)

Horowitz, P.; Bowman, S.

1987-01-01

For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [ 35 S]SO 3 (2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states

mathFISH, a Web Tool That Uses Thermodynamics-Based Mathematical Models for In Silico Evaluation of Oligonucleotide Probes for Fluorescence In Situ Hybridization▿ †

OpenAIRE

Yilmaz, L. Safak; Parnerkar, Shreyas; Noguera, Daniel R.

2010-01-01

Mathematical models of RNA-targeted fluorescence in situ hybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc.edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design.

Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

DEFF Research Database (Denmark)

Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno

2015-01-01

acid (LNA)/ 2' O-methyl RNA (2'OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization...... step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion......In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo...

Sequential Events in the Irreversible Thermal Denaturation of Human Brain-Type Creatine Kinase by Spectroscopic Methods

Directory of Open Access Journals (Sweden)

Yan-Song Gao

2010-06-01

Full Text Available The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK thermal denaturation were studied by differential scanning calorimetry (DSC, CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK. The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities.

Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

Science.gov (United States)

Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

2010-03-01

Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

Hemichrome formation during hemoglobin Zurich denaturation

International Nuclear Information System (INIS)

Zago, M.A.; Costa, F.F.; Botura, C.; Baffa, O.

1988-01-01

Electron paramagnetic resonance (EPR)spectrum of hemoglobin Zurich, after oxidation, storage and heating, showed several absorption derives in the high field region (g ≅ 2) which are indicative of hemichrome formation. Characteristic visible spectra of hemichromes were observed for oxidized Hb Zurich and for its spontaneous precipitate. The proportional increase of EPR signals at g ≅ 2 and decrease at g = 6.37, the constant ratio of absorbance at 540 nm to 280 nm during heating, and the similarity of this ratio for spontaneously precipitated HbA and for Hb Zurich indicate that heme is not lost during the first steps of Hb Zurich denaturation. (author) [pt

Chemical denaturation of globular proteins at the air/water interface: an x-ray and neutron reflectometry study

International Nuclear Information System (INIS)

Perriman, A.W.; Henderson, M.J.; White, J.W.

2003-01-01

Full text: X-ray and neutron reflectometry has been used to probe the equilibrium surface structure of hen egg white lysozyme (lysozyme) and bovine β -lactoglobulin (β -lactoglobulin) under denaturing conditions at the air-water interface. This was achieved by performing experiments on 10 mg mL -1 protein solutions containing increasing concentrations of the chemical denaturant guanidinium hydrochloride (G.HCl). For solutions containing no G.HCl, the surface structure of the proteins was represented by a two-layer model with total thicknesses of 48 Angstroms and 38 Angstroms for lysozyme and β -lactoglobulin, respectively. The total volume of a single protein molecule and the associated water molecules was evaluated to be approximately 45 (0.3) nm 3 for lysozyme, and 60 (0.3) nm 3 for β-lactoglobulin. The thickness dimensions and the total volumes compared favourably with the crystal dimensions of 45 x 30 x 30 Angstroms (40.5 nm 3 ),1 and 36 x 36 x 36 Angstroms (47 nm 3 ) 2 for lysozyme and β -lactoglobulin, respectively. This comparison suggests that when no denaturant was present, the structures of lysozyme and β -lactoglobulin were near to their native conformations at the air-water interface. The response to the presence of the chemical denaturant was different for each protein. The surface layer of β-lactoglobulin expanded at very low concentrations (0.2 mol dm -3 ) of G.HCl. In contrast, the lysozyme layer contracted. At higher concentrations, unfolding of both the proteins led to the formation of a third diffuse layer. In general, lysozyme appeared to be less responsive to the chemical denaturant, which is most likely a result of the higher disulfide content of lysozyme. A protocol allowing quantitative thermodynamic analysis of the contribution from the air-water interface to the chemical denaturation of a protein was developed

EGFR status in oral squamous cell carcinoma: comparing immunohistochemistry, FISH and CISH detection in a case series study

OpenAIRE

Bernardes, Vanessa F?tima; Gleber-Netto, Frederico Omar; de Sousa, S?lvia Ferreira; Rocha, Rafael Malagoli; de Aguiar, Maria C?ssia Ferreira

2013-01-01

Objectives To compare the immunohistochemistry (IHC) expression of epidermal growth factor receptor (EGFR) in oral squamous cell carcinomas (OSCC) with the gene amplification evaluated by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) and their association with clinicopathological parameters. Additionally, we tested the sensibility and specificity of CISH in comparison with FISH. Design Case series study Setting Oral surgery and pathology department in ...

Exact method for numerically analyzing a model of local denaturation in superhelically stressed DNA

International Nuclear Information System (INIS)

Fye, R.M.; Benham, C.J.

1999-01-01

Local denaturation, the separation at specific sites of the two strands comprising the DNA double helix, is one of the most fundamental processes in biology, required to allow the base sequence to be read both in DNA transcription and in replication. In living organisms this process can be mediated by enzymes which regulate the amount of superhelical stress imposed on the DNA. We present a numerically exact technique for analyzing a model of denaturation in superhelically stressed DNA. This approach is capable of predicting the locations and extents of transition in circular superhelical DNA molecules of kilobase lengths and specified base pair sequences. It can also be used for closed loops of DNA which are typically found in vivo to be kilobases long. The analytic method consists of an integration over the DNA twist degrees of freedom followed by the introduction of auxiliary variables to decouple the remaining degrees of freedom, which allows the use of the transfer matrix method. The algorithm implementing our technique requires O(N 2 ) operations and O(N) memory to analyze a DNA domain containing N base pairs. However, to analyze kilobase length DNA molecules it must be implemented in high precision floating point arithmetic. An accelerated algorithm is constructed by imposing an upper bound M on the number of base pairs that can simultaneously denature in a state. This accelerated algorithm requires O(MN) operations, and has an analytically bounded error. Sample calculations show that it achieves high accuracy (greater than 15 decimal digits) with relatively small values of M (M<0.05N) for kilobase length molecules under physiologically relevant conditions. Calculations are performed on the superhelical pBR322 DNA sequence to test the accuracy of the method. With no free parameters in the model, the locations and extents of local denaturation predicted by this analysis are in quantitatively precise agreement with in vitro experimental measurements

Growth of monosex hybrid tilapia in the labortory and sewage oxidation ponds

International Nuclear Information System (INIS)

Suffern, J.S.; Adams, S.M.; Blaylock, B.G.; Coutant, C.C.; Guthrie, C.A.

1978-01-01

Studies were conducted to evaluate the potential of monosex hybrid tilapia (female T. mossambica x male T. hornorum) in waste-heat polyculture systems. The optimum growth temperature for this hybrid was found to be 32 0 C in laboratory experiments. Experiments in sewage pond cage culture in the temperature range of 23 to 33 0 C at stocking densities of approximately 53 fish/m 3 were also conducted. At fish sizes between 5 and 12 cm TL, estimated annual production is approximately 50,000 kg/ha/yr (50,000 lb/acre/yr). Fish in the sewage oxidation ponds grew significantly faster than fish fed trout chow at optimum temperature in the laboratory, even though temperatures in the sewage ponds averaged below the optimum growth temperature. Techniques to accelerate growth rates are being explored. Exposure to gamma radiation (500 rads), known to cause significant increases in channel catfish growth rate, was found to have a similar effect on tilapia. After a 20-week growth period, exposed fish weighed an average of 20% more than controls

Reliability of chromogenic in situ hybridization for epidermal growth factor receptor gene copy number detection in non-small-cell lung carcinomas: a comparison with fluorescence in situ hybridization study.

Science.gov (United States)

Yoo, Seol Bong; Lee, Hyun Ju; Park, Jung Ok; Choe, Gheeyoung; Chung, Doo Hyun; Seo, Jeong-Wook; Chung, Jin-Haeng

2010-03-01

Fluorescence in situ hybridization (FISH) has been known to be the most representative and standardized test for assessing gene amplification. However, FISH requires a fluorescence microscope, the signals are labile and rapidly fade over time. Recently, chromogenic in situ hybridization (CISH) has emerged as a potential alternative to FISH. The aim of this study is to test the reliability of CISH technique for the detection of epidermal growth factor receptor (EGFR) gene amplification in non-small-cell lung carcinomas (NSCLC), to compare CISH results with FISH. A total of 277 formalin-fixed and paraffin embedded NSCLC tissue samples were retrieved from the surgical pathology archives at Seoul National University Bundang Hospital. CISH and FISH examinations were performed to test EGFR gene amplification status. There was high concordance in the assessment of EGFR gene copy number between CISH and FISH tests (Kappa coefficient=0.83). Excellent concordance was shown between two observers on the interpretation of the CISH results (Kappa coefficient=0.90). In conclusion, CISH result is highly reproducible, accurate and practical method to determine EGFR gene amplification in NSCLC. In addition, CISH allows a concurrent analysis of histological features of the tumors and gene copy numbers.

In situ hybridization; principles and applications: review article

Directory of Open Access Journals (Sweden)

Zahra Nozhat

2015-06-01

Full Text Available In situ hybridization (ISH is a method that uses labeled complementary single strand DNA or RNA to localize specific DNA or RNA sequences in an intact cell or in a fixed tissue section. The main steps of ISH consist of: probe selection, tissue or sample preparation, pre-hybridization treatment, hybridization and washing, detection and control procedure. Probe selection is one of the important aspects of successful hybridization. ISH sensitivity and specificity can be influenced by: probe construct, efficiency of labeling, percentage of GC, probe length and signal detection systems. Different methods such as nick translation, random priming, end tailing and T4 DNA polymerase replacement are used for probe generation. Both radioactive and non-radioactive labels can be used in order to probe labeling. Nucleic acid maintenance in samples, prevention of morphological changes of samples and probe penetration into tissue section are the main aims of sample preparation step. Then, a small amount of solution containing probe, is added on slides containing tissue sections for hybridization process, then slides are incubated overnight. Next day, washes are carried out to remove the probes which are not bound to target DNA or RNA. Finally, in order to be sure that the observed labeling is specific to the target sequence, using several control procedures is very important. Various techniques based on ISH consist of: Fluorescence in situ hybridization (FISH, chromogenic in situ hybridization (CISH, genomic in situ hybridization (GISH, comparative genomic hybridization (CGH, spectral karyotyping (SKY and multiplex fluorescence in situ hybridization (MFISH. One of the most common techniques of ISH is fluorescence in situ hybridization. FISH can be used to: 1 detect small deletions and duplications that are not visible using microscope analysis, 2 detect how many chromosomes of a certain type are present in each cell and 3 confirm rearrangements that are

Intrinsic alterations in the partial molar volume on the protein denaturation: surficial Kirkwood-Buff approach.

Science.gov (United States)

Yu, Isseki; Takayanagi, Masayoshi; Nagaoka, Masataka

2009-03-19

The partial molar volume (PMV) of the protein chymotrypsin inhibitor 2 (CI2) was calculated by all-atom MD simulation. Denatured CI2 showed almost the same average PMV value as that of native CI2. This is consistent with the phenomenological question of the protein volume paradox. Furthermore, using the surficial Kirkwood-Buff approach, spatial distributions of PMV were analyzed as a function of the distance from the CI2 surface. The profiles of the new R-dependent PMV indicate that, in denatured CI2, the reduction in the solvent electrostatic interaction volume is canceled out mainly by an increment in thermal volume in the vicinity of its surface. In addition, the PMV of the denatured CI2 was found to increase in the region in which the number density of water atoms is minimum. These results provide a direct and detailed picture of the mechanism of the protein volume paradox suggested by Chalikian et al.

Fluorescent in situ hybridization (FISH) on corneal impression cytology specimens (CICS): study of epithelial cell survival after keratoplasty.

Science.gov (United States)

Catanese, Muriel; Popovici, Cornel; Proust, Hélène; Hoffart, Louis; Matonti, Frédéric; Cochereau, Isabelle; Conrath, John; Gabison, Eric E

2011-02-22

To assess corneal epithelial cell survival after keratoplasty. Corneal impression cytology (CIC) was performed on sex-mismatched corneal transplants. Fluorescent in situ hybridization (FISH) with sex chromosome-specific probes was performed to identify epithelial cell mosaicism and therefore allocate the donor or recipient origin of the cells. Twenty-four samples of corneal epithelial cells derived from 21 transplanted patients were analyzed. All patients received post-operative treatment using dexamethasone eye drops, with progressive tapering over 18 months, and nine patients also received 2% cyclosporine eye drops. Out of the 24 samples reaching quality criteria, sex mosaicism was found in 13, demonstrating the presence of donor-derived cells at the center of the graft for at least 211 days post keratoplasty. Kaplan-Meier analysis established a median survival of donor corneal epithelial cells of 385 days. Although not statistically significant, the disappearance of donor cells seemed to be delayed and the average number of persistent cells appeared to be greater when 2% cyclosporine was used topically as an additional immunosuppressive therapy. The combination of corneal impressions and FISH analysis is a valuable tool with negligible side effects to investigate the presence of epithelial cell mosaicism in sex-mismatched donor transplants. Epithelial cells survived at the center of the graft with a median survival of more than one year, suggesting slower epithelial turnover than previously described.

Effect of Stocking Density on Growth Performance of Hybrids of ...

African Journals Online (AJOL)

Fingerlings of Oreochromis niloticus♀ and Oreochromis urolepis urolepis♂ hybrids were reared at stocking densities of control, 5, 10, 15 and 20 fish/m3 at 15 Practical Salinity Units (PSU) in 1m3 plastic tanks for 63 days. They were kept at low, intermediate and high densities respectively. All hybrids were fed on a ...

Dispersal and selection mediate hybridization between a native and invasive species

Science.gov (United States)

Kovach, Ryan P.; Muhlfeld, Clint C.; Boyer, Matthew C.; Lowe, Winsor H.; Allendorf, Fred W.; Luikart, Gordon

2015-01-01

Hybridization between native and non-native species has serious biological consequences, but our understanding of how dispersal and selection interact to influence invasive hybridization is limited. Here, we document the spread of genetic introgression between a native (Oncorhynchus clarkii) and invasive (Oncorhynchus mykiss) trout, and identify the mechanisms influencing genetic admixture. In two populations inhabiting contrasting environments, non-native admixture increased rapidly from 1984 to 2007 and was driven by surprisingly consistent processes. Individual admixture was related to two phenotypic traits associated with fitness: size at spawning and age of juvenile emigration. Fish with higher non-native admixture were larger and tended to emigrate at a younger age—relationships that are expected to confer fitness advantages to hybrid individuals. However, strong selection against non-native admixture was evident across streams and cohorts (mean selection coefficient against genotypes with non-native alleles (s) ¼ 0.60; s.e. ¼ 0.10). Nevertheless, hybridization was promoted in both streams by the continuous immigration of individuals with high levels of non-native admixture from other hybrid source populations. Thus, antagonistic relationships between dispersal and selection are mediating invasive hybridization between these fish, emphasizing that data on dispersal and natural selection are needed to fully understand the dynamics of introgression between native and non-native species. .

Fluorescence in situ hybridization of old G-banded and mounted chromosome preparations

DEFF Research Database (Denmark)

Gerdes, A M; Pandis, N; Bomme, L

1997-01-01

the coverslips detach spontaneously; any mechanical manipulation will jeopardize the results. The success of chromosome painting is improved by excluding the regular RNase treatment step prior to hybridization. Additional changes compared with standard FISH protocols are that the 2 x SSC step is omitted......An improved method for fluorescence in situ hybridization (FISH) investigation of old, previously G-banded, mounted chromosome preparations with chromosome specific painting probes and centromere-specific probes is described. Before hybridization, the slides are incubated in xylene until......, that the amount of added probe is increased approximately 2.5 times, and that the amplification of signals is performed twice. The applicability of the method, which allows double painting with two differently labeled probes using two differently fluorescing colors, was tested on 11 cases involving different...

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance.

Science.gov (United States)

Vogtt, K; Winter, R

2005-08-01

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80 degrees C) and under high pressure conditions at low temperature (3.75 kbar, -13 degrees C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

Directory of Open Access Journals (Sweden)

K. Vogtt

2005-08-01

Full Text Available COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC and under high pressure conditions at low temperature (3.75 kbar, -13ºC. Moreover, the influence of co-solvents (sorbitol, urea on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Urea and Guanidinium Induced Denaturation of a Trp-Cage Miniprotein

Czech Academy of Sciences Publication Activity Database

Heyda, Jan; Kožíšek, Milan; Bednárová, Lucie; Thompson, G.; Konvalinka, Jan; Vondrášek, Jiří; Jungwirth, Pavel

2011-01-01

Roč. 115, č. 28 (2011), s. 8910-8924 ISSN 1520-6106 R&D Projects: GA MŠk LC512; GA ČR GA203/08/0114 Institutional research plan: CEZ:AV0Z40550506 Keywords : trp-cage denaturation * urea * guanidinium * molecular dynamics Subject RIV: CC - Organic Chemistry Impact factor: 3.696, year: 2011

A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

Energy Technology Data Exchange (ETDEWEB)

Castronuovo, Giuseppina, E-mail: giuseppina.castronuovo@unina.it [Department of Chemistry, University Federico II of Naples, Complesso Universitario Monte S. Angelo, via Cintia, 80126 Naples (Italy); Niccoli, Marcella [Department of Chemistry, University Federico II of Naples, Complesso Universitario Monte S. Angelo, via Cintia, 80126 Naples (Italy)

2012-09-10

Highlights: Black-Right-Pointing-Pointer A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Black-Right-Pointing-Pointer The enthalpic interaction coefficients are very useful parameters to gain information about the mechanism through which two hydrated molecules interact in solution. Black-Right-Pointing-Pointer Hypotheses are proposed about the mechanism underlying the denaturation of lysozyme induced by high concentrations of urea or ethanol. - Abstract: A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg{sup -1}, the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules. The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on

A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

International Nuclear Information System (INIS)

Castronuovo, Giuseppina; Niccoli, Marcella

2012-01-01

Highlights: ► A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. ► The enthalpic interaction coefficients are very useful parameters to gain information about the mechanism through which two hydrated molecules interact in solution. ► Hypotheses are proposed about the mechanism underlying the denaturation of lysozyme induced by high concentrations of urea or ethanol. - Abstract: A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg −1 , the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules. The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on the possible mode of action of the two cosolvents on the stability of proteins

Acetic acid denaturing pulsed field capillary electrophoresis for RNA separation.

Science.gov (United States)

Li, Zhenqing; Dou, Xiaoming; Ni, Yi; Sumitomo, Keiko; Yamaguchi, Yoshinori

2010-10-01

Based on our previous work of in-capillary denaturing polymer electrophoresis, we present a study of RNA molecular separation up to 6.0 kilo nucleotide by pulsed field CE. This is the first systematic investigation of electrophoresis of a larger molecular mass RNA in linear hydroxyethylcellulose (HEC) under pulsed field conditions. The parameters that may influence the separation performance, e.g. gel polymer concentration, modulation depth and pulse frequency, are analyzed in terms of resolution and mobility. For denaturing and separating RNA in the capillary simultaneously, 2 M acetic acid was added into the HEC polymer to serve as separation buffer. Result shows that (i) in pulsed field conditions, RNA separation can be achieved in a wide range of concentration of HEC polymer, and RNA fragments between 0.3 and 0.6 kilo nucleotide are sensitive to the polymer concentration; (ii) under certain pulsed field conditions, RNA fragments move linearly as the modulation depth increases; (iii) 12.5 Hz is the resonance frequency for RNA reorientation time and applied frequency.

Equilibrium unfolding of A. niger RNase: pH dependence of chemical and thermal denaturation.

Science.gov (United States)

Kumar, Gundampati Ravi; Sharma, Anurag; Kumari, Moni; Jagannadham, Medicherla V; Debnath, Mira

2011-08-01

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A(MG1)) into another (I(MG2)).

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

Science.gov (United States)

Malhotra, Pooja; Jethva, Prashant N; Udgaonkar, Jayant B

2017-08-08

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

CARD-FISH for Environmental Microorganisms: Technical Advancement and Future Applications

OpenAIRE

Kubota, Kengo

2012-01-01

Fluorescence in situ hybridization (FISH) has become a standard technique in environmental microbiology. More than 20 years have passed since this technique was first described, and it is currently used for the detection of ribosomal RNA, messenger RNA, and functional genes encoded on chromosomes. This review focuses on the advancement and applications of FISH combined with catalyzed reporter deposition (CARD, also known as tyramide signal amplification or TSA), in the detection of environmen...

Radioimmunoassay and heat denaturation enzyme assay for the detection of Tay-Sachs heterozygotes during pregnancy

International Nuclear Information System (INIS)

Nguyen, C.; Gold, R.J.M.; Mahuran, D.; Lowden, J.A.

1981-01-01

Tay-Sachs disease results from a loss of activity of hexosaminidase A (HEX A) in body tissues and fluids. Heterozygotes for the disease are usually identified by their relatively low ratio of heat-labile HEX A to total hexosaminidase. During pregnancy an intermediate isoenzyme (HEX I) increases in activity in serum and obscures the heterozygote status. HEX I does not increase in leucocytes, tears and other body tissues but because of technical difficulties in these assays the authors examined the feasibility of using a radioimmunoassay for HEX A. By univariate analysis, the heat denaturation assay gave a lower cost of misclassification for non-pregnant normals while RIA did so for pregnant normals. A combination of both tests led to reduced cost of misclassification compared to either alone. Bayesian analysis of bivariate gaussian density functions for heat denaturation and for radioimmunoassays of HEX isoenzymes was employed to calculate misclassification frequencies. Among the parameters examined, HEX A measured by RIA and % HEX A by heat-denaturation assay were the two having the best discriminatory power. (Auth.)

T1ρ is superior to T2 mapping for the evaluation of articular cartilage denaturalization with osteoarthritis: radiological-pathological correlation after total knee arthroplasty.

Science.gov (United States)

Takayama, Yukihisa; Hatakenaka, Masamitsu; Tsushima, Hidetoshi; Okazaki, Ken; Yoshiura, Takashi; Yonezawa, Masato; Nishikawa, Kei; Iwamoto, Yukihide; Honda, Hiroshi

2013-04-01

We compared the diagnostic performance of T1ρ and T2 mappings in the evaluation of denatured articular cartilage with osteoarthritis of the knee. 2D-Sagittal T1ρ and T2 mappings of the knee were obtained from 16 patients before total knee arthroplasty. After surgery, specimens of the femur and tibia were regionally segmented according to a 5-point scale of the severity of denaturalization. The T1ρ and T2 values in the full thickness of the articular cartilage in each region were measured by two observers. The two mappings were compared for their ability to differentiate between normal and denatured articular cartilage and also for their usefulness in grading the severity of the denaturalization using the area under receiver operating characteristic curves (Az). A pT2 mapping for the differentiation between normal and denatured articular cartilage (pT2 mapping could not. However, there were no significant differences between the two mappings in the discrimination of mild versus moderate denaturalization or of moderate versus severe denaturalization. The two observers showed good agreement in the results (intraclass correlation coefficient=0.81 for T1ρ and 0.92 for T2). T1ρ mapping is superior to T2 mapping for the evaluation of denatured articular cartilage with osteoarthritis of the knee. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Shoal bass hybridization in the Chattahoochee River Basin near Atlanta, Georgia

Science.gov (United States)

Taylor, Andrew T.; Tringali, Michael D.; O'Rourke, Patrick M.; Long, James M.

2018-01-01

The shoal bass (Micropterus cataractae) is a sportfish endemic to the Apalachicola-Chattahoochee-Flint Basin of the southeastern United States. Introgression with several non-native congeners poses a pertinent threat to shoal bass conservation, particularly in the altered habitats of the Chattahoochee River. Our primary objective was to characterize hybridization in shoal bass populations near Atlanta, Georgia, including a population inhabiting Big Creek and another in the main stem Chattahoochee River below Morgan Falls Dam (MFD). A secondary objective was to examine the accuracy of phenotypic identifications below MFD based on a simplified suite of characters examined in the field. Fish were genotyped with 16 microsatellite DNA markers, and results demonstrated that at least four black bass species were involved in introgressive hybridization. Of 62 fish genotyped from Big Creek, 27% were pure shoal bass and 65% represented either F1 hybrids of shoal bass x smallmouth bass (M. dolomieu) or unidirectional backcrosses towards shoal bass. Of 29 fish genotyped below MFD and downstream at Cochran Shoals, 45% were pure shoal bass. Six hybrid shoal bass included both F1 hybrids and backcrosses with non-natives including Alabama bass (M. henshalli), spotted bass (M. punctulatus), and smallmouth bass. Shoal bass alleles comprised only 21% of the overall genomic composition in Big Creek and 31% below MFD (when combined with Cochran Shoals). Phenotypic identification below MFD resulted in an overall correct classification rate of 86% when discerning pure shoal bass from all other non-natives and hybrids. Results suggest that although these two shoal bass populations feature some of the highest introgression rates documented, only a fleeting opportunity may exist to conserve pure shoal bass in both populations. Continued supplemental stocking of pure shoal bass below MFD appears warranted to thwart increased admixture among multiple black bass taxa, and a similar stocking

Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species.

Science.gov (United States)

Prado, E A; Faivre-Rampant, P; Schneider, C; Darmency, M A

1996-10-01

Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.

Biotechnology applied to fish reproduction: tools for conservation.

Science.gov (United States)

de Siqueira-Silva, Diógenes Henrique; Saito, Taiju; Dos Santos-Silva, Amanda Pereira; da Silva Costa, Raphael; Psenicka, Martin; Yasui, George Shigueki

2018-04-29

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.

Annual trends in catchability and fish stock assessments

DEFF Research Database (Denmark)

Marchal, P.; Ulrich, Clara; Korsbrekke, K.

2003-01-01

. The performances of the new and traditional XSA assessments are compared using criteria based on the precision of catchability estimates, stationarity of Log-catchability residuals and retrospective patterns relative to fishing mortality, spawning stock biomass and recruitment estimates. The performances....... A range of catchability trends, including values derived from the "Hybrid" method, is then implemented to standardise the fishing effort of some tuning fleets used in the stock assessments performed by XSA (eXtended Survivors Analysis). Stocks being assessed are the North Sea cod, saithe, plaice and sole...

Fast-FISH Detection and Semi-Automated Image Analysis of Numerical Chromosome Aberrations in Hematological Malignancies

Directory of Open Access Journals (Sweden)

Arif Esa

1998-01-01

Full Text Available A new fluorescence in situ hybridization (FISH technique called Fast-FISH in combination with semi-automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML and chronic lymphocytic leukemia (CLL. Commercially available α-satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of the Fast-FISH protocol, the microscopic images of the fluorescence-labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with a standard FISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling and Fast-FISH was found. Automated spot counting after Fast-FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility of Fast-FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since the Fast-FISH technique requires a hybridization time as low as 1/20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours.

Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system.

Science.gov (United States)

Inoue, H; Hirobe, M

1987-05-29

The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.

Anisakid nematodes as possible markers to trace fish products

Directory of Open Access Journals (Sweden)

Vincenzo Ferrantelli

2015-03-01

Full Text Available In this work a total of 949 fish samples were analysed for the identification of nematode larvae belonging to the Anisakidae family. Biomolecular application for the identification of Anisakidae larvae can be an optimal instrument for the traceability of fish products, described on the Reg. EC 178/2002. Results confirm a correlation between geographical distribution of fishes and presence of specific Anisakid larvae. FAO 37 zone (Mediterranean sea showed a prevailing distribution of Anisakis pegreffii and a minimal presence of A. simplex s.s. in hybrid form with Anisakis pegreffii. FAO 27 zone showed a prevailing distribution of A. simplex s.s. in fish like Brosme (Brosme brosme and infestation prevalence of Pseudoterranova krabbei and P. decipiens s.s. in Gadus morhua. Obtained results validate the hypothesis that molecular biology methods for identifying Anisakidae larvae are effective traceability markers of fish products.

Feasibility of using fluorescence in situ hybridization (FISH) to detect early gene changes in sputum cells from uranium miners

Energy Technology Data Exchange (ETDEWEB)

Neft, R.E.; Rogers, J.L.; Belinsky, S.A. [and others

1995-12-01

Epidemiological studies have shown that combined exposure to radon progeny and tobacco smoke produce a greater than additive or synergistic increase in lung cancer risk. Lung cancer results from multiple genetic changes over a long period of time. An early change that occurs in lung cancer is trisomy 7 which is found in 50% of non-small cell lung cancer and in the far margins of resected lung tumors. The 80% mortality associated with lung cancer is in part related to the high proportion of patients who present with an advanced, unresectable tumor. Therefore, early detection of patients at risk for tumor development is critical to improve treatment of this disease. Currently, it is difficult to detect lung cancer early while it is still amendable by surgery. Saccomanno, G. has shown that premalignant cytologic changes in sputum cells collected from uranium miners can be detected by a skilled, highly trained cytopathologist. A more objective alternative for identifying premalignant cells in sputum may be to determine whether an early genetic change such as trisomy 7 is present in these cells. Fluorescence in situ hybridization (FISH) can be used to identify cells with trisomy 7. The results of this investigation indicate that FISH may prove to be an accurate, efficient method to test at-risk individuals for genetic alterations in bronchial epithelial cells from sputum.

Feasibility of using fluorescence in situ hybridization (FISH) to detect early gene changes in sputum cells from uranium miners

International Nuclear Information System (INIS)

Neft, R.E.; Rogers, J.L.; Belinsky, S.A.

1995-01-01

Epidemiological studies have shown that combined exposure to radon progeny and tobacco smoke produce a greater than additive or synergistic increase in lung cancer risk. Lung cancer results from multiple genetic changes over a long period of time. An early change that occurs in lung cancer is trisomy 7 which is found in 50% of non-small cell lung cancer and in the far margins of resected lung tumors. The 80% mortality associated with lung cancer is in part related to the high proportion of patients who present with an advanced, unresectable tumor. Therefore, early detection of patients at risk for tumor development is critical to improve treatment of this disease. Currently, it is difficult to detect lung cancer early while it is still amendable by surgery. Saccomanno, G. has shown that premalignant cytologic changes in sputum cells collected from uranium miners can be detected by a skilled, highly trained cytopathologist. A more objective alternative for identifying premalignant cells in sputum may be to determine whether an early genetic change such as trisomy 7 is present in these cells. Fluorescence in situ hybridization (FISH) can be used to identify cells with trisomy 7. The results of this investigation indicate that FISH may prove to be an accurate, efficient method to test at-risk individuals for genetic alterations in bronchial epithelial cells from sputum

Widespread hybridization and bidirectional introgression in sympatric species of coral reef fish

KAUST Repository

Harrison, Hugo B.; Berumen, Michael L.; Saenz-Agudelo, Pablo; Salas, Eva; Williamson, David H.; Jones, Geoffrey P.

2017-01-01

interspecific hybrids from a collection of 2,991 coral trout sampled in inshore and mid-shelf reefs of the southern Great Barrier Reef. Hybrids were ubiquitous among reefs, fertile and spanned multiple generations suggesting both ecological and evolutionary

Experimental observation of G banding verifying X-ray workers' chromosome translocation detected by FISH

International Nuclear Information System (INIS)

Sun Yuanming; Li Jin; Wang Qin; Tang Weisheng; Wang Zhiquan

2002-01-01

Objective: FISH is the most effective way of detecting chromosome aberration and many factors affect its accuracy. G-banding is used to verify the results of early X-ray workers' chromosome translocation examined by FISH. Methods: The chromosome translocations of early X-ray workers have been analysed by FISH (fluorescence in situ hybridization) and G-banding, yields of translocation treated with statistics. Results: The chromosome aberrations frequencies by tow methods are closely related. Conclusion: FISH is a feasible way to analyse chromosome aberrations of X-ray workers and reconstruct dose

Theoretical aspects of pressure and solute denaturation of proteins: A Kirkwood-buff-theory approach

Science.gov (United States)

Ben-Naim, Arieh

2012-12-01

A new approach to the problem of pressure-denaturation (PD) and solute-denaturation (SD) of proteins is presented. The problem is formulated in terms of Le Chatelier principle, and a solution is sought in terms of the Kirkwood-Buff theory of solutions. It is found that both problems have one factor in common; the excluded volumes of the folded and the unfolded forms with respect to the solvent molecules. It is shown that solvent-induced effects operating on hydrophilic groups along the protein are probably the main reason for PD. On the other hand, the SD depends on the preferential solvation of the folded and the unfolded forms with respect to solvent and co-solvent molecules.

Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

International Nuclear Information System (INIS)

Olive, P.L.; Vanderbyl, S.; MacPhail, S.H.

1991-01-01

Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though constraints to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to round up and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing

Evaluation of hybrid neutralization/biosorption process for zinc ions removal from automotive battery effluent by dolomite and fish scales.

Science.gov (United States)

Ribeiro, C; Scheufele, F B; Alves, H J; Kroumov, A D; Espinoza-Quiñones, F R; Módenes, A N; Borba, C E

2018-02-26

This work focused in the evaluation of Oreochromis niloticus fish scales (FS) as biosorbent material in the removal of Zn from a synthetic effluent based on automotive battery industry effluent and, further, a hybrid neutralization/biosorption process, aiming at a high-quality treated effluent, by a cooperative use of dolomite and FS. For this, a physicochemical and morphological characterization (i.e. SEM-EDX, FTIR, XRD, and TXRF) was performed, which helped to clarify a great heterogeneity of active sites (phosphate, carbonate, amide, and hydroxyl) on the biosorbent; also the inorganic constituents (apatites) leaching from the FS was identified. Biosorption results pointed out to a pH-dependent process due to changes in the functional group's anionic character (i.e. electrostatic interactions), where an initial pH = 3 favored the Zn uptake. Kinetic and equilibrium studies confirmed the heterogeneous surface and cooperative sorption, wherein experimental data were described by Generalized Elovich kinetic model and the favorable isotherm profile by Langmuir-Freundlich isotherm ([Formula: see text] = 15.38 mg g -1 and [Formula: see text]). Speciation diagram of Zn species along with the leached species demonstrated that, for the studied pH range, the biosorption was the most likely phenomena rather than precipitation. Finally, the hybrid neutralization/biosorption process showed great potential since both the Zn concentration levels and the pH reached the legislation standards (C Zn  = 4 mg L -1 ; pH = 5). Hence, based on the characterization and biosorption results, a comprehensive evaluation of the involved mechanisms in such complex system helped to verify the prospective of FS biosorbent for the Zn treatment from solution, in both individual and hybrid processes.

Isoenergic modification of whey protein structure by denaturation and crosslinking using transglutaminase

DEFF Research Database (Denmark)

Stender, Emil G. P.; Koutina, Glykeria; Almdal, Kristoffer

2018-01-01

Transglutaminase (TG) catalyzes formation of covalent bonds between lysine and glutamine side chains and has applications in manipulation of food structure. Physical properties of a whey protein mixture (SPC) denatured either at elevated pH or by heat-treatment and followed by TG catalyzed...

A Role For Ca 2+ in the Thermal and Urea Denaturation of ...

African Journals Online (AJOL)

Giant African snail (Achatina achatina) becomes dormant (aestivate) under harsh environmental conditions like dry seasons. During this period the animal accumulates urea and is faced with thermal death. The stability towards thermal and urea denaturation of haemocyanin from aestivating and nonaestivating A. achatina ...

Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota.

Science.gov (United States)

Hoyles, Lesley; Clear, Jessica A; McCartney, Anne L

2013-08-01

With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intensity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content.

Science.gov (United States)

Fazi, Stefano; Amalfitano, Stefano; Pizzetti, Ilaria; Pernthaler, Jakob

2007-09-01

We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased from dry to wet sediments, showing a positive correlation with the community activity measured via incorporation of (3)H leucine. FISH and signal amplification by catalyzed reporter deposition (CARD-FISH) could significantly better detect cells with low activity in dried sediments. Through the application of an optimized cell permeabilization protocol, the percentage of hybridized cells by CARD-FISH showed comparable values in dry and wet conditions. This approach was unrelated to (3)H leucine incorporation rates. Moreover, the optimized protocol allowed a significantly better visualization of Gram-positive Actinobacteria in the studied samples. CARD-FISH is, therefore, proposed as an effective technique to compare bacterial communities residing in sediments with contrasting water content, irrespective of differences in the activity state of target cells. Considering the increasing frequencies of flood and drought cycles in European temporary rivers, our approach may help to better understand the dynamics of microbial communities in such systems.

Uranium production in thorium/denatured uranium fueled PWRs

International Nuclear Information System (INIS)

Arthur, W.B.

1977-01-01

Uranium-232 buildup in a thorium/denatured uranium fueled pressurized water reactor, PWR(Th), was studied using a modified version of the spectrum-dependent zero dimensional depletion code, LEOPARD. The generic Combustion Engineering System 80 reactor design was selected as the reactor model for the calculations. Reactors fueled with either enriched natural uranium and self-generated recycled uranium or uranium from a thorium breeder and self-generated recycled uranium were considered. For enriched natural uranium, concentrations of 232 U varied from about 135 ppM ( 232 U/U weight basis) in the zeroth generation to about 260 ppM ( 232 U/U weight basis) at the end of the fifth generation. For the case in which thorium breeder fuel (with its relatively high 232 U concentration) was used as reactor makeup fuel, concentrations of 232 U varied from 441 ppM ( 232 U/U weight basis) at discharge from the first generation to about 512 ppM ( 232 U/U weight basis) at the end of the fifth generation. Concentrations in freshly fabricated fuel for this later case were 20 to 35% higher than the discharge concentration. These concentrations are low when compared to those of other thorium fueled reactor types (HTGR and MSBR) because of the relatively high 238 U concentration added to the fuel as a denaturant. Excellent agreement was found between calculated and existing experimental values. Nevertheless, caution is urged in the use of these values because experimental results are very limited, and the relevant nuclear data, especially for 231 Pa and 232 U, are not of high quality

Effects of high pressure freezing (HPF) on denaturation of natural actomyosin extracted from prawn (Metapenaeus ensis).

Science.gov (United States)

Cheng, Lina; Sun, Da-Wen; Zhu, Zhiwei; Zhang, Zhihang

2017-08-15

Effects of protein denaturation caused by high pressure freezing, involving Pressure-Factors (pressure, time) and Freezing-Factors (temperature, phase transition, recrystallization, ice crystal types), are complicated. In the current study, the conformation and functional changes of natural actomyosin (NAM) under pressure assisted freezing (PAF, 100,150,300,400,500MPa P -20°C/25min ), pressure shift freezing (PSF, 200MPa P -20°C/25min ), and immersion freezing ( 0.1MPa P -20°C/5min ) after pressure was released to 0.1MPa, as compared to normal immersion freezing process (IF, 0.1MPa P -20°C/30min ). Results indicated that PSF ( 200MPa P -20°C/30min ) could reduce the denaturation of frozen NAM and a pressure of 300MPa was the critical point to induce such a denaturation. During the periods of B→D in PSF or B→C→D in PAF, the generation and growth of ice crystals played an important role on changing the secondary and tertiary structure of the treated NAM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Clinical consequences of using PNA-FISH in Staphylococcal bacteraemia

DEFF Research Database (Denmark)

Laub, R R; Knudsen, Inge Jenny Dahl

2014-01-01

To optimize patient treatment and rational use of antimicrobials, it is important to provide fast information on findings in blood-cultures (BCs). The purpose of this study was to evaluate the impact of using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) on positive BCs conta...

Cytogenetics of Festulolium (Festuca x Lolium hybrids)

Czech Academy of Sciences Publication Activity Database

Kopecký, David; Lukaszewski, A.J.; Doležel, Jaroslav

2008-01-01

Roč. 120, 3-4 (2008), s. 370-383 ISSN 1424-8581 R&D Projects: GA ČR GP521/07/P479; GA MZe QH71267 Institutional research plan: CEZ:AV0Z50380511 Keywords : Grasses * hybrids * FISH Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.965, year: 2008

Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis.

Science.gov (United States)

Nikolakakis, K; Lehnert, E; McFall-Ngai, M J; Ruby, E G

2015-07-01

The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

Science.gov (United States)

WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

Preparation of denatured protein bone sterilized with gamma radiation; Preparacion de hueso desproteinizado esterilizado con radiacion gamma

Energy Technology Data Exchange (ETDEWEB)

Luna Z, D [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

2005-07-01

The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

Preparation of denatured protein bone sterilized with gamma radiation; Preparacion de hueso desproteinizado esterilizado con radiacion gamma

Energy Technology Data Exchange (ETDEWEB)

Luna Z, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)]. e-mail: dlz@nuclear.inin.mx

2005-07-01

The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

Science.gov (United States)

Kim, Jeongyong; Song, Hugeun; Park, Inho; Carlisle, Christine R; Bonin, Keith; Guthold, Martin

2011-03-01

Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers. Copyright © 2010 Wiley-Liss, Inc.

Teaching what one does not know: strangeness and denaturation in (autobiographical narrations

Directory of Open Access Journals (Sweden)

Jorge Luiz da Cunha

2014-01-01

Full Text Available The thematic focus in this text are the estrangement/denaturation processes in (autobiographical narrations. The aim of this study was to reflect on the possibility to promote estrangement/denatura - tion in (autobiographical writings made by teenagers in the space/ time of the classroom environment. The methodological proposal consisted on developing (autobiographical writings by students from sociology classes in High School. A total of 138 teenagers from a public school, attending the first school trimester in the year 2013, have participated in the study. The concepts of estrangement/de - naturation are located in the anthropology field and, the work with (autobiographical narrations is located in the socio-clinic perspec - tives and of biographization processes. The results indicate that (autobiographical narrations provide estrangements/denaturation and go towards teaching what one does not know. We can, then, conclude that this possibility, as an educational act, may generate knowledge suspension to self-inventiveness.

Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation.

Science.gov (United States)

Visentin, Jonathan; Guidicelli, Gwendaline; Moreau, Jean-François; Lee, Jar-How; Taupin, Jean-Luc

2015-07-01

Anti-HLA donor-specific antibodies are deleterious for organ transplant survival. Class I HLA donor-specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti-denatured HLA antibodies (anti-dHLAs). Anti-dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti-dHLAs to be discriminated from anti-nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid-treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti-dHLAs. However, some anti-dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti-nHLAs conserved significant reactivity toward acid-treated LSAB. After depleting serum anti-nHLA reactivity with HLA-typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid-treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti-nHLAs and anti-dHLAs, or anti-nHLAs recognizing acid-resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti-HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti-HLA antibodies in human serum. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of Fluorescence In Situ Hybridization and Chromogenic In Situ Hybridization for Low and High Throughput HER2 Genetic Testing

Science.gov (United States)

Poulsen, Tim S.; Espersen, Maiken L. M.; Kofoed, Vibeke; Dabetic, Tanja; Høgdall, Estrid; Balslev, Eva

2013-01-01

The purpose was to evaluate and compare 5 different HER2 genetic assays with different characteristics that could affect the performance to analyze the human epidermal growth factor 2 (HER2) gene copy number under low and high throughput conditions. The study included 108 tissue samples from breast cancer patients with HER2 immunohistochemistry (IHC) results scored as 0/1+, 2+, and 3+. HER2 genetic status was analysed using chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH). Scoring results were documented through digital image analysis. The cancer region of interest was identified from a serial H&E stained slide following tissue cores were transferred to a tissue microarrays (TMA). When using TMA in a routine flow, all patients will be tested for HER2 status with IHC followed by CISH or FISH, thereby providing individual HER2 results. In conclusion, our results show that the differences between the HER2 genetic assays do not have an effect on the analytic performance and the CISH technology is superior to high throughput HER2 genetic testing due to scanning speed, while the IQ-FISH may still be a choice for fast low throughput HER2 genetic testing. PMID:24383005

Establishment of 60Co dose calibration curve using fluorescent in situ hybridization assay technique: Result of preliminary study

International Nuclear Information System (INIS)

Rahimah Abdul Rahim; Noriah Jamal; Noraisyah Mohd Yusof; Juliana Mahamad Napiah; Nelly Bo Nai Lee

2010-01-01

This study aims at establishing an in-vitro 60 Co dose calibration curve using Fluorescent In-Situ Hybridization assay technique for the Malaysian National Bio dosimetry Laboratory. Blood samples collected from a female healthy donor were irradiated with several doses of 60 Co radiation. Following culturing of lymphocytes, microscopic slides are prepared, denatured and hybridized. The frequencies of translocation are estimated in the metaphases. A calibration curve was then generated using a regression technique. It shows a good fit to a linear-quadratic model. The results of this study might be useful in estimating absorbed dose for the individual exposed to ionizing radiation retrospectively. This information may be useful as a guide for medical treatment for the assessment of possible health consequences. (author)

Sugar Cane Genome Numbers Assumption by Ribosomal DNA FISH Techniques

NARCIS (Netherlands)

Thumjamras, S.; Jong, de H.; Iamtham, S.; Prammanee, S.

2013-01-01

Conventional cytological method is limited for polyploidy plant genome study, especially sugar cane chromosomes that show unstable numbers of each cultivar. Molecular cytogenetic as fluorescent in situ hybridization (FISH) techniques were used in this study. A basic chromosome number of sugar cane

San Juanico Hybrid System Technical and Institutional Assessment: Preprint

Energy Technology Data Exchange (ETDEWEB)

Corbus, D.; Newcomb, C.; Yewdall, Z.

2004-07-01

San Juanico is a fishing village of approximately 120 homes in the Municipality of Comondu, Baja California. In April, 1999, a hybrid power system was installed in San Juanico to provide 24-hour power, which was not previously available. Before the installation of the hybrid power system, a field study was conducted to characterize the electrical usage and institutional and social framework of San Juanico. One year after the installation of the hybrid power system a''post-electrification'' study was performed to document the changes that had occurred after the installation. In December of 2003, NREL visited the site to conduct a technical assessment of the system.

Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

Science.gov (United States)

Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A

2016-01-01

Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (β-glucosidase, β-d-cellobiosidase, N-acetyl-β-glucosaminidase, phosphatase, leucine aminopeptidase, β-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and β-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for β-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar. Copyright © 2015 Elsevier Ltd. All rights reserved.

Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA

Science.gov (United States)

Mielke, Steven P.; Grønbech-Jensen, Niels; Krishnan, V. V.; Fink, William H.; Benham, Craig J.

2005-09-01

The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

Science.gov (United States)

Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno; Madureira, Pedro; Ferreira, Rui Manuel; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

2015-01-01

In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization. PMID:25915865

Towards Fluorescence In Vivo Hybridization (FIVH Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes.

Directory of Open Access Journals (Sweden)

Sílvia Fontenete

Full Text Available In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA/ 2' O-methyl RNA (2'OMe probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH. In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.

Micro-CT imaging of denatured chitin by silver to explore honey bee and insect pathologies.

Directory of Open Access Journals (Sweden)

Peter R Butzloff

Full Text Available BACKGROUND: Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term "denatured chitin" calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. METHODOLOGY/PRINCIPAL FINDINGS: A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT. Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi, at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. CONCLUSIONS/SIGNIFICANCE: The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may

Micro-CT Imaging of Denatured Chitin by Silver to Explore Honey Bee and Insect Pathologies

Science.gov (United States)

Butzloff, Peter R.

2011-01-01

Background Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term “denatured chitin” calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. Methodology/Principal Findings A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT). Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi), at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. Conclusions/Significance The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may provide an

Evaluation of Myc Gene Amplification in Prostate Cancer Using a Dual Color Chromogenic in-Situ Hybridization (Dual CISH) Assay

OpenAIRE

Daniel Lerda; Marta Cabrera; Jorge Flores; Luis Gutierrez; Armando Chierichetti; Martin Revol; Hernan Garcia Onto

2013-01-01

Objetive: The overall purpose of the study was to demonstrate applicability of the Dako dual-color chromogenic in situ hybridization (CISH) assay (DAKO Denmark, Glostrup) with respect to fluorescence in situ hybridization (FISH) probes MYC-C. Methods: MYC gene amplification by FISH and Dako dual-color CISH Results: The study showed that the dual-color CISH assay can convert Texas red and fluorescein isothiocyanate (FITC) signals into chromogenic signals. The dual –color CISH assay was p...

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

Science.gov (United States)

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

Fish sarcoplasmic proteins as a high value marine material for wound dressing applications.

Science.gov (United States)

Vieira, Sara; Franco, Albina R; Fernandes, Emanuel M; Amorim, Sara; Ferreira, Helena; Pires, Ricardo A; Reis, Rui L; Martins, Albino; Neves, Nuno M

2018-07-01

Fish sarcoplasmic proteins (FSP) constitute around 25-30% of the total fish muscle protein. As the FSP are water soluble, FSP were isolated from fresh cod (Gadus morhua) by centrifugation. By SDS-PAGE, it was possible to determine the composition of FSP extracts (FSP-E). The FSP-E undergo denaturation at 44.12 ± 2.34° C, as characterized by differential scanning calorimetry thermograms (DSC). The secondary structure of FSP-E is mainly composed by α-helix structure, as determined by circular dichroism. The cytocompatibility of FSP-E, at concentrations ranging from 5 to 20 mg/mL, was investigated. Concentrations lower than 10 mg/mL have no cytotoxicity cultures of fibroblasts over 72 h. Further on, FSP membranes (FSP-M) were produced by spin coating to evaluate its properties. FSP-M shown having uniform surface as analyzed by Scanning Electron Microscopy (SEM). The relative amount of α-helix structures is higher when compared with the FSP-E. The FSP-M have higher temperature stability than the FSP-E, since they presented a denaturation temperature of 58.88 ± 3.36° C, according to the DSC analysis. FSP-M shown distinctive mechanical properties, with a stiffness of 16.57 ± 3.95 MPa and a yield strength of 23.85 ± 5.97 MPa. Human lung fibroblasts cell lines (MRC-5) were cultured in direct contact with FSP-M, demonstrating its cytocompatibility for 48 h. Based on these results, FSP can be considered a potential biomaterial recovered from nature, for wound dressing applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Immunoglobulin heavy-chain fluorescence in situ hybridization-chromogenic in situ hybridization DNA probe split signal in the clonality assessment of lymphoproliferative processes on cytological samples.

Science.gov (United States)

Zeppa, Pio; Sosa Fernandez, Laura Virginia; Cozzolino, Immacolata; Ronga, Valentina; Genesio, Rita; Salatiello, Maria; Picardi, Marco; Malapelle, Umberto; Troncone, Giancarlo; Vigliar, Elena

2012-12-25

The human immunoglobulin heavy-chain (IGH) locus at chromosome 14q32 is frequently involved in different translocations of non-Hodgkin lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B-cell NHL. The split-signal IGH fluorescence in situ hybridization-chromogenic in situ hybridization (FISH-CISH) DNA probe is a mixture of 2 fluorochrome-labeled DNAs: a green one that binds the telomeric segment and a red one that binds the centromeric segment, both on the IGH breakpoint. In the current study, the authors tested the capability of the IGH FISH-CISH DNA probe to detect IGH translocations and diagnose B-cell lymphoproliferative processes on cytological samples. Fifty cytological specimens from cases of lymphoproliferative processes were tested using the split-signal IGH FISH-CISH DNA probe and the results were compared with light-chain assessment by flow cytometry (FC), IGH status was tested by polymerase chain reaction (PCR), and clinicohistological data. The signal score produced comparable results on FISH and CISH analysis and detected 29 positive, 15 negative, and 6 inadequate cases; there were 29 true-positive cases (66%), 9 true-negative cases (20%), 6 false-negative cases (14%), and no false-positive cases (0%). Comparing the sensitivity of the IGH FISH-CISH DNA split probe with FC and PCR, the highest sensitivity was obtained by FC, followed by FISH-CISH and PCR. The split-signal IGH FISH-CISH DNA probe is effective in detecting any translocation involving the IGH locus. This probe can be used on different samples from different B-cell lymphoproliferative processes, although it is not useful for classifying specific entities. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society. Copyright © 2012 American Cancer Society.

Thermal denaturation of A-DNA

International Nuclear Information System (INIS)

Valle-Orero, J; Wildes, A R; Theodorakopoulos, N; Cuesta-López, S; Peyrard, M; Garden, J-L; Danilkin, S

2014-01-01

The DNA molecule can take various conformational forms. Investigations focus mainly on the so-called ‘B-form’, schematically drawn in the famous paper by Watson and Crick [1]. This is the usual form of DNA in a biological environment and is the only form that is stable in an aqueous environment. Other forms, however, can teach us much about DNA. They have the same nucleotide base pairs for ‘building blocks’ as B-DNA, but with different relative positions, and studying these forms gives insight into the interactions between elements under conditions far from equilibrium in the B-form. Studying the thermal denaturation is particularly interesting because it provides a direct probe of those interactions which control the growth of the fluctuations when the ‘melting’ temperature is approached. Here we report such a study on the ‘A-form’ using calorimetry and neutron scattering. We show that it can be carried further than a similar study on B-DNA, requiring the improvement of thermodynamic models for DNA. (paper)

A hybrid ARIMA and neural network model applied to forecast catch volumes of Selar crumenophthalmus

Science.gov (United States)

Aquino, Ronald L.; Alcantara, Nialle Loui Mar T.; Addawe, Rizavel C.

2017-11-01

The Selar crumenophthalmus with the English name big-eyed scad fish, locally known as matang-baka, is one of the fishes commonly caught along the waters of La Union, Philippines. The study deals with the forecasting of catch volumes of big-eyed scad fish for commercial consumption. The data used are quarterly caught volumes of big-eyed scad fish from 2002 to first quarter of 2017. This actual data is available from the open stat database published by the Philippine Statistics Authority (PSA)whose task is to collect, compiles, analyzes and publish information concerning different aspects of the Philippine setting. Autoregressive Integrated Moving Average (ARIMA) models, Artificial Neural Network (ANN) model and the Hybrid model consisting of ARIMA and ANN were developed to forecast catch volumes of big-eyed scad fish. Statistical errors such as Mean Absolute Errors (MAE) and Root Mean Square Errors (RMSE) were computed and compared to choose the most suitable model for forecasting the catch volume for the next few quarters. A comparison of the results of each model and corresponding statistical errors reveals that the hybrid model, ARIMA-ANN (2,1,2)(6:3:1), is the most suitable model to forecast the catch volumes of the big-eyed scad fish for the next few quarters.

Preparation of denatured sup(99m)Tc labeled HSA aerosols of different median diameters for various imaging studies

Energy Technology Data Exchange (ETDEWEB)

Raghunath, B.; Kotrappa, P.; Soni, P.S.; Ganatra, R.D. (Bhabha Atomic Research Centre, Bombay (India))

1982-02-01

The preparation of denatured sup(99m)Tc-labelled human serum albumin (HSA) aerosols of different median diameters is described using the BARC (Bhabha Atomic Research Centre) dry aerosol generation and delivery system. The applications of these radioactive aerosols are demonstrated in aerosol scintigraphy of lungs, mucociliary movement studies and lymphoscintigraphy in rabbits. It is concluded that the BARC system gives a simplified, rapid and versatile procedure for generation of denatured volume tagged HSA aerosols for a variety of clinical applications.

Preparation of denatured sup(99m)Tc labeled HSA aerosols of different median diameters for various imaging studies

International Nuclear Information System (INIS)

Raghunath, B.; Kotrappa, P.; Soni, P.S.; Ganatra, R.D.

1982-01-01

The preparation of denatured sup(99m)Tc-labelled human serum albumin (HSA) aerosols of different median diameters is described using the BARC (Bhabha Atomic Research Centre) dry aerosol generation and delivery system. The applications of these radioactive aerosols are demonstrated in aerosol scintigraphy of lungs, mucociliary movement studies and lymphoscintigraphy in rabbits. It is concluded that the BARC system gives a simplified, rapid and versatile procedure for generation of denatured volume tagged HSA aerosols for a variety of clinical applications. (U.K.)

On the mobility of partially denatured DNA in gel electrophoresis: a theoretical investigation

Science.gov (United States)

Sean, David

There are technologies which exploit a rapid reduction of the gel electrophoretic mobility of DNA arising from partial denaturation. The underlying phenomenon behind these experiments---the mechanisms which reduce the mobility---are not very well understood. Such is the purpose of my thesis. The first chapter provides a brief introduction to the field of polymer physics. The subjects covered are carefully chosen to directly relate to the forthcoming research. There is a published semi-empirical formula used to model the rapid decrease of mobility which is largely considered to be consistent with experimental data. The second chapter of this thesis demonstrates that there is a fundamental confusion in the literature regarding the fitting parameter Lr, in the said formula. By going back to the original derivation, a physical interpretation can be given to L r. This interpretation yields theoretical values which are consistent with what has been published. However, we find that an underlying assumption---that the effect of the denaturation does not depend on its position along the DNA fragment---may systematically overestimate experimental observations of Lr. To measure the impact of this assumption, a simulation model of DNA is presented. The article presented in the third chapter reveals that indeed, the position of the denatured region affects the migration of the DNA fragment. A refined version of the formula which takes these factors into account is proposed. The simulations also reveal that, for certain fields, an unexpected conformation completely dominates during migration of the fragment. This surprising result: a squid-like conformation, is explored in chapter four.

A novel hybridization approach for detection of citrus viroids.

Science.gov (United States)

Murcia, N; Serra, P; Olmos, A; Duran-Vila, N

2009-04-01

Citrus plants are natural hosts of several viroid species all belonging to the family Pospiviroidae. Previous attempts to detect viroids from field-grown species and cultivars yielded erratic results unless analyses were performed using Etrog citron a secondary bio-amplification host. To overcome the use of Etrog citron a number of RT-PCR approaches have been proposed with different degrees of success. Here we report the suitability of an easy to handle northern hybridization protocol for viroid detection of samples collected from field-grown citrus species and cultivars. The protocol involves: (i) Nucleic acid preparations from bark tissue samples collected from field-grown trees regardless of the growing season and storage conditions; (ii) Separation in 5% PAGE or 1% agarose, blotting to membrane and fixing; (iii) Hybridization with viroid-specific DIG-labelled probes and detection with anti-DIG-alkaline phosphatase conjugate and autoradiography with the CSPD substrate. The method has been tested with viroid-infected trees of sweet orange, lemon, mandarin, grapefruit, sour orange, Swingle citrumello, Tahiti lime and Mexican lime. This novel hybridization approach is extremely sensitive, easy to handle and shortens the time needed for reliable viroid indexing tests. The suitability of PCR generated DIG-labelled probes and the sensitivity achieved when the samples are separated and blotted from non-denaturing gels are discussed.

Denatured protein-coated docetaxel nanoparticles: Alterable drug state and cytosolic delivery.

Science.gov (United States)

Zhang, Li; Xiao, Qingqing; Wang, Yiran; Zhang, Chenshuang; He, Wei; Yin, Lifang

2017-05-15

Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

Science.gov (United States)

Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

2015-02-01

In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet. Copyright © 2015 Elsevier Inc. All rights reserved.

EGFR status in oral squamous cell carcinoma: comparing immunohistochemistry, FISH and CISH detection in a case series study.

Science.gov (United States)

Bernardes, Vanessa Fátima; Gleber-Netto, Frederico Omar; Sousa, Sílvia Ferreira de; Rocha, Rafael Malagoli; Aguiar, Maria Cássia Ferreira de

2013-01-28

To compare the immunohistochemistry (IHC) expression of epidermal growth factor receptor (EGFR) in oral squamous cell carcinomas (OSCC) with the gene amplification evaluated by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) and their association with clinicopathological parameters. Additionally, we tested the sensibility and specificity of CISH in comparison with FISH. Case series study Oral surgery and pathology department in a school of dentistry. 52 patients with histopathological diagnosis of OSCC. Tumour tissue samples from 52 patients with OSCC were evaluated by IHC, FISH and CISH using tissue microarray technology. Clinicopathological data from all patients were collected. EGFR+ rates were 53.8% (28/52) by IHC, 5.8% (3/52) by CISH and 15.4% (8/52) by FISH. Amplification detected by CISH and FISH with IHC negative occurred in 3.8% (2/52), and one case (1.9%) showed amplification detected by CISH and FISH and protein overexpression concomitantly. There were 9.6% FISH+ cases with IHC and CISH negative rates and 6/8 (75%) FISH+ and also EGFR+ cases; however, an association between protein expression and gene amplification was not found for both techniques. IHC and FISH rates were not associated with clinicopathological features. CISH+ rates were associated with T3-T4 status. Compared with FISH assay, CISH reached a sensitivity of 37.5% and specificity of 100%. There is no association between EGFR expression and gene amplification in OSCC when the IHC is driven to external epitopes of the protein. Although CISH demonstrates specificity, technical problems may influence sensibility when compared with FISH.

Strategies for denaturing the weapons-grade plutonium stockpile

International Nuclear Information System (INIS)

Buckner, M.R.; Parks, P.B.

1992-10-01

In the next few years, approximately 50 metric tons of weapons-grade plutonium and 150 metric tons of highly-enriched uranium (HEU) may be removed from nuclear weapons in the US and declared excess. These materials represent a significant energy resource that could substantially contribute to our national energy requirements. HEU can be used as fuel in naval reactors, or diluted with depleted uranium for use as fuel in commercial reactors. This paper proposes to use the weapons-grade plutonium as fuel in light water reactors. The first such reactor would demonstrate the dual objectives of producing electrical power and denaturing the plutonium to prevent use in nuclear weapons

Single Molecular Level Probing of Structure and Dynamics of Papain Under Denaturation.

Science.gov (United States)

Sengupta, Bhaswati; Chaudhury, Apala; Das, Nilimesh; Sen, Pratik

2017-01-01

Papain is a cysteine protease enzyme present in papaya and known to help in digesting peptide. Thus the structure and function of the active site of papain is of interest. The objective of present study is to unveil the overall structural transformation and the local structural change around the active site of papain as a function of chemical denaturant. Papain has been tagged at Cys-25 with a thiol specific fluorescence probe N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA). Guanidine hydrochloride (GnHCl) has been used as the chemical denaturant. Steady state, time-resolved, and single molecular level fluorescence techniques was applied to map the change in the local environment. It is found that papain undergoes a two-step denaturation in the presence of GnHCl. Fluorescence correlation spectroscopic (FCS) data indicate that the size (hydrodynamic diameter) of native papain is ~36.8 Å, which steadily increases to ~53 Å in the presence of 6M GnHCl. FCS study also reveals that the conformational fluctuation time of papain is 6.3 µs in its native state, which decreased to 2.7 µs in the presence of 0.75 M GnHCl. Upon further increase in GnHCl concentration the conformational fluctuation time increase monotonically till 6 M GnHCl, where the time constant is measured as 14 µs. On the other hand, the measurement of ellipticity, hence the helical structure, by circular dichroism spectroscopy is found to be incapable to capture such structural transformation. It is concluded that in the presence of small amount of GnHCl the active site of papain takes up a more compact structure (although the overall size increases) than in the native state, which has been designated as the intermediate state. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Fluorescence in situ hybridization (FISH screening for the 22q11.2 deletion in patients with clinical features of velocardiofacial syndrome but without cardiac anomalies

Directory of Open Access Journals (Sweden)

Paula Sandrin-Garcia

2007-01-01

Full Text Available The velocardiofacial syndrome (VCFS, a condition associated with 22q11.2 deletions, is characterized by a typical facies, palatal anomalies, learning disabilities, behavioral disturbances and cardiac defects. We investigated the frequency of these chromosomal deletions in 16 individuals with VCFS features who presented no cardiac anomalies, one of the main characteristics of VCFS. Fluorescent in situ hybridization (FISH with the N25 (D22S75; 22q11.2 probe revealed deletions in ten individuals (62%. Therefore, even in the absence of cardiac anomalies testing for the 22q11.2 microdeletions in individuals showing other clinical features of this syndrome is recommended.

Phylogeny and FISH probe analysis of the “Candidatus Competibacter”-lineage in wastewater treatment systems

DEFF Research Database (Denmark)

Nittami, Tadashi; McIlroy, Simon Jon; Kanai, Eri

Our understanding of the microbial ecology of enhanced biological phosphorus removal (EBPR) wastewater treatment systems has been greatly advanced through the application of molecular methods such as fluorescence in situ hybridization (FISH). Considerable attention has been directed at the identi......Our understanding of the microbial ecology of enhanced biological phosphorus removal (EBPR) wastewater treatment systems has been greatly advanced through the application of molecular methods such as fluorescence in situ hybridization (FISH). Considerable attention has been directed...... at the identification and characterization of the glycogen accumulating organisms (GAO), a phenotypic group thought to compete with the polyphosphate accumulating organisms (PAO) for resources at the theoretical expense of EBPR efficiency. Demonstrated candidates for members of the GAO phenotype include...... the gammaproteobacterial “Candidatus Competibacter”-lineage. The group is currently delineated by 8 FISH probe defined phylotypes, although further undescribed phylogenetic diversity beyond what is covered by these probes is evident. Where studied, marked differences in physiology between members are observed, including...

Cooperative unfolding of apolipoprotein A-1 induced by chemical denaturation.

Science.gov (United States)

Eckhardt, D; Li-Blatter, X; Schönfeld, H-J; Heerklotz, H; Seelig, J

2018-05-25

Apolipoprotein A-1 (Apo A-1) plays an important role in lipid transfer and obesity. Chemical unfolding of α-helical Apo A-1 is induced with guanidineHCl and monitored with differential scanning calorimetry (DSC) and CD spectroscopy. The unfolding enthalpy and the midpoint temperature of unfolding decrease linearly with increasing guanidineHCl concentration, caused by the weak binding of denaturant. At room temperature, binding of 50-60 molecules guanidineHCl leads to a complete Apo A-1 unfolding. The entropy of unfolding decreases to a lesser extent than the unfolding enthalpy. Apo A-1 chemical unfolding is a dynamic multi-state equilibrium that is analysed with the Zimm-Bragg theory modified for chemical unfolding. The chemical Zimm-Bragg theory predicts the denaturant binding constant K D and the protein cooperativity σ. Chemical unfolding of Apo A-1 is two orders of magnitude less cooperative than thermal unfolding. The free energy of thermal unfolding is ~0.2 kcal/mol per amino acid residue and ~1.0 kcal/mol for chemical unfolding at room temperature. The Zimm-Bragg theory calculates conformational probabilities and the chemical Zimm-Bragg theory predicts stretches of α-helical segments in dynamic equilibrium, unfolding and refolding independently and fast. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Supernumerary ring chromosome 20 characterized by fluorescence in situ hybridization

NARCIS (Netherlands)

Van Langen, Irene M.; Otter, Mariëlle A.; Aronson, Daniël C.; Overweg-Plandsoen, W.C.G.; Hennekam, Raoul C.M.; Leschot, Nico J.; Hoovers, Jan M.N.

1996-01-01

We report on a boy with mild dysmorphic features and developmental delay, in whom karyotyping showed an additional minute ring chromosome in 60% of metaphases. Fluorescence in situ hybridization (FISH) with a centromere specific probe demonstrated that the ring chromosome contained the centromeric

Vibrational energy relaxation: proposed pathway of fast local chromatin denaturation

International Nuclear Information System (INIS)

Harder, D.; Greinert, R.

2002-01-01

The molecular mechanism responsible for the a component of exchange-type chromosome aberrations, of chromosome fragmentation and of reproductive cell death is one of the unsolved issues of radiation biology. Under review is whether vibrational energy relaxation in the constitutive biopolymers of chromatin, induced by inelastic energy deposition events and mediated via highly excited vibrational states, may provide a pathway of fast local chromatin denaturation, thereby producing the severe DNA lesion able to interact chemically with other, non-damaged chromatin. (author)

Simulated pressure denaturation thermodynamics of ubiquitin.

Science.gov (United States)

Ploetz, Elizabeth A; Smith, Paul E

2017-12-01

Simulations of protein thermodynamics are generally difficult to perform and provide limited information. It is desirable to increase the degree of detail provided by simulation and thereby the potential insight into the thermodynamic properties of proteins. In this study, we outline how to analyze simulation trajectories to decompose conformation-specific, parameter free, thermodynamically defined protein volumes into residue-based contributions. The total volumes are obtained using established methods from Fluctuation Solution Theory, while the volume decomposition is new and is performed using a simple proximity method. Native and fully extended ubiquitin are used as the test conformations. Changes in the protein volumes are then followed as a function of pressure, allowing for conformation-specific protein compressibility values to also be obtained. Residue volume and compressibility values indicate significant contributions to protein denaturation thermodynamics from nonpolar and coil residues, together with a general negative compressibility exhibited by acidic residues. Copyright © 2017 Elsevier B.V. All rights reserved.

Denaturing gradient gel electrophoresis

International Nuclear Information System (INIS)

Kocherginskaya, S.A.; Cann, I.K.O.; Mackie, R.I.

2005-01-01

It is worthwhile considering that only some 30 species make up the bulk of the bacterial population in human faeces at any one time based on the classical cultivation-based approach. The situation in the rumen is similar. Thus, it is practical to focus on specific groups of interest within the complex community. These may be the predominant or the most active species, specific physiological groups or readily identifiable (genetic) clusters of phylogenetically related organisms. Several 16S rDNA fingerprinting techniques can be invaluable for selecting and monitoring sequences or phylogenetic groups of interest and are described below. Over the past few decades, considerable attention was focussed on the identification of pure cultures of microbes on the basis of genetic polymorphisms of DNA encoding rRNA such as ribotyping, amplified fragment length polymorphism and randomly amplified polymorphic DNA. However, many of these methods require prior cultivation and are less suitable for use in analysis of complex mixed populations although important in describing cultivated microbial diversity in molecular terms. Much less attention was given to molecular characterization of complex communities. In particular, research into diversity and community structure over time has been revolutionized by the advent of molecular fingerprinting techniques for complex communities. Denaturing or temperature gradient gel electrophoresis (DGGE/TGGE) methods have been successfully applied to the analysis of human, pig, cattle, dog and rodent intestinal populations

Raman spectral markers of collagen denaturation and hydration in human cortical bone tissue are affected by radiation sterilization and high cycle fatigue damage.

Science.gov (United States)

Flanagan, Christopher D; Unal, Mustafa; Akkus, Ozan; Rimnac, Clare M

2017-11-01

Thermal denaturation and monotonic mechanical damage alter the organic and water-related compartments of cortical bone. These changes can be detected using Raman spectroscopy. However, less is known regarding Raman sensitivity to detect the effects of cyclic fatigue damage and allograft sterilization doses of gamma radiation. To determine if Raman spectroscopic biomarkers of collagen denaturation and hydration are sensitive to the effects of (a) high cycle fatigue damage and (b) 25kGy irradiation. Unirradiated and gamma-radiation sterilized human cortical bone specimens previously tested in vitro under high-cycle (> 100,000 cycles) fatigue conditions at 15MPa, 25MPa, 35MPa, 45MPa, and 55MPa cyclic stress levels were studied. Cortical bone Raman spectral profiles from wavenumber ranges of 800-1750cm -1 and 2700-3800cm -1 were obtained and compared from: a) non-fatigue vs fatigue fracture sites and b) radiated vs. unirradiated states. Raman biomarker ratios 1670/1640 and 3220/2949, which reflect collagen denaturation and organic matrix (mainly collagen)-bound water, respectively, were assessed. One- and two-way ANOVA analyses were utilized to identify differences between groups along with interaction effects between cyclic fatigue and radiation-induced damage. Cyclic fatigue damage resulted in increases in collagen denaturation (1670/1640: 1.517 ± 0.043 vs 1.579 ± 0.021, p Raman spectroscopy can detect the effects of cyclic fatigue damage and 25kGy irradiation via increases in organic matrix (mainly collagen)-bound water. A Raman measure of collagen denaturation was sensitive to cyclic fatigue damage but not 25kGy irradiation. Collagen denaturation was correlated with organic matrix-bound water, suggesting that denaturation of collagen to gelatinous form may expose more binding sites to water by unwinding the triple alpha chains. This research may eventually be useful to help identify allograft quality and more appropriately match donors to recipients. Copyright �

Potential for bias in using hybrids between common carp (Cyprinus carpio) and goldfish (Carassius auratus) in endocrine studies: a first report of hybrids in Lake Mead, Nevada, U.S.A

Science.gov (United States)

Goodbred, Steven L.; Patino, Reynaldo; Orsak, Erik; Sharma, Prakash; Ruessler, Shane

2013-01-01

During a 2008 study to assess endocrine and reproductive health of common carp (Cyprinus carpio) in Lake Mead, Nevada (U.S.A.) we identified two fish, one male and one female, as hybrids with goldfish (Carassius auratus) based on morphology, lateral line scale count, and lack of anterior barbels. Gross examination of the female hybrid ovaries indicated presence of vitellogenic ovarian follicles; whereas histological evaluation of the male hybrid testes showed lobule-like structures with open lumens but without germ cells, suggesting it was sterile. Because common carp/goldfish hybrids are more susceptible to gonadal tumors and may have different endocrine profiles than common carp, researchers using common carp as a model for endocrine/reproductive studies should be aware of the possible presence of hybrids.

Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations.

Science.gov (United States)

Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei

2013-05-01

In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Copyright © 2013 Wiley Periodicals, Inc.

Effects of salinity on gastric emptying time in hybrid grouper, Epinephelus fuscoguttatus x E. lanceolattus juveniles

Science.gov (United States)

Noor, Noorashikin Md.; Das, Simon Kumar; Cob, Zaidi Che; Ghaffar, Mazlan Abd.

2018-04-01

The newly developed hybrid grouper: tiger grouper (Epinephelus fuscoguttatus) × giant grouper (Epinephelus fuscoguttatus) (TG×GG), has a high resistance towards different environmental condition (eg. in euryhaline environment) due to its genetic improvement. This study aims to investigate the effects of different salinities (10, 15, 20, 25 and 30 ppt) on the gastric emptying time (GET) of the TG×GG hybrid grouper juveniles. The fish were fed with commercial pellet over a 30 days experimental period under controlled laboratory conditions. The GET was determined by X-radiographic method, using barium sulfate (BaSO4) as an inert food marker. The X-radiography images showed that the shortest GET (12 h) was observed in the 15 ppt group, whereas the longest GET (18 h) in 30 ppt group. The results suggests to culture TG×GG hybrid grouper juveniles in 15 ppt with commercial pellet diet as this salinity proliferates faster digestion process which may contribute faster growth rate of this important fish species. Overall, these findings would be useful for the betterment of TG×GG hybrid grouper aquaculture which will eventually boost up the production of this newly developed hybrid grouper species.

Mate Choice Drives Evolutionary Stability in a Hybrid Complex.

Directory of Open Access Journals (Sweden)

Miguel Morgado-Santos

Full Text Available Previous studies have shown that assortative mating acts as a driver of speciation by countering hybridization between two populations of the same species (pre-zygotic isolation or through mate choice among the hybrids (hybrid speciation. In both speciation types, assortative mating promotes speciation over a transient hybridization stage. We studied mate choice in a hybrid vertebrate complex, the allopolyploid fish Squalius alburnoides. This complex is composed by several genomotypes connected by an intricate reproductive dynamics. We developed a model that predicts the hybrid complex can persist when females exhibit particular mate choice patterns. Our model is able to reproduce the diversity of population dynamic outcomes found in nature, namely the dominance of the triploids and the dominance of the tetraploids, depending on female mate choice patterns and frequency of the parental species. Experimental mate choice trials showed that females exhibit the preferences predicted by the model. Thus, despite the known role of assortative mating in driving speciation, our findings suggest that certain mate choice patterns can instead hinder speciation and support the persistence of hybrids over time without speciation or extinction.

Detection of AGEs as markers for carbohydrate metabolism and protein denaturation.

Science.gov (United States)

Nagai, Ryoji; Shirakawa, Jun-Ichi; Fujiwara, Yukio; Ohno, Rei-Ichi; Moroishi, Narumi; Sakata, Noriyuki; Nagai, Mime

2014-07-01

Approximately 100 years have passed since the Maillard reaction was first reported in the field of food chemistry as a condensation reaction between reducing sugars and amino acids. This reaction is thought to progress slowly primarily from glucose with proteins in vivo. An early-stage product, called the "Amadori product", is converted into advanced glycation end products. Those accumulate in the body in accordance with age, with such accumulation being enhanced by lifestyle-related diseases that result in the denaturation of proteins. Recent studies have demonstrated that intermediate carbonyls are generated by several pathways, and rapidly generate many glycation products. However, accurate quantification of glycation products in vivo is difficult due to instability and differences in physicochemical properties. In this connection, little is known about the relationship between the structure of glycation products and pathology. Furthermore, the interaction between proteins modified by glycation and receptors for advanced glycation end products is also known to induce the production of several inflammatory cytokines. Therefore, those inhibitors have been developed over the world to prevent lifestyle-related diseases. In this review, we describe the process of protein denaturation induced by glycation and discuss the possibility of using the process as a marker of age-related diseases.

Identification of MYCN gene amplification in neuroblastoma using chromogenic in situ hybridization (CISH): an alternative and practical method.

Science.gov (United States)

Bhargava, Rohit; Oppenheimer, Orit; Gerald, William; Jhanwar, Suresh C; Chen, Beiyun

2005-06-01

Chromogenic in situ hybridization (CISH) is a recently developed technique, which utilizes the general principles of in situ hybridization and a detection system similar to immunohistochemistry. To assess the utility of CISH for analysis of MYCN gene amplification, we compared this assay with established diagnostic assays such as Southern blot analysis (SB) and fluorescent in situ hybridization (FISH). CISH was performed on 67 cases of neuroblastoma using tissue microarray (65 cases) and whole tissue sections (2 cases). Unequivocal, high-level amplification (> or =10 gene copies per tumor nucleus) was identified in 19 of 67 (28.4%) tumors. Two (3%) tumors showed low-level amplification (6-9 gene copies per tumor nucleus). No amplification was seen in 46 of 67 (68.6%) tumors. SB data were available in 44 tumors. Forty-one of the 44 tumors (93%) showed concordant results between CISH and SB. Three tumors showed MYCN amplification by CISH but no amplification by SB, most likely due to dilution effect of nonneoplastic tissue in the test samples. Two of these three tumors also showed MYCN amplification by FISH, and the third tumor was not analyzed by FISH. FISH data were available in total of 30 tumors. All 30 tumors showed concordant results between CISH and FISH for classifying a tumor as MYCN amplified or not amplified. We conclude that CISH is an accurate method for determining MYCN gene amplification, with added advantages that make it a more practically useful method.

Genetic investigation of natural hybridization between rainbow and coastal cutthroat trout in the copper River Delta, Alaska

Science.gov (United States)

Williams, I.; Reeves, G.H.; Graziano, S.L.; Nielsen, J.L.

2007-01-01

Molecular genetic methods were used to quantify natural hybridization between rainbow trout Oncorhynchus mykiss or steelhead (anadromous rainbow trout) and coastal cutthroat trout O. clarkii clarkii collected in the Copper River delta, Southeast Alaska. Eleven locations were sampled to determine the extent of hybridization and the distribution of hybrids. Four diagnostic nuclear microsatellite loci and four species-specific simple sequence repeat markers were used in combination with restriction fragment length polymorphism analyses of NADH dehydrogenase 5/6 (ND5/6) mitochondrial DNA (mtDNA) to investigate the genetic structure of trout from both species and identify putative interspecific hybrids. Hybrids were found in 7 of the 11 streams sampled in the Copper River delta, the extent of hybridization across all streams varying from 0% to 58%. Hybrid trout distribution appeared to be nonrandom, most individuals of mixed taxonomic ancestry being detected in streams containing rainbow trout rather than in streams containing coastal cutthroat trout. Genotypic disequilibrium was observed among microsatellite loci in populations with high levels of hybridization. We found no significant correlation between unique stream channel process groups and the number of hybrid fish sampled. Eighty-eight percent of fish identified as first-generation hybrids (F1) in two populations contained coastal cutthroat trout mtDNA, suggesting directionality in hybridization. However, dominance of coastal cutthroat trout mtDNA was not observed at a third location containing F1 hybrids, indicating that interspecific mating behavior varied among locations. Backcrossed individuals were found in drainages lacking F1 hybrids and in populations previously thought to contain a single species. The extent and distribution of backcrossed individuals suggested that at least some hybrids are reproductively viable and backcrossed hybrid offspring move throughout the system.

Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

NARCIS (Netherlands)

Selz, O. M.; Thommen, R.; Maan, M. E.; Seehausen, O.

Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post-mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid

Dynamic and structural study of neocarzinostatin native and denatured states, by differential microcalorimetry, optical spectroscopies and X-ray and neutron scattering

International Nuclear Information System (INIS)

Russo, Daniela

2000-01-01

A structural and dynamic characterization of proteins denatured states is essential to the understanding of mechanisms which control proteins folding. It is in this framework that this study has been undertaken in taking as model the neocarzinostatin globular protein. It is formed with seven cell-layers which form a barrel pattern maintained by two bi-sulfur bonds. A full characterization of native and denatured states, both from structural and dynamic point of view, has been implemented with several techniques able to bring data at different levels. During the experiments, ncs has been stabilized by temperature and by the use of a chaotropic agent: the guanidinium chloride (gdmcl). Small angle x-ray and neutron scattering have allowed us to obtain data on the variation of the protein compactness in terms of gdmcl temperature and concentration. The diffusion spectra show that ncs loses completely its globular structure above 80 C or in presence of about 5 m of gdmcl. Temperature and concentration of half denaturation are tm= 70 C and cm=3.5 m (in heavy water), respectively. Spectra analysis of strongly denatured protein has allowed us to obtain values of its chain length and of its persistence length which are in agreement with those theoretically estimated. Experiments have been carried out too to measure the radius of gyration to zero concentration and the second virial coefficient of the solution in order to estimate the interactions between the molecules. A full characterization has been performed in terms of gdmcl temperature and concentration by fluorescence and circular dichroism. These two techniques reveal the variations of the local three-dimensional structure and secondary structure of the protein respectively. Microcalorimetry measurements have shown that thermal denaturation of ncs is completely reversible and has been used to measure the enthalpy variation during the transition. At last, it has been possible to study ncs intramolecular dynamics in

Automated Image Analysis for Quantitative Fluorescence In Situ Hybridization with Environmental Samples▿ †

OpenAIRE

Zhou, Zhi; Pons, Marie Noëlle; Raskin, Lutgarde; Zilles, Julie L.

2007-01-01

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An au...

Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

Science.gov (United States)

Meek, Megan E; Van Dolah, Frances M

2016-05-01

Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

A hybrid genetic linkage map of two ecologically and morphologically divergent Midas cichlid fishes (Amphilophus spp.) obtained by massively parallel DNA sequencing (ddRADSeq).

Science.gov (United States)

Recknagel, Hans; Elmer, Kathryn R; Meyer, Axel

2013-01-01

Cichlid fishes are an excellent model system for studying speciation and the formation of adaptive radiations because of their tremendous species richness and astonishing phenotypic diversity. Most research has focused on African rift lake fishes, although Neotropical cichlid species display much variability as well. Almost one dozen species of the Midas cichlid species complex (Amphilophus spp.) have been described so far and have formed repeated adaptive radiations in several Nicaraguan crater lakes. Here we apply double-digest restriction-site associated DNA sequencing to obtain a high-density linkage map of an interspecific cross between the benthic Amphilophus astorquii and the limnetic Amphilophus zaliosus, which are sympatric species endemic to Crater Lake Apoyo, Nicaragua. A total of 755 RAD markers were genotyped in 343 F(2) hybrids. The map resolved 25 linkage groups and spans a total distance of 1427 cM with an average marker spacing distance of 1.95 cM, almost matching the total number of chromosomes (n = 24) in these species. Regions of segregation distortion were identified in five linkage groups. Based on the pedigree of parents to F(2) offspring, we calculated a genome-wide mutation rate of 6.6 × 10(-8) mutations per nucleotide per generation. This genetic map will facilitate the mapping of ecomorphologically relevant adaptive traits in the repeated phenotypes that evolved within the Midas cichlid lineage and, as the first linkage map of a Neotropical cichlid, facilitate comparative genomic analyses between African cichlids, Neotropical cichlids and other teleost fishes.

Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae▿

Science.gov (United States)

Nakamura, Kohei ; Terada, Takeshi; Sekiguchi, Yuji; Shinzato, Naoya; Meng, Xian-Ying; Enoki, Miho; Kamagata, Yoichi

2006-01-01

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H2-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe. PMID:16950902

27 CFR 20.178 - Marks and brands on containers of specially denatured spirits.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Marks and brands on... Dealers § 20.178 Marks and brands on containers of specially denatured spirits. (a) Required marks. Each... officer, or (2) Consist of a brand name, or consist of caution notices, or consist of other material...

Analysis of the Ceratitis capitata y chromosome using in situ hybridization to mitotic chromosomes

International Nuclear Information System (INIS)

Willhoeft, U.; Franz, G.

1998-01-01

In Ceratitis capitata the Y chromosome is responsible for sex-determination. We used fluorescence in situ hybridization (FISH) for cytogenetic analysis of mitotic chromosomes. FISH with the wild-type strain EgyptII and two repetitive DNA probes enabled us to differentiate between the short and the long arm of the Y chromosome and gives a much better resolution than C-banding of mitotic chromosomes. We identified the Y-chromosomal breakpoints in Y-autosome translocations using FISH. Even more complex rearrangements i.e. deletions and insertions in some translocation strains were detected by this method. A strategy for mapping the primary sex determination factor in Ceratitis capitata by FISH is presented. (author)

Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis

NARCIS (Netherlands)

Wu, Y; Hayes, VM; Osinga, J; Mulder, IM; Looman, MWG; Buys, CHCM; Hofstra, RMW

1998-01-01

Denaturing gradient gel electrophoresis (DGGE) is one of the most powerful methods for mutation detection currently available. For successful application the appropriate selection of PCR fragments and PCR primers is crucial. The sequence of interest should always be within the domain with the lowest

EGFR mutation is a better predictor of response to tyrosine kinase inhibitors in non-small cell lung carcinoma than FISH, CISH, and immunohistochemistry.

Science.gov (United States)

Sholl, Lynette M; Xiao, Yun; Joshi, Victoria; Yeap, Beow Y; Cioffredi, Leigh-Anne; Jackman, David M; Lee, Charles; Jänne, Pasi A; Lindeman, Neal I

2010-06-01

About 10% of patients with non-small cell lung carcinoma (NSCLC) respond to epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors (TKIs). More than 75% of "responders" have activating mutations in EGFR. However, mutation analysis is not widely available, and proposed alternatives (in situ hybridization and immunohistochemical analysis) have shown inconsistent associations with outcome. Fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemical analysis, and DNA sequencing were compared in this study of 40 NSCLC samples from TKI-treated patients. Response rates were 12 of 19 in EGFR-mutant vs 1 of 20 EGFR wild-type tumors (P = .0001), 7 of 19 FISH+ vs 4 of 17 FISH- tumors (not significant [NS]), 5 of 16 CISH+ vs 6 of 21 CISH- tumors (NS), and 3 of 9 immunohistochemically positive vs 7 of 22 immunohistochemically negative tumors (NS). EGFR mutation was associated with improved progression-free survival (P = .0004). Increased copy number (FISH or CISH) and protein expression (immunohistochemical) did not independently predict outcome. Thus, EGFR sequence analysis was the only method useful for predicting response and progression-free survival following TKI therapy in NSCLC.

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample.

Science.gov (United States)

Ootsubo, M; Shimizu, T; Tanaka, R; Sawabe, T; Tajima, K; Ezura, Y

2003-01-01

A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.

FISH as A method for detection of radiation Induced genetic damage

International Nuclear Information System (INIS)

Lakatosova, M.; Holeckova, B.

2006-01-01

Fluorescence in situ hybridization (FISH) has been considered as a suitable method for rapid and easy detection of chromosome aberrations. In contrast to the standard conventional staining procedure, this technique enables the detection and specification of stable chromosomal re-arrangements, which are compatible with cellular division and thus, they could be transmitted from common ancestral to next cell generations. FISH chromosome - specific painting probes have been effectively applied for the detection of chromosomal damage after exposure to radiation. During last years, several specific fluorescent labeled probes were performed that allowed precise detection of centromeres, sub-telomeres or other regions (sequences) in genome. Our paper deals with describing of different types of FISH probes and their possibilities for application in radiobiology. (authors)

Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas.

Science.gov (United States)

Haugg, Anke M; Rennspiess, Dorit; zur Hausen, Axel; Speel, Ernst-Jan M; Cathomas, Gieri; Becker, Jürgen C; Schrama, David

2014-12-15

The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumor-specific mutations in the large T antigen are strong arguments that MCPyV is a human tumor virus. However, the relationship between viral presence and cancer induction remains discussed controversially. Since almost all studies on virus prevalence are based on PCR techniques, we performed MCPyV fluorescence in situ hybridization (FISH) on MCC to gain information about the quality of the viral presence on the single cell level. MCPyV-FISH was performed on tissue microarrays containing 62 formalin-fixed and paraffin-embedded tissue samples including all tumor grades of 42 patients. The hybridization patterns were correlated to the qPCR data determined on corresponding whole tissue sections. Indeed, MCPyV-FISH and qPCR data were highly correlated, i.e. 83% for FISH-positive and 93% for FISH-negative cores. Accordingly, the mean of the qPCR values of all MCPyV-positive cores differed significantly from the mean of the negative cores (p = 0.0076). Importantly, two hybridization patterns were definable in the MCPyV-FISH: a punctate pattern (85%) indicating viral integration, which correlated with a moderate viral abundance and a combination of the punctate with a diffuse pattern (15%), suggesting a possible coexistence of integrated and episomal virus which was associated with very high viral load and VP1 expression. Thus, MCPyV-FISH adds important information on the single cell level within the histomorphological context and could therefore be an important tool to further elucidate MCPyV related carcinogenesis. © 2014 UICC.

Detection of ALK gene rearrangement in non-small cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression.

Science.gov (United States)

Kim, Hyojin; Yoo, Seol-Bong; Choe, Ji-Young; Paik, Jin Ho; Xu, Xianhua; Nitta, Hiroaki; Zhang, Wenjun; Grogan, Thomas M; Lee, Choon-Taek; Jheon, Sanghoon; Chung, Jin-Haeng

2011-08-01

Accurate determination of ALK rearrangement is important in lung cancer patients, especially in determining their eligibility for crizotinib therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting ALK rearrangement. However, FISH requires a fluorescence microscope, and the signals are labile and rapidly fade over time. This study evaluates the concordance between ALK gene rearrangement in non-small cell lung cancer assessed by ALK FISH and a newly developed ALK chromogenic in situ hybridization (CISH) and correlates the results with ALK protein expression assessed by immunohistochemistry. A total of 465 formalin-fixed, paraffin-embedded non-small cell lung cancer samples were analyzed by ALK FISH (PathVysion, Vysis, Abbott) and ALK CISH. For comparison, all specimens were stained by immunohistochemistry (clone 5A4, Novocastra) and interobserver reproducibility was assessed. We found that agreement between the pathologists on the CISH-determined ALK status was achieved in 449 patients (96.6%), and ALK rearrangement was identified in 18 patients (4.0%) in CISH method. Among these cases, 443 cases (95.3%) had results matching the corresponding FISH results: 17 rearranged, 425 wild types, and 1 discordant case. There was high concordance in the assessment of ALK gene rearrangement between FISH and CISH techniques (κ = 0.92) and between observers (κ = 0.97). In addition, there was high concordance in the ALK gene status and ALK protein expression between CISH and IHC tests (κ = 0.82). CISH is a highly reproducible and practical method to detect ALK gene rearrangement and correlated well with ALK protein expression. Here, we present a diagnostic algorithm (Chung's SNUBH ALK protocol) to detect lung cancer with ALK rearrangements using IHC, FISH and CISH. Because CISH allows a concurrent analysis of histological features of the tumors and gene rearrangement, it appears to be a useful method in determining ALK gene

RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

Science.gov (United States)

Moffitt, Jeffrey R.; Zhuang, Xiaowei

2016-01-01

Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

Identification of hemiclonal reproduction in three species of Hexagrammos marine reef fishes.

Science.gov (United States)

Kimura-Kawaguchi, M R; Horita, M; Abe, S; Arai, K; Kawata, M; Munehara, H

2014-08-01

Natural hybrids between the boreal species Hexagrammos octogrammus and two temperate species Hexagrammos agrammus and Hexagrammos otakii were observed frequently in southern Hokkaido, Japan. Previous studies revealed that H. octogrammus is a maternal ancestor of both hybrids; the hybrids are all fertile females and they frequently breed with paternal species. Although such rampant hybridization occurs, species boundaries have been maintained in the hybrid zone. Possible explanations for the absence of introgressions, despite the frequent backcrossing, might include clonal reproduction: parthenogenesis, gynogenesis and hybridogenesis. The natural hybrids produced haploid eggs that contained only the H. octogrammus genome (maternal ancestor) with discarded paternal genome and generated F1 -hybrid type offspring by fertilization with the haploid sperm of H. agrammus or H. otakii (paternal ancestor). This reproductive mode was found in an artificial backcross hybrid between the natural hybrid and a male of the paternal ancestor. These findings indicate that the natural hybrids adopt hybridogenesis with high possibility and produce successive generations through hybridogenesis by backcrossing with the paternal ancestor. These hybrids of Hexagrammos represent the first hybridogenetic system found from marine fishes that widely inhabit the North Pacific Ocean. In contrast with other hybridogenetic systems, these Hexagrammos hybrids coexist with all three ancestral species in the hybrid zone. The coexistence mechanism is also discussed. © 2014 The Fisheries Society of the British Isles.

Large-scale polymorphism near the ends of several human chromosomes analyzed by using fluorescence in situ hybridization (FISH)

Energy Technology Data Exchange (ETDEWEB)

Trask, B.J.; Friedman, C. [Univ. of Washington, Seattle, WA (United States); Giorgi, D. [CNRS, Montpelier (France)] [and others

1994-09-01

We have discovered a large DNA segment that is polymorphically present at the ends of several human chromosomes. The segment, f7501, was originally derived form a human chromosome 19-specific cosmid library. FISH was used to determine the cosmid`s chromosomal distribution on 44 unrelated humans and several closely related primates. The human subjects represent a diversity of reproductively isolated ethnic populations. FISH analysis revealed that sequences highly homologous to the cosmid`s insert are present on both homologs at 3q, 15q,. and 19p in almost all individuals (88, 85, and 87 of 88 homologs, respectively). Other chromosomes sites were labeled much more rarely in the sampled individuals. For example, 56 of the 88 analyzed chromosomes 11 were labeled (18+/+, 6-/-, and 20+/- individuals). In contrast, 2q was labeled on only 1/88 sampled chromosomes. The termini of 2q, 5q, 6p, 6q, 7p, 8p, 9p, 9q, 11p, 12q, 16p, 19q, and 20q and an interstitial site at 2q13-14 were labeled in at least one individual of the set. EcoR1-fragments derived from the cosmid showed the same hybridization pattern as the entire cosmid, indicating that at least 40 kbp is shared by these chromosome ends. Ethnic differences in the allele frequency of these polymorphic variants was observed. For example, signals were observed on 8/10 and 7/10 of the chromosomes 7p and 16q, respectively, derived form Biakan Pygmies, but these sites were infrequently labeled in non-Pygmy human populations (2/68, respectively). This region has undergone significant changes in chromosome location during human evolution. Strong signal was seen on chimpanzee and gorilla chromosome 3, which is homologous to human chromosome 4, a chromosome unlabeled in any of the humans we have analyzed.

Helical Propensity Affects the Conformational Properties of the Denatured State of Cytochrome c'.

Science.gov (United States)

Danielson, Travis A; Bowler, Bruce E

2018-01-23

Changing the helical propensity of a polypeptide sequence might be expected to affect the conformational properties of the denatured state of a protein. To test this hypothesis, alanines at positions 83 and 87 near the center of helix 3 of cytochrome c' from Rhodopseudomonas palustris were mutated to serine to decrease the stability of this helix. A set of 13 single histidine variants in the A83S/A87S background were prepared to permit assessment of the conformational properties of the denatured state using histidine-loop formation in 3 M guanidine hydrochloride. The data are compared with previous histidine-heme loop formation data for wild-type cytochrome c'. As expected, destabilization of helix 3 decreases the global stabilities of the histidine variants in the A83S/A87S background relative to the wild-type background. Loop stability versus loop size data yields a scaling exponent of 2.1 ± 0.2, similar to the value of 2.3 ± 0.2 obtained for wild-type cytochrome c'. However, the stabilities of all histidine-heme loops, which contain the helix 3 sequence segment, are increased in the A83S/A87S background compared to the wild-type background. Rate constants for histidine-heme loop breakage are similar for the wild-type and A83S/A87S variants. However, for histidine-heme loops that contain the helix 3 sequence segment, the rate constants for loop formation increase in the A83S/A87S background compared to the wild-type background. Thus, residual helical structure appears to stiffen the polypeptide chain slowing loop formation in the denatured state. The implications of these results for protein folding mechanisms are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Cloning, characterization and chromosomal location of a satellite DNA from the Pacific oyster, Crassostrea gigas

Digital Repository Service at National Institute of Oceanography (India)

Clabby, C.; Goswami, U.; Flavin, F.; Wilkins, N.P.; Houghton, J.A.; Powell, R.

F2.18 (includ- ing pBGS8 vector) was labelled with fluor-12-dUTP and hybridized to the chromosome slides. The fluorescence signals were directly observed without additional immu- nological enhancement. No fluorescence signals were obtained when...-it-Fluor, Stratagene, La Jolla, CA, USA). Chromosomes were denatured at 72°C in 70% formamide/2 × SSC and the denatured labelled Cgl70 probe was added and hybridization performed at 37°C overnight. Post-hybridization washes were according to manufacturer...

A comparative genomic hybridization study in a 46,XX male.

Science.gov (United States)

Rigola, M Angels; Carrera, Marta; Ribas, Isabel; Egozcue, Josep; Miró, Rosa; Fuster, Carme

2002-07-01

To identify Y chromosome material in an azoospermic male with an XX karyotype. Case report. Faculty of medicine and Centro de Patologia Celular (CPC) medical center. A 33-year-old man with infertility. G-banding, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and comparative genomic hybridization (CGH). FISH for X and Y chromosomes, PCR for the SRYgene and amelogenin gene in the Xp (AMGX) and (AMGY), and losses or gains with CGH. FISH analysis using X and Y chromosome-specific probes showed an X chromosome containing Y chromosome sequences on the top of the short arm; this Y chromosome region was not visible by conventional cytogenetic analysis. PCR amplification of DNA showed the presence of the sex-determining region of the Y chromosome (SRY) and the amelogenin gene in the pseudoautosomal boundary of the X chromosome (AMGX). CGH confirmed the presence of the chromosome region Yp11.2-pter and detected the presence of the two otherwise normal X chromosomes. The two Xpter (XPAR1) pseudoautosomal regions present in this XX male suggest the need to reevaluate XX males using CGH and PCR to characterize the clinical variability in XX males due to genes other than those located on the Y chromosome.

Monosomy 3 by FISH in uveal melanoma: variability in techniques and results.

Science.gov (United States)

Aronow, Mary; Sun, Yang; Saunthararajah, Yogen; Biscotti, Charles; Tubbs, Raymond; Triozzi, Pierre; Singh, Arun D

2012-09-01

Tumor monosomy 3 confers a poor prognosis in patients with uveal melanoma. We critically review the techniques used for fluorescence in situ hybridization (FISH) detection of monosomy 3 in order to assess variability in practice patterns and to explain differences in results. Significant variability that has likely affected reported results was found in tissue sampling methods, selection of FISH probes, number of cells counted, and the cut-off point used to determine monosomy 3 status. Clinical parameters and specific techniques employed to report FISH results should be specified so as to allow meta-analysis of published studies. FISH-based detection of monosomy 3 in uveal melanoma has not been performed in a standardized manner, which limits conclusions regarding its clinical utility. FISH is a widely available, versatile technology, and when performed optimally has the potential to be a valuable tool for determining the prognosis of uveal melanoma. Copyright © 2012 Elsevier Inc. All rights reserved.

Analysis of Chromothripsis by Combined FISH and Microarray Analysis.

Science.gov (United States)

MacKinnon, Ruth N

2018-01-01

Fluorescence in situ hybridization (FISH) to metaphase chromosomes, in conjunction with SNP array, array CGH, or whole genome sequencing, can help determine the organization of abnormal genomes after chromothripsis and other types of complex genome rearrangement. DNA microarrays can identify the changes in copy number, but they do not give information on the organization of the abnormal chromosomes, balanced rearrangements, or abnormalities of the centromeres and other regions comprised of highly repetitive DNA. Many of these details can be determined by the strategic use of metaphase FISH. FISH is a single-cell technique, so it can identify low-frequency chromosome abnormalities, and it can determine which chromosome abnormalities occur in the same or different clonal populations. These are important considerations in cancer. Metaphase chromosomes are intact, so information about abnormalities of the chromosome homologues is preserved. Here we describe strategies for working out the organization of highly rearranged genomes by combining SNP array data with various metaphase FISH methods. This approach can also be used to address some of the uncertainties arising from whole genome or mate-pair sequencing data.

Impact of fishing and stocking practices on Coregonid diversity

Science.gov (United States)

Anneville, Orlane; Lasne, Emilien; Guillard, Jean; Eckmann, Reiner; Stockwell, Jason D.; Gillet, Christian; Yule, Daniel

2015-01-01

Fish species diversity can be lost through interacting stressors including habitat loss, stocking and overfishing. Although a multitude of stressors have played a role in the global decline of coregonid (Coregonus spp.) diversity, a number of contemporary studies have identified habitat loss stemming from eutrophication as the primary cause. Unfortunately, reconstructing the role of fishing and stocking practices can be difficult, because these records are incomplete or appear only in hard-to-access historic grey literature. Based on an illustrative set of historic and contemporary studies, we describe how fisheries management practices may have contributed to coregonid diversity loss in European and North American lakes. We provide case studies examining how fishing and stocking may reduce coregonid diversity through demographic decline and introgressive hybridization. In some lakes, fisheries management practices may have led to a loss of coregonid diversity well before issues with habitat degradation manifested. Our review suggests that fish conservation policies could beneficially consider the relative importance of all stressors, including management practices, as potential drivers of diversity loss.

Protective role of microRNA-29a in denatured dermis and skin fibroblast cells after thermal injury

Directory of Open Access Journals (Sweden)

Jie Zhou

2016-03-01

Full Text Available Our previous study has suggested that downregulated microRNA (miR-29a in denatured dermis might be involved in burn wound healing. However, the exact role of miR-29a in healing of burn injury still remains unclear. Here, we found that expression of miR-29a was notably upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury, and thereafter gradually downregulated compared with control group. By contrast, the expression of collagen, type I, alpha 2 (COL1A2 and vascular endothelial growth factor (VEGF-A were first reduced and subsequently upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury. We further identified COL1A2 as a novel target of miR-29a, which is involved in type I collagen synthesis, and showed that miR-29a negatively regulated the expression level of COL1A2 in skin fibroblast cells. In addition, VEGF-A, another target gene of miR-29a, was also negatively mediated by miR-29a in skin fibroblast cells. Inhibition of miR-29a expression significantly promoted the proliferation and migration of skin fibroblast cells after thermal injury, and knockdown of COL1A2 and VEGF-A reversed the effects of miR-29a on the proliferation and migration of skin fibroblast cells. Furthermore, we found that Notch2/Jagged2 signaling was involved in miR-29a response to burn wound healing. Our findings suggest that downregulated miR-29a in denatured dermis may help burn wound healing in the later phase, probably via upregulation of COL1A2 and VEGF-A expression, which can further enhance type I collagen synthesis and angiogenesis.

Heterogeneity of equilibrium molten globule state of cytochrome c induced by weak salt denaturants under physiological condition.

Directory of Open Access Journals (Sweden)

Hamidur Rahaman

Full Text Available While many proteins are recognized to undergo folding via intermediate(s, the heterogeneity of equilibrium folding intermediate(s along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD, ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS were used to study the structural and thermodynamic characteristics of the native (N, denatured (D and intermediate state (X of goat cytochorme c (cyt-c induced by weak salt denaturants (LiBr, LiCl and LiClO4 at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε400 and CD ([θ]409, is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO4 and acidic pH. Our observations suggest: (1 that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III axial bond and Trp59-propionate interactions; (2 that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO4 and 0.5 M NaCl at pH 2.0; and (3 that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1, classical (X2 and disordered (X3, i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.

Contribution of fluorescence in situ hybridization to biological dosimetry

International Nuclear Information System (INIS)

Sorokine-Durm, I.; Roy, L.; Durand, V.; Voisin, P.

1995-01-01

Fluorescence in situ hybridization with composite whole chromosome specific DNA probes for human chromosomes 2, 4 and 12 an α-satellite centromeric DNA probe labelled with biotin were used to measure symmetrical and terminal translocations (dose rate 0.5 Gy/min) and dicentrics (0.1 Gy/min) induced in vitro by 60 Co γ-irradiation (0-5 Gy). The suitability of fluorescence in situ hybridization (F.I.S.H.) technique for dicentrics detection is compared with the conventional technique. Dose-response curves for γ-rays ( 60 Co) for two dose rates are shown (dicentrics and translocations). (authors). 10 refs., 2 figs

speciation through asexuality in fish: postzygotic reproductive isolation may be completed in spite of fertility of hybrids

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Karel Janko

2015-12-01

Altogether, it appears that initiation of hybrid asexuality and the completion of speciation process through formation of postRIMs are interconnected phenomena. Both processes are linked to the genetic divergence of hybridizing taxa: initially, hybridization between little diverged species leads to recombinant and fertile hybrids allowing intensive gene flow. As the hybridizing taxa continue to diverge, clonally reproducing hybrid females and sterile males become dominant and the gene flow ceases. The speciation may therefore be completed through asexuality of hybrids The work was supported by grant no. 13-12580S provided by the Czech Science Foundation (www.gacr.cz. Further support was provided by the Academy of Sciences of the Czech Republic (www.cas.cz by the grant no. RVO 67985904

Divergent mating preferences and nuptial coloration in sibling species of cichlid fish

NARCIS (Netherlands)

Sluijs, Inke van der

2008-01-01

Mate choice by female cichlid fish from Lake Victoria plays an important role in speciation and the maintenance of species. Females are expected to select against males that are intermediate in their phenotype during the process of speciation driven by sexual selection. To test this, we hybridized

Genome constitution and evolution in Lolium X Festuca hybrid cultivars (Festulolium)

Czech Academy of Sciences Publication Activity Database

Kopecký, David; Loureiro, J.; Zwierzykowski, Z.; Ghesquière, M.; Doležel, Jaroslav

2006-01-01

Roč. 113, - (2006), s. 731-742 ISSN 0040-5752 R&D Projects: GA AV ČR IBS5038104 Institutional research plan: CEZ:AV0Z50380511 Keywords : Festulolium hybrids * GISH * FISH Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.715, year: 2006

A study of the thermal denaturation of the S-layer protein from Lactobacillus salivarius

Science.gov (United States)

Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian

2012-09-01

Surface layer (S-layer) proteins display an intrinsic self-assembly property, forming monomolecular crystalline arrays, identified in outermost structures of the cell envelope in many organisms, such as bacteria and archaea. Isolated S-layer proteins also possess the ability to recrystallize into regular lattices, being used in biotechnological applications, such as controlling the architecture of biomimetic surfaces. To this end, the stability of the S-layer proteins under high-temperature conditions is very important. In this study, the S-layer protein has been isolated from Lactobacillus salivarius 16 strain of human origin, and purified by cation-exchange chromatography. Using circular dichroism (CD) spectroscopy, we have investigated the thermal denaturation of the S-layer protein. The far- and near-UV CD spectra have been collected, and the temperature dependence of the CD signal in these spectral domains has been analyzed. The variable temperature results show that the secondary and tertiary structures of the S-layer protein change irreversibly due to the heating of the sample. After the cooling of the heated protein, the secondary and tertiary structures are partially recovered. The denaturation curves show that the protein unfolding depends on the sample concentration and on the heating rate. The secondary and tertiary structures of the protein suffer changes in the same temperature range. We have also detected an intermediate state in the protein denaturation pathway. Our results on the thermal behavior of the S-layer protein may be important for the use of S-layer proteins in biotechnological applications, as well as for a better understanding of the structure and function of S-layer proteins.

Three sympatric karyomorphs in the fish Astyanax fasciatus (Teleostei, Characidae do not seem to hybridize in natural populations

Directory of Open Access Journals (Sweden)

Maressa Ferreira-Neto

2012-01-01

Full Text Available Ninety individuals of the characid fish Astyanax fasciatus (Cuvier, 1819 were collected at Água da Madalena stream (Botucatu, São Paulo, Brazil and analyzed for diploid chromosome number 2n and karyotype composition as well as for the chromosomal location of the 5S and 18S ribosomal DNA (rDNA. Whereas no chromosome differences were associated with sex, three different karyomorphs with diploid chromosome numbers 2n=46, 2n=48 and 2n=50 were found. No intermediate 2n numbers were discovered. The 2n=50 karyomorph showed some differences in 18S rDNA location compared to the two other karyomorphs. Finally, all specimens with the 2n=46 karyomorph showed the presence of a partly heterochromatic macro supernumerary chromosome, which was absent in all individuals with the two other karyomorphs. All these results suggest that indviduals of the three different karyomorphs are not likely to hybridize in the examined populations. Our findings strongly suggest the presence of three separate species (sensu biological species concept easily diagnosed on the basis of differences in the diploid chromosome numbers and other chromosomal markers.

Evaluation of upper urinary tract tumors by FISH in Chinese patients.

Science.gov (United States)

Shan, Zhengfei; Wu, Peng; Zheng, Shaobin; Tan, Wanlong; Zhou, Haikuan; Zuo, Yi; Qi, Huan; Zhang, Peng; Peng, Hongmei; Wang, Yanfen

2010-12-01

Upper urinary tract tumor (UUTT) usually presents a high grade and stage, and recurs frequently. The aim of this study was to evaluate the utility of a fluorescence in situ hybridization (FISH) assay on chromosomes 3, 7, 9, and 17 as a reliable and noninvasive method for the diagnosis of Chinese patients with UUTT. Urine specimens from 50 patients with UUTT and 25 donors without evidence of urothelial tumors were analyzed by cytology and FISH. Voided urine samples from 20 normal individuals were used to establish the cut-off values for FISH assay. The McNemar test was applied for sensitivity and specificity. The overall sensitivity of FISH was statistically significantly greater than that of cytology (84.0 vs. 40.0%, P = 0.000). The overall specificities of FISH and urine cytology were all 96.0% (P = 1.000). Polysomy in chromosomes 3, 7, and 17 were 38, 42, and 30%, respectively. Heterozygous and homozygous loss of the p16 locus was found in 36 and 32%, respectively. FISH analysis performed on cells collected from voided urine is feasible, and FISH could prove to be a reliable and less invasive ancillary test and improve the sensitivity of urine cytology in the diagnosis of UUTT. Copyright © 2010 Elsevier Inc. All rights reserved.

Structural and dynamical study about denatured states of yeast phosphoglycerate kinase by neutrons scattering and X-rays; Etude structurale et dynamique des etats denatures de la phosphoglycerate kinase de levure par diffusion des neutrons et des rayons X

Energy Technology Data Exchange (ETDEWEB)

Receveur, V

1997-04-28

During a long time, the neutron scattering and X-rays techniques have not been used for the studies bearing on the folding of proteins. The compactness and the globularness of a protein are two structural characteristics describing the denatured states and the intermediate states of folding, and the neutrons and x-rays scattering are probably the two techniques the most appropriate to give this kind of information; they are sensible to the spatial extent and to the molecules compactness, and to their general shape. For these three or four last years, the works using these techniques are increasing, giving precious knowledge on the different steps of folding and on the interactions stabilizing the denatured or intermediate states. This thesis falls into this category. (N.C.).

Using copper sulfate to control egg fungus at Keo Fish Farm

Science.gov (United States)

Keo Fish Farm is the biggest producer of hybrid striped bass fry in the world. The hatchery manager asked about treatments to control fungus on eggs which occurred fairly often. Our lab has been working on gaining FDA-approval to use copper sulfate to control fungus on catfish eggs, so we were con...

Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment.

Science.gov (United States)

Pavlekovic, Marko; Schmid, Markus C; Schmider-Poignee, Nadja; Spring, Stefan; Pilhofer, Martin; Gaul, Tobias; Fiandaca, Mark; Löffler, Frank E; Jetten, Mike; Schleifer, K-H; Lee, Natuschka M

2009-08-01

Fluorescence in situ hybridization (FISH) using fluorochrome-labeled DNA oligonucleotide probes has been successfully applied for in situ detection of anaerobic ammonium oxidizing (anammox) bacteria. However, application of the standard FISH protocols to visualize anammox bacteria in biofilms from a laboratory-scale wastewater reactor produced only weak signals. Increased signal intensity was achieved either by modifying the standard FISH protocol, using peptide nucleic acid probes (PNA FISH), or applying horse radish peroxidase- (HRP-) labeled probes and subsequent catalyzed reporter deposition (CARD-FISH). A comparative analysis using anammox biofilm samples and suspended anammox biomass from different laboratory wastewater bioreactors revealed that the modified standard FISH protocol and the PNA FISH probes produced equally strong fluorescence signals on suspended biomass, but only weak signals were obtained with the biofilm samples. The probe signal intensities in the biofilm samples could be enhanced by enzymatic pre-treatment of fixed cells, and by increasing the hybridization time of the PNA FISH protocol. CARD-FISH always produced up to four-fold stronger fluorescent signals but unspecific fluorescence signals, likely caused by endogenous peroxidases as reported in several previous studies, compromised the results. Interference of the development of fluorescence intensity with endogenous peroxidases was also observed in cells of aerobic ammonium oxidizers like Nitrosomonas europea, and sulfate-reducers like Desulfobacter postgatei. Interestingly, no interference was observed with other peroxidase-positive microorganisms, suggesting that CARD-FISH is not only compromised by the mere presence of peroxidases. Pre-treatment of cells to inactivate peroxidase with HCl or autoclavation/pasteurization failed to inactive peroxidases, but H(2)O(2) significantly reduced endogenous peroxidase activity. However, for optimal inactivation, different H(2)O(2

Implementation and importance of fluorescence in situ hybridization (fish) in paraffin tissues for categorization of B-cell lymphoma unclassifiable, with features intermediate between Burkitt lymphoma and diffuse large B-cell lymphoma

International Nuclear Information System (INIS)

Carvajal Cuenca, Alejandra

2011-01-01

The diagnostic criteria have been defined based on the tools that the country has acquired and international guidelines for pure entities: the LB, LDCGB, and the new entity of B lymphoma unclassifiable with features intermediate LDCGB and LB. The fluorescence in situ hybridization for the translocation (8;14) has been implemented in paraffin tissues for proper categorization. A total of 21 cases have been studied: the characteristics of patients, morphology, immunohistochemistry and the presence or absence of the translocation (8;14). Twelve of the cases have been classified as B-cell lymphoma unclassifiable with features intermediate between LDCGB and LB. Furthermore, nine of the cases were classified in LB. Fluorescence in situ hybridization (FISH) has been negative in 5 of the 21 cases. The diagnosis of lymphoma with features bordering between the LB and the LDCGB has been imperative for the survival of the patient and the corresponding treatment [es

An improved method for detecting genetic variation in DNA using denaturing gradient gel electrophoresis

International Nuclear Information System (INIS)

Takahashi, Norio; Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko.

1990-05-01

We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that use of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than use of DNA:DNA heteroduplexes, because preparation of RNA probes is easier than that of DNA probes. Three different 32 P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of a mismatch(es) was detected as a difference in the mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and three thalassemic human β-globin genes. The results from experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human β-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was undertaken in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for detecting heritable variation and should be a powerful tool for detecting fresh mutations in DNA, which occur outside the known restriction sites. (author)

Structural and dynamical study about denatured states of yeast phosphoglycerate kinase by neutrons scattering and X-rays

International Nuclear Information System (INIS)

Receveur, V.

1997-01-01

During a long time, the neutron scattering and X-rays techniques have not been used for the studies bearing on the folding of proteins. The compactness and the globularness of a protein are two structural characteristics describing the denatured states and the intermediate states of folding, and the neutrons and x-rays scattering are probably the two techniques the most appropriate to give this kind of information; they are sensible to the spatial extent and to the molecules compactness, and to their general shape. For these three or four last years, the works using these techniques are increasing, giving precious knowledge on the different steps of folding and on the interactions stabilizing the denatured or intermediate states. This thesis falls into this category. (N.C.)

A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1.

Science.gov (United States)

Brewer, J M; Wampler, J E

2001-03-14

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.

Denaturing gradient gel electrophoresis profiling of bacterial communities composition in Arabian Sea

Digital Repository Service at National Institute of Oceanography (India)

Singh, S.K.; Ramaiah, N.

of Environmental Biology circleshadowdwnMay 2011circleshadowdwn Introduction The bacteria play a major role in carbon dynamics of marine ecosystems and, the importance of heterotrophic bacteria in marine ecosystem functioning is very well recognized (Azam et al..., 2008). Denaturing gradient gel-electrophoressis (DGGE) based fingerprinting helps estimate the numbers of dominant phylotype in a given sample (Muyzer et al., 1993). Very diverse bacterial assemblages such as those in the soils present many bands...

Molecular cytogenetic analysis of human blastocysts andcytotrophoblasts by multi-color FISH and Spectra Imaging analyses

Energy Technology Data Exchange (ETDEWEB)

Weier, Jingly F.; Ferlatte, Christy; Baumgartner, Adolf; Jung,Christine J.; Nguyen, Ha-Nam; Chu, Lisa W.; Pedersen, Roger A.; Fisher,Susan J.; Weier, Heinz-Ulrich G.

2006-02-08

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing technologies to score all 24 chromosomes in single cells within a 3 day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found aneuploidy, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.

Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

International Nuclear Information System (INIS)

Bjoerheim, Jens; Abrahamsen, Torveig Weum; Kristensen, Annette Torgunrud; Gaudernack, Gustav; Ekstroem, Per O.

2003-01-01

Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification

A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC and the Identification of 9 New Mutations Previously Unidentified by DGGE

Directory of Open Access Journals (Sweden)

Meldrum Cliff J

2003-12-01

Full Text Available Abstract Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE with denaturing high performance liquid chromatography (DHPLC in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE. From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.

Impact of a widely cultivated tree (moringa oleifera) on the health of commercially important hybrid catfish

International Nuclear Information System (INIS)

Adamu, K.M.; Ahmed, I.M.

2014-01-01

Plantations of the tree Moringa oleifera often lead to increase levels of leaf dust in nearby freshwater environments, and there is concern that, this could have a negative impact on catfish, which are important for aquaculture. Therefore, this study, determined the biochemical alterations in serum, liver and kidney of hybrid catfish (Clarias gariepinus ( ) Heterobranchus bidorsalis ( ) exposed to sub-lethal concentrations of M. oleifera leaf dust in a static renewable bioassay system during a medium term exposure period. The fish (mean length, 16.33 cm, mean weight, 9.90 g) were exposed to 0.16, 0.12, 0.08, 0.04 and 0.00 mg/L concentrations of the plant leaf dust in triplicate exposure. After 21-days of exposure period, the fish were sacrificed for the biochemical parameters: glucose, total protein, albumin, globulin, cholesterol, triglyceride, g-glutamyltransferase, alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase. Fish exposed to leaf dust showed significant differences (P 0.05), in the liver and kidney, respectively, in the test fish. Other parameters were not significantly different (P>0.05) in their respective tissue-organs. Ipso-facto, the alteration in biochemical parameters of hybrid catfish exposed to M. oleifera leaf dust was concentration dependent with 0.16 mg/L showing the highest negative alterations thus fish exposed to concentrations above 0.16 mg/L for longer durations may suffer impaired health effect. (author)

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Energy Technology Data Exchange (ETDEWEB)

Rosa, F.E.; Santos, R.M. [Departamento de Patologia, Hospital das Clínicas, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Rogatto, S.R. [Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Hospital A.C. Camargo, CIPE, São Paulo, SP (Brazil); Domingues, M.A.C. [Departamento de Patologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil)

2013-03-19

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Science.gov (United States)

Rosa, F.E.; Santos, R.M.; Rogatto, S.R.; Domingues, M.A.C.

2013-01-01

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer. PMID:23558859

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

International Nuclear Information System (INIS)

Rosa, F.E.; Santos, R.M.; Rogatto, S.R.; Domingues, M.A.C.

2013-01-01

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer

Evaluation of Myc Gene Amplification in Prostate Cancer Using a Dual Color Chromogenic in-Situ Hybridization (Dual CISH Assay

Directory of Open Access Journals (Sweden)

Daniel Lerda

2013-04-01

Full Text Available Objetive: The overall purpose of the study was to demonstrate applicability of the Dako dual-color chromogenic in situ hybridization (CISH assay (DAKO Denmark, Glostrup with respect to fluorescence in situ hybridization (FISH probes MYC-C. Methods: MYC gene amplification by FISH and Dako dual-color CISH Results: The study showed that the dual-color CISH assay can convert Texas red and fluorescein isothiocyanate (FITC signals into chromogenic signals. The dual –color CISH assay was performed on 40 cases of prostate cancer. Amplification was identified in 12 of 40 (30% tumors. No amplification was seen in 28 of 40 (70% tumors. FISH data were available in total of 40 tumors. All tumors showed concordant results between dual-color CISH and FISH for classifying a tumor as MYC amplified or not amplified. Conclusions: We conclude that dual-color Dako CISH assay is an accurate method for determining MYC gene amplification with added advantages that make it a more practically useful method. [J Interdiscipl Histopathol 2013; 1(2.000: 81-84

FISH analysis for diagnostic evaluation of challenging melanocytic lesions.

Science.gov (United States)

Zimmermann, A K; Hirschmann, A; Pfeiffer, D; Paredes, B E; Diebold, J

2010-09-01

The differential diagnosis of malignant melanomas and atypical melanocytic nevi is still a diagnostic challenge. The currently accepted morphologic criteria show substantial interobserver variability, likewise immunohistochemical studies are often not able to discriminate these lesions reliably. Techniques that support diagnostic accuracy are of the greatest importance considering the growing incidence of malignant melanomas and their increase in younger patients. In this study we analyzed the feasibility of fluorescence in situ hybridization (FISH) analysis for the discrimination of malignant and benign melanocytic tumors. A panel of DNA probes was used to detect chromosomal aberrations of chromosomes 6 and 11. On a series of 5 clearly malignant and benign melanocytic tumors we confirmed the applicability of the test. Then we focused on examination of ambiguous melanocytic lesions, where atypical cells are often difficult to relocalize in the 4',6-Diamidino-2-phenylindol (DAPI)-fluorescence stain. FISH analyses were conducted on destained H&E-stained slides. By comparison of the DAPI-image with photos taken from the H&E stain, unambiguous assignment of the FISH results to the conspicuous groups of cells was possible. The results of FISH analysis were consistent with the conventional diagnosis in 11 of 14 small ambiguous lesions. Of the remaining 3 cases, 2 showed FISH-results close to the cut-off level. Comparison of FISH results on thin and thick sections revealed that the cut-off values have to be adapted for 2 microm destained sections. In conclusion, FISH analysis is a useful and applicable tool for assessment of even smallest melanocytic neoplasms, although there will remain unclear cases that cannot be solved even after additional FISH evaluation.

Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using Fluorescence in situ Hybridization (FISH) and immunohistochemistry

International Nuclear Information System (INIS)

Sato-Kuwabara, Yukie; Neves, José I; Fregnani, José HTG; Sallum, Rubens A; Soares, Fernando A

2009-01-01

Esophageal squamous cell carcinoma (ESCC) is the sixth most frequent neoplasia in Brazil. It is usually associated with a poor prognosis because it is often at an advanced stage when diagnosed and there is a high frequency of lymph node metastases. It is important to know what prognostic factors can facilitate diagnosis, optimize therapeutic decisions, and improve the survival of these patients. A member of the epidermal growth factor receptor (EGFR) family, c-erbB-2, has received much attention because of its therapeutic implications; however, few studies involving fluorescence in situ hybridization (FISH) analysis of HER-2/neu gene amplification and protein expression in ESCC have been conducted. The aim of this study was to verify the presence of HER-2/neu gene amplification using FISH, and to correlate the results with immunohistochemical expression and clinical-pathological findings. One hundred and ninety-nine ESCC cases were evaluated using the Tissue Microarray (TMA) technique. A polyclonal antibody against c-erbB-2 was used for immunohistochemistry. Analyses were based on the membrane staining pattern. The results were classified according to the Herceptest criteria (DAKO): negative (0/1+), potential positive (2+) and positive (3+). The FISH reactions were performed according to the FISH HER2 PharmDx (DAKO) protocol. In each case, 100 tumor nuclei were evaluated. Cases showing a gene/CEN17 fluorescence ratio ≥ 2 were considered positive for gene amplification. The c-erbB-2 expression was negative in 117/185 cases (63.2%) and positive in 68 (36.8%), of which 56 (30.3%) were 2+ and 12 (6.5%) were 3+. No significant associations were found among protein expression, clinicopathological data and overall survival. Among the 47 cases analyzed, 38 (80.9%) showed no gene amplification while 9 (19.1%) showed amplification, as demonstrated by FISH. Cases that were negative (0/1+) and potential positive (2+) for c-erbB-2 expression by immunohistochemistry showed no

Fish allergy and fish allergens

DEFF Research Database (Denmark)

Kuehn, A; Hilger, Christiane; Ollert, Markus

2016-01-01

Fish is one of the main elicitors for food allergies. For a long time, the clinical picture of fish allergy was reduced to the following features. First, fish-allergic patients suffer from a high IgE cross-reactivity among fishes so that they have to avoid all species. Second, clinically relevant...... symptoms are linked to the presence of IgE-antibodies recognizing parvalbumin, the fish panallergen. This view was challenged by results from recent studies as follows. 1. Allergic reactions which are limited to single or several fish species (mono-or oligosensitisations) apply not only to single cases...... but patients with this phenotype constitute an important sub-group among fish-allergic individuals. 2. Newly identified fish allergens, enolases, aldolases, and fish gelatin, are of high relevance as the majority of the fish-allergic individuals seem to develop specific IgE against these proteins. The present...

Correlation among genetic variations of c-MET in Chinese patients with non-small cell lung cancer.

Science.gov (United States)

Duan, Jianchun; Yang, Xiaodan; Zhao, Jun; Zhuo, Minglei; Wang, Zhijie; An, Tongtong; Bai, Hua; Wang, Jie

2018-01-05

The purpose of our research was to determine the correlation of amplification, protein expression and somatic mutation of c-MET in IIIb-IV stage NSCLC (Non-small cell lung cancer). We also explored correlation of c-MET variation with clinical outcome. c-MET expression was observed in 28.6% (56/196) cases, and among those 13.8% (27/196) were shown to be FISH positive. Only 2.67% patients in this study carried the c-MET mutation. Cases with c-MET FISH positive were all IHC positive ,but in IHC positive cases, only half were FISH positive. Among patients with IHC 2+ staining, 35.5% was FISH positive, while cases with IHC 3+ staining,64% was FISH positive. Both protein expression and copy number of c-MET did not significantly correlate with clinical prognosis in these patients treated with EGFR-TKIs. IHC could be used as a preliminary screening method for c-MET copy number amplification and should be confirmed by FISH only in IHC positive case which facilitate selection of ALK or MET inhibitor therapy. c-MET gene copy number, protein expression and somatic mutation for exon 14 were detected by fluorescent- In-Situ -Hybridization (FISH), Immunohistochemistry (IHC), and Denaturing-High-Performance-Liquid-Chromatography (DHPLC), respectively, in 196 NSCLC patients. The relationship between c-MET abnormalities and clinical outcome of targeted therapy was analyzed by McNemar's test.

Analysis of the nitrifying bacterial community in BioCube sponge media using fluorescent in situ hybridization (FISH) and microelectrodes.

Science.gov (United States)

Chae, Kyu-Jung; Rameshwar, T; Jang, Am; Kim, Sung H; Kim, In S

2008-09-01

There is growing interest in the development of more cost-effective and retrofit technologies for the upgrade and expansion of existing wastewater treatment plants with extreme space constraints. A free-floating sponge media (BioCube) process, using a 24 L lab scale reactor, was operated to study the nitrification profiles and microbial community. The COD removal efficiencies were maintained, at an average of 95%, with the mixed liquor suspended solids (MLSS) inside the BioCube sponge media maintained at 12,688 mg/L. The nitrification removal efficiencies were between 92% and 100%, with an average value of 99%. From the results of microelectrode measurements, the ammonium ion concentration was found to rapidly decrease from the surface of the BioCube sponge media to a depth of 2mm due to chemical reactions carried out by ammonia oxidizing bacteria (AOB) species. Multi-fluorescence in situ hybridization (FISH) has been used to investigate the spatial distributions of various microbial activities within reactors. Microbial communities were targeted using different oligonucleotide probes specific to AOB and nitrite oxidizing bacteria (NOB). There were a large number of AOB populations, but these were not uniformly distributed in the biofilm compared to the NOB populations.

Identification of embryonic chromosomal abnormality using FISH-based preimplantaion genetic diagnosis

Institute of Scientific and Technical Information of China (English)

叶英辉; 徐晨明; 金帆; 钱羽力

2004-01-01

Objective: Embryonic chromosomal abnormality is one of the main reasons for in vitro fertilization (IVF) failure. This study aimed at evaluating the value of Fluorescence in-situ Hybridization (FISH)-based Preimplantation Genetic Diagnosis (PGD) in screening for embryonic chromosomal abnormality to increase the successful rate of IVF. Method: Ten couples, four with high risk of chromosomal abnormality and six infertile couples, underwent FISH-based PGD during IVF procedure. At day 3, one or two blastomeres were aspirated from each embryo. Biopsied blastomeres were examined using FISH analysis to screen out embryos with chromosomal abnormalities. At day 4, embryos without detectable chromosomal abnormality were transferred to the mother bodies as in regular IVF. Results: Among 54 embryos screened using FISH-based PGD, 30 embryos were detected to have chromosomal abnormalities. The 24 healthy embryos were implanted, resulting in four clinical pregnancies, two of which led to successful normal birth of two healthy babies; one to ongoing pregnancy during the writing of this article; and one to ectopic pregnancy. Conclusion: FISH-based PGD is an effective method for detecting embryonic chromosomal abnormality, which is one of the common causes of spontaneous miscarriages and chromosomally unbalanced offsprings.

Identification of embryonic chromosomal abnormality using FISH-based preimplantaion genetic diagnosis

Institute of Scientific and Technical Information of China (English)

叶英辉; 徐晨明; 金帆; 钱羽力

2004-01-01

Objective: Embryonic chromosomal abnormality is one of the main reasons for in vitro fertilization (IVF)failure. This study aimed at evaluating the value of Fluorescence in-situ Hybridization (FISH)-based Preimplantation Genetic Diagnosis (PGD) in screening for embryonic chromosomal abnormality to increase the successful rate of IVF. Method:Ten couples, four with high risk of chromosomal abnormality and six infertile couples, underwent FISH-based PGD during IVF procedure. At day 3, one or two blastomeres were aspirated from each embryo. Biopsied blastomeres were examined using FISH analysis to screen out embryos with chromosomal abnormalities. At day 4, embryos without detectable chromosomal abnormality were transferred to the mother bodies as in regular IVF. Results: Among 54 embryos screened using FISH-based PGD, 30 embryos were detected to have chromosomal abnormalities. The 24 healthy embryos were implanted,resulting in four clinical pregnancies, two of which led to successful normal birth of two healthy babies; one to ongoing pregnancy during the writing of this article; and one to ectopic pregnancy. Conclusion: FISH-based PGD is an effective method for detecting embryonic chromosomal abnormality, which is one of the common causes of spontaneous miscarriages and chromosomally unbalanced offsprings.

Growth of monosex hybrid tilapia in the labortory and sewage oxidation ponds. [Effects of water temperature, nutrient level, and. gamma. rays on growth

Energy Technology Data Exchange (ETDEWEB)

Suffern, J.S.; Adams, S.M.; Blaylock, B.G.; Coutant, C.C.; Guthrie, C.A.

1978-01-01

Studies were conducted to evaluate the potential of monosex hybrid tilapia (female T. mossambica x male T. hornorum) in waste-heat polyculture systems. The optimum growth temperature for this hybrid was found to be 32/sup 0/C in laboratory experiments. Experiments in sewage pond cage culture in the temperature range of 23 to 33/sup 0/C at stocking densities of approximately 53 fish/m/sup 3/ were also conducted. At fish sizes between 5 and 12 cm TL, estimated annual production is approximately 50,000 kg/ha/yr (50,000 lb/acre/yr). Fish in the sewage oxidation ponds grew significantly faster than fish fed trout chow at optimum temperature in the laboratory, even though temperatures in the sewage ponds averaged below the optimum growth temperature. Techniques to accelerate growth rates are being explored. Exposure to gamma radiation (500 rads), known to cause significant increases in channel catfish growth rate, was found to have a similar effect on tilapia. After a 20-week growth period, exposed fish weighed an average of 20% more than controls.

Assessment of DNA damage and Chromosome aberration in human lymphocyte exposed to low dose radiation detected by FISH(Fluorescence In Situ Hybridization) and SCGE(Single Cell Gel Electrophoresis)

International Nuclear Information System (INIS)

Chung, Hai Won; Kim, Su Young; Kim, Byung Mo; Kim, Sun Jin; Ha, Sung Whan; Kim, Tae Hwan; Cho, Chul Koo

2000-01-01

Comparative study was performed for the assessment of DNA damage and Chromosomal aberration in human lymphocyte exposed to low dose radiation using Fluorescence In Situ Hybridization(FISH) and Single Cell Gel Electrophoresis(SCGE). Chromosomal aberrations in human lymphocyte exposed to radiation at doses of 5, 10, 30 and 50cGy were analysed with whole chromosome-specific probes by human chromosome 1, 2 and 4 according to PAINT system. FISH with chromosome-specific probe has been used to be a valid and rapid method for detection of chromosome rearrangements induced by low dose radiation. The frequencies of stable translocation per cell equivalents were 0.0116, 0.0375, 0.0407, 0.0727 and 0.0814 for 0, 5, 10, 30 and 50cGy, respectively, and those of dicentric were 0.00, 0.0125, 0.174, 0.0291 and 0.0407 respectively. Radiation induced DNA damage in human lymphocyte in a dose-dependent manner at low doses from 5cGy to 50cGy, which were analysed by single Cell Gel Electrophoresis(SCGE). From above results, FISH seemed to be useful for radiation biodosimetry by which the frequencies of stable aberrations in human lymphocyte can be observed more easily than by conventional method and SCGE also seemed to be sensitive method for detecting DNA damage by low dose radiation exposure, so that those methods will improve our technique to perform meaningful biodosimetry for radiation at low doses

Comparative evaluation of non-informative HER-2 immunoreactions (2+) in breast carcinomas with FISH, CISH and QRT-PCR.

Science.gov (United States)

Kostopoulou, Evanthia; Vageli, Dimitra; Kaisaridou, Despina; Nakou, Marianna; Netsika, Maria; Vladica, Natalia; Daponte, Alexandros; Koukoulis, George

2007-12-01

The routine assessment of HER-2 expression can be affected by many immunohistological preanalytical and analytical variables. The evaluation of non-informative HER-2 tests, because of 2(+) scores, has been addressed in studies using in situ hybridization (fluorescent in situ hybridization (FISH) or chromogenic in situ hybridization (CISH)). There are very few studies that additionally checked 2(+) cases by quantitative reverse transcription-PCR (QRT-PCR). We analyzed totally 195 breast carcinoma cases, 70 of them showing 2(+) immunoreaction, with FISH/CISH and QRT-PCR. Confirmed amplification in 2(+) cases fell within the reported range (12.8% vs. 8-44%) and some of them showed lower mRNA levels indicating a genuine decrease of HER-2 protein as a mechanism for the non-informative score. In other cases, increased mRNA levels could be ascribed to HER-2 polysomy, verifying previous observations of immunohistologically detectable HER-2 polysomy. A remarkable subset of the 2(+) cases showed "normal" mRNA levels without amplification or polysomy and technical parameters as well as heterogeneity could be incriminated. The overall concordance of QRT-PCR and FISH was 93.8%, highest than most previously reported. Yet, the lack of clear cut-off mRNA values and the challenge of sample microdissection hinder QRT-PCR from claiming the status of a gold standard test for HER-2 evaluation.

Nonnative Fishes in the Upper Mississippi River System

Science.gov (United States)

Irons, Kevin S.; DeLain, Steven A.; Gittinger, Eric; Ickes, Brian S.; Kolar, Cindy S.; Ostendort, David; Ratcliff, Eric N.; Benson, Amy J.; Irons, Kevin S.

2009-01-01

The introduction, spread, and establishment of nonnative species is widely regarded as a leading threat to aquatic biodiversity and consequently is ranked among the most serious environmental problems facing the United States today. This report presents information on nonnative fish species observed by the Long Term Resource Monitoring Program on the Upper Mississippi River System a nexus of North American freshwater fish diversity for the Nation. The Long Term Resource Monitoring Program, as part of the U.S. Army Corps of Engineers' Environmental Management Plan, is the Nation's largest river monitoring program and stands as the primary source of standardized ecological information on the Upper Mississippi River System. The Long Term Resource Monitoring Program has been monitoring fish communities in six study areas on the Upper Mississippi River System since 1989. During this period, more than 3.5 million individual fish, consisting of 139 species, have been collected. Although fish monitoring activities of the Long Term Resource Monitoring Program focus principally on entire fish communities, data collected by the Program are useful for detecting and monitoring the establishment and spread of nonnative fish species within the Upper Mississippi River System Basin. Sixteen taxa of nonnative fishes, or hybrids thereof, have been observed by the Long Term Resource Monitoring Program since 1989, and several species are presently expanding their distribution and increasing in abundance. For example, in one of the six study areas monitored by the Long Term Resource Monitoring Program, the number of established nonnative species has increased from two to eight species in less than 10 years. Furthermore, contributions of those eight species can account for up to 60 percent of the total annual catch and greater than 80 percent of the observed biomass. These observations are critical because the Upper Mississippi River System stands as a nationally significant pathway for

New comprehensive denaturing-gradient-gel-electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours

NARCIS (Netherlands)

Hayes, VM; Westra, JL; Verlind, E; Bleeker, W; Plukker, JT; Hofstra, RMW; Buys, CHCM

2000-01-01

A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within

Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes

Czech Academy of Sciences Publication Activity Database

Ostatná, Veronika; Černocká, Hana; Kurzatkowska, K.; Paleček, Emil

2012-01-01

Roč. 735, JUL (2012), s. 31-36 ISSN 0003-2670 R&D Projects: GA AV ČR(CZ) KJB100040901; GA ČR(CZ) GAP301/11/2055; GA MŠk(CZ) ME09038 Institutional research plan: CEZ:AV0Z50040702 Keywords : protein denaturation * carbon electrodes * edge plane pyrolytic graphite Subject RIV: BO - Biophysics Impact factor: 4.387, year: 2012

Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life.

Directory of Open Access Journals (Sweden)

M Luisa Romero-Romero

Full Text Available The relationship between the denaturation temperatures of proteins (Tm values and the living temperatures of their host organisms (environmental temperatures: TENV values is poorly understood. Since different proteins in the same organism may show widely different Tm's, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm's are oftentimes found to correlate with TENV's but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm's for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

Molecular confirmation of hybridization between Dascyllus reticulatus × Dascyllus aruanus from the Great Barrier Reef

KAUST Repository

He, Song; Johansen, Jacob L.; Hoey, Andrew S.; Pappas, Melissa; Berumen, Michael L.

2017-01-01

To date, more than 81 species of tropical coral reef fish have been reported to hybridize in nature, spanning multiple families, including the Chaetodontidae, Pomacanthidae, and Labridae. Hybridization, however, is seemingly rare among benthic nesting species that engage in pair spawning, such as the Pomacentridae. Here, we present evidence for the first molecularly confirmed record of hybridization within the genus Dascyllus; D. aruanus and D. reticulatus. Interestingly, although many hybridization events are attributed to peripheral range effects or areas of limited overlap among otherwise allopatric species, this hybrid individual was collected from the northern Great Barrier Reef, centrally located within the distribution ranges of both species. The hybrid exhibited coloration and meristic counts intermediate between D. aruanus and D. reticulatus. Diagnostic genetic markers and subsequent microsatellites analysis confirmed that this individual was a hybrid offspring of D. aruanus and D. reticulatus, with the latter providing the maternal contribution. The occurrence of the D. aruanus × D. reticulatus hybrid on the Great Barrier Reef represents an exception to the otherwise species-specific haplotypes. The nuclear diagnostic marker which was identified during this study could serve as a hybrid indicator and benefit future hybrid investigations for hybridization between these two species.

Molecular confirmation of hybridization between Dascyllus reticulatus × Dascyllus aruanus from the Great Barrier Reef

KAUST Repository

He, Song

2017-11-17

To date, more than 81 species of tropical coral reef fish have been reported to hybridize in nature, spanning multiple families, including the Chaetodontidae, Pomacanthidae, and Labridae. Hybridization, however, is seemingly rare among benthic nesting species that engage in pair spawning, such as the Pomacentridae. Here, we present evidence for the first molecularly confirmed record of hybridization within the genus Dascyllus; D. aruanus and D. reticulatus. Interestingly, although many hybridization events are attributed to peripheral range effects or areas of limited overlap among otherwise allopatric species, this hybrid individual was collected from the northern Great Barrier Reef, centrally located within the distribution ranges of both species. The hybrid exhibited coloration and meristic counts intermediate between D. aruanus and D. reticulatus. Diagnostic genetic markers and subsequent microsatellites analysis confirmed that this individual was a hybrid offspring of D. aruanus and D. reticulatus, with the latter providing the maternal contribution. The occurrence of the D. aruanus × D. reticulatus hybrid on the Great Barrier Reef represents an exception to the otherwise species-specific haplotypes. The nuclear diagnostic marker which was identified during this study could serve as a hybrid indicator and benefit future hybrid investigations for hybridization between these two species.

Cross-species BAC-FISH painting of the tomato and potato chromosome 6 reveals undescribed chromosomal rearrangements

NARCIS (Netherlands)

Tang, X.; Szinay, D.; Ramanna, M.S.; Vossen, van der E.A.G.; Datema, E.; Klein Lankhorst, R.M.; Boer, de J.M.; Peters, S.A.; Bachem, C.W.B.; Stiekema, W.J.; Visser, R.G.F.; Jong, de J.H.; Bai, Y.

2008-01-01

Ongoing genomics projects of tomato (Solanum lycopersicum ) and potato (Solanum tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, BACs can be positioned on pachytene comple-ments by fluorescent in situ hybridization (FISH) on homoeologous

Formation of diploid and triploid hybrid groupers (hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂) and their 5S gene analysis.

Science.gov (United States)

Huang, Wen; Qin, Qinbo; Yang, Huirong; Li, Shuisheng; Hu, Chaoqun; Wang, Yude; Zhang, Yong; Liu, Shaojun; Lin, Haoran

2016-10-07

Interspecies hybridization is widely used to achieve heterosis or hybrid vigor, which has been observed and harnessed by breeders for centuries. Natural allopolyploid hybrids generally exhibit more superior heterosis than both the diploid progenies and their parental species. However, polyploid formation processes have been long ignored, the genetic basis of heterosis in polyploids remains elusive. In the present study, triploid hybrids had been demonstrated to contain two sets of chromosomes from mother species and one set from father species. Cellular polyploidization process in the embryos had been traced. The triploid hybrids might be formed by failure formation of the second polarized genome during the second meiosis stage. Four spindle centers were observed in anaphase stage of the first cell division. Three spindle centers were observed in side of cell plate after the first cell division. The 5S rDNA genes of four types of groupers were cloned and analyzed. The diploid and triploid hybrids had been proved to contain the tandem chimera structures which were recombined by maternal and paternal monomer units. The results indicated that genome re-fusion had occurred in the hybrid progenies. To further elucidate the genetic patterns of diploid and triploid hybrids, fluorescence chromosome location had been carried out, maternal 5S gene (M-386) were used as the probe. The triploid hybrids contained fewer fluorescence loci numbers than the maternal species. The results indicated that participation of paternal 5S gene in the triploid hybrid genome had degraded the match rates of M-386 probe. Our study is the first to investigate the cellular formation processes of natural allopolyploids in hybrid fish, the cellular polyploidization process may be caused by failure formation of the second polarized genome during the meiosis, and our results will provide the molecular basis of hybrid vigor in interspecies hybridization.

50 CFR 23.43 - What are the requirements for a wildlife hybrid?

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false What are the requirements for a wildlife hybrid? 23.43 Section 23.43 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF... IMPORTATION OF WILDLIFE AND PLANTS (CONTINUED) CONVENTION ON INTERNATIONAL TRADE IN ENDANGERED SPECIES OF WILD...

Spatial segregation of spawning habitat limits hybridization between sympatric native Steelhead and Coastal Cutthroat Trout

Science.gov (United States)

Buehrens, T.W.; Glasgow, J.; Ostberg, Carl O.; Quinn, T.P.

2013-01-01

Native Coastal Cutthroat Trout Oncorhynchus clarkii clarkii and Coastal Steelhead O. mykiss irideus hybridize naturally in watersheds of the Pacific Northwest yet maintain species integrity. Partial reproductive isolation due to differences in spawning habitat may limit hybridization between these species, but this process is poorly understood. We used a riverscape approach to determine the spatial distribution of spawning habitats used by native Coastal Cutthroat Trout and Steelhead as evidenced by the distribution of recently emerged fry. Molecular genetic markers were used to classify individuals as pure species or hybrids, and individuals were assigned to age-classes based on length. Fish and physical habitat data were collected in a spatially continuous framework to assess the relationship between habitat and watershed features and the spatial distribution of parental species and hybrids. Sampling occurred in 35 reaches from tidewaters to headwaters in a small (20 km2) coastal watershed in Washington State. Cutthroat, Steelhead, and hybrid trout accounted for 35%, 42%, and 23% of the fish collected, respectively. Strong segregation of spawning areas between Coastal Cutthroat Trout and Steelhead was evidenced by the distribution of age-0 trout. Cutthroat Trout were located farther upstream and in smaller tributaries than Steelhead were. The best predictor of species occurrence at a site was the drainage area of the watershed that contributed to the site. This area was positively correlated with the occurrence of age-0 Steelhead and negatively with the presence of Cutthroat Trout, whereas hybrids were found in areas occupied by both parental species. A similar pattern was observed in older juveniles of both species but overlap was greater, suggesting substantial dispersal of trout after emergence. Our results offer support for spatial reproductive segregation as a factor limiting hybridization between Steelhead and Coastal Cutthroat Trout.

Detection of iron-depositing Pedomicrobium species in native biofilms from the Odertal National Park by a new, specific FISH probe.

Science.gov (United States)

Braun, Burga; Richert, Inga; Szewzyk, Ulrich

2009-10-01

Iron-depositing bacteria play an important role in technical water systems (water wells, distribution systems) due to their intense deposition of iron oxides and resulting clogging effects. Pedomicrobium is known as iron- and manganese-oxidizing and accumulating bacterium. The ability to detect and quantify members of this species in biofilm communities is therefore desirable. In this study the fluorescence in situ hybridization (FISH) method was used to detect Pedomicrobium in iron and manganese incrusted biofilms. Based on comparative sequence analysis, we designed and evaluated a specific oligonucleotide probe (Pedo 1250) complementary to the hypervariable region 8 of the 16S rRNA gene for Pedomicrobium. Probe specificities were tested against 3 different strains of Pedomicrobium and Sphingobium yanoikuyae as non-target organism. Using optimized conditions the probe hybridized with all tested strains of Pedomicrobium with an efficiency of 80%. The non-target organism showed no hybridization signals. The new FISH probe was applied successfully for the in situ detection of Pedomicrobium in different native, iron-depositing biofilms. The hybridization results of native bioflims using probe Pedo_1250 agreed with the results of the morphological structure of Pedomicrobium bioflims based on scanning electron microscopy.

[Cytogenetic Abnormalities and Outcomes of 117 Patients with Multiple Myeloma Detected by FISH].

Science.gov (United States)

Zhai, Bing; Zou, Dan-Dan; Yan, Jian-Jun; Wang, Nan; Wang, Li-Li; Zhu, Hong-Li; Huang, Wen-Rong; Yu, Li

2016-02-01

To analyze the cytogenetic abnormalities and prognostic outcomes of patients with multiple myeloma (MM) detected by fluorescence in situ hybridization (FISH). The clinical record of 117 newly-diagnosed patients with MM treated in department of hematology and geriatric hematology of our hospital for 7 years were collected, and their molecular cytogenetic abnormalities detected by FISH and the clinical outcome were analyzed retrospectively. The detected rate of cytogenetic abnormality was 76.9%(90/117), the most common abnormality deteted by FISH was 1q21+ (71.1%), followed by 13q- (56.6%). The cross comparison method showed that 13q- and 17p13-, t(11;14) and t(4;14) were related respectively. All the patients with cytogenetic abnormalities showed no significant difference in the overall survival from cytogenetic normal patients. The positive rate of molecular cytogenetic abnormalities detected by FISH in MM patients is high, but data from larger and longer studies are needed to evaluate the prognostic outcomes.

Natural selection interacts with recombination to shape the evolution of hybrid genomes.

Science.gov (United States)

Schumer, Molly; Xu, Chenling; Powell, Daniel L; Durvasula, Arun; Skov, Laurits; Holland, Chris; Blazier, John C; Sankararaman, Sriram; Andolfatto, Peter; Rosenthal, Gil G; Przeworski, Molly

2018-05-11

To investigate the consequences of hybridization between species, we studied three replicate hybrid populations that formed naturally between two swordtail fish species, estimating their fine-scale genetic map and inferring ancestry along the genomes of 690 individuals. In all three populations, ancestry from the "minor" parental species is more common in regions of high recombination and where there is linkage to fewer putative targets of selection. The same patterns are apparent in a reanalysis of human and archaic admixture. These results support models in which ancestry from the minor parental species is more likely to persist when rapidly uncoupled from alleles that are deleterious in hybrids. Our analyses further indicate that selection on swordtail hybrids stems predominantly from deleterious combinations of epistatically interacting alleles. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Detection in a Japanese population of a length polymorphism in the 5' flanking region of the human β-globin gene with denaturing gradient gel electrophoresis

International Nuclear Information System (INIS)

Takahashi, Noria; Hiyama, Keiko; Kodaira, Mieko; Satoh, Chiyoko

1992-10-01

An analysis of the ATTTT repeat polymorphism located approximately 1,400 base pairs upstream from the β-globin structural gene was carried out by denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes. Genomic or cloned DNAs were digested with restriction enzymes and hybridized with 32 P-labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. A difference in the number of repeat units was recognized by differences in duplex mobility on the DGGE gel. In this study of 81 unrelated Japanese from Hiroshima, a sequence heteromorphism was observed at this site. Alleles with 5 and 6 repeats of the ATTTT unit, which had already been reported, were found in polymorphic proportions. In addition, two unreported alleles, one having 7 repeats and the other having an A-to-G nucleotide substitution in the 5th repeat, were detected. Family study data showed that the segregation of these four types of variants is consistent with an autosomal codominant mode of inheritance. This study also demonstrated that DGGE of RNA:DNA duplexes is a sensitive tool for detecting variations in DNA. (author)

Native and denatured bovine serum albumin. D.c. polarography, stripping voltammetry and constant current chronopotentiometry

Czech Academy of Sciences Publication Activity Database

Ostatná, Veronika; Uslu, B.; Dogan, B.; Ozkan, S.; Paleček, Emil

2006-01-01

Roč. 593, č. 1-2 (2006), s. 172-178 ISSN 0022-0728 R&D Projects: GA AV ČR(CZ) IAA500040513; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507 Keywords : protein electrochemistry * bovine serum albumin * native and denatured proteins Subject RIV: BO - Biophysics Impact factor: 2.339, year: 2006

The development of FISH tools for genetic, phylogenetic and breeding studies in tomato (Solanum lycopersicum)

NARCIS (Netherlands)

Szinay, D.

2010-01-01

In this thesis various fluorescence in situ hybridization (FISH) technologies are described to support genome projects, plant breeding and phylogenetic analysis on tomato (Solanum lycopersicum, 2n=24). Its genome is 980 Mb and only 30 % are single copy sequences, which are mostly found in the

Detection of dengue group viruses by fluorescence in situ hybridization

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Raquin Vincent

2012-10-01

Full Text Available Abstract Background Dengue fever (DF and dengue hemorrhagic fever (DHF represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus that comprises four distinct serotypes (DENV-1 to DENV-4. Fluorescence in situ hybridization (FISH has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae. The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use

Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification.

Science.gov (United States)

Agersborg, Sally; Mixon, Christopher; Nguyen, Thanh; Aithal, Sramila; Sudarsanam, Sucha; Blocker, Forrest; Weiss, Lawrence; Gasparini, Robert; Jiang, Shiping; Chen, Wayne; Hess, Gregory; Albitar, Maher

2018-03-22

While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases. Between 2014 and 2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of the patients tested by FISH, 505/3908 (12.9%) also had IHC data. Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. However, 50.5% of equivocal cases were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe: 78% of positive cases by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (p alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH. The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine the benefit of anti-HER2 therapy in these patients are urgently needed.

Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

Science.gov (United States)

Anumalla, Bramhini; Prabhu, N Prakash

2018-01-25

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two

Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in archival breast carcinoma.

Science.gov (United States)

Zhao, Jianxin; Wu, Rina; Au, Alfred; Marquez, Abbey; Yu, Yibing; Shi, Zuorong

2002-06-01

To compare the efficacy of chromogenic in situ hybridization (CISH(TM)) with fluorescence in situ (FISH) hybridization and immunohistochemistry (IHC) in determination of the HER2 status in human breast cancer. HER2 gene amplification was determined on formalin-fixed paraffin-embedded (FFPE) sections of 62 invasive breast cancers by FISH and followed by CISH using a digoxigenin (DIG)-labeled HER2 DNA probe generated by Subtraction Probe Technology (SPT(TM)), and a biotin-labeled chromosome 17 centromeric (chr.17cen) probe. The sections were heat treated and enzyme digested. After in situ hybridization, the HER2 probe was detected with fluorescein (FITC)-anti-DIG for FISH, followed by peroxidase-anti-FITC and diaminobenzidine (DAB) for CISH. The chr.17cen probe was detected with peroxidase-streptavidin and DAB. For CISH application, HER2 gene copies or chromosome 17 centromeres and morphology of cells were easily visualized simultaneously with a 40x objective under bright-field microscope in hematoxylin-counterstained sections. IHC study of HER2 overexpression was performed on adjacent sections using a panel of three HER2 antibodies (TAB 250, CB11, A0485), and staining was scored according to the criteria specified in the HercepTest. HER2 gene amplification detected by CISH was visualized typically as large DAB-stained clusters or by many dots in the nucleus. FISH and CISH identified HER2 gene amplification in 19% of the tumors. Chromosome 17 polysomy was detected in 31% of the tumors. HER2 overexpression was demonstrated in 19% (TAB 250), 23% (CB11), and 36% (A0485) of the tumors. Complete concordance between the results of CISH with FISH, TAB 250, CB11, and A0485 was seen in 100%, 97%, 94%, and 84% of the cases, respectively. By permitting observation of morphology using a bright-field microscope, CISH is an accurate, practical, and economical approach to screen HER2 status in breast cancers. It is a useful methodology for confirming ambiguous IHC results.

Multifractal-based nuclei segmentation in fish images.

Science.gov (United States)

Reljin, Nikola; Slavkovic-Ilic, Marijeta; Tapia, Coya; Cihoric, Nikola; Stankovic, Srdjan

2017-09-01

The method for nuclei segmentation in fluorescence in-situ hybridization (FISH) images, based on the inverse multifractal analysis (IMFA) is proposed. From the blue channel of the FISH image in RGB format, the matrix of Holder exponents, with one-by-one correspondence with the image pixels, is determined first. The following semi-automatic procedure is proposed: initial nuclei segmentation is performed automatically from the matrix of Holder exponents by applying predefined hard thresholding; then the user evaluates the result and is able to refine the segmentation by changing the threshold, if necessary. After successful nuclei segmentation, the HER2 (human epidermal growth factor receptor 2) scoring can be determined in usual way: by counting red and green dots within segmented nuclei, and finding their ratio. The IMFA segmentation method is tested over 100 clinical cases, evaluated by skilled pathologist. Testing results show that the new method has advantages compared to already reported methods.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Directory of Open Access Journals (Sweden)

F.E. Rosa

Full Text Available Human epidermal growth factor receptor 2 (HER2 has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC. HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH in moderate immunoexpression (IHC 2+ cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH, which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Directory of Open Access Journals (Sweden)

F.E. Rosa

2013-03-01

Full Text Available Human epidermal growth factor receptor 2 (HER2 has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC. HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH in moderate immunoexpression (IHC 2+ cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH, which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

Specific Fluorescence in Situ Hybridization (FISH) Test to Highlight Colonization of Xylem Vessels by Xylella fastidiosa in Naturally Infected Olive Trees (Olea europaea L.)

Science.gov (United States)

Cardinale, Massimiliano; Luvisi, Andrea; Meyer, Joana B.; Sabella, Erika; De Bellis, Luigi; Cruz, Albert C.; Ampatzidis, Yiannis; Cherubini, Paolo

2018-01-01

The colonization behavior of the Xylella fastidiosa strain CoDiRO, the causal agent of olive quick decline syndrome (OQDS), within the xylem of Olea europaea L. is still quite controversial. As previous literature suggests, even if xylem vessel occlusions in naturally infected olive plants were observed, cell aggregation in the formation of occlusions had a minimal role. This observation left some open questions about the whole behavior of the CoDiRO strain and its actual role in OQDS pathogenesis. In order to evaluate the extent of bacterial infection in olive trees and the role of bacterial aggregates in vessel occlusions, we tested a specific fluorescence in situ hybridization (FISH) probe (KO 210) for X. fastidiosa and quantified the level of infection and vessel occlusion in both petioles and branches of naturally infected and non-infected olive trees. All symptomatic petioles showed colonization by X. fastidiosa, especially in the larger innermost vessels. In several cases, the vessels appeared completely occluded by a biofilm containing bacterial cells and extracellular matrix and the frequent colonization of adjacent vessels suggested a horizontal movement of the bacteria. Infected symptomatic trees had 21.6 ± 10.7% of petiole vessels colonized by the pathogen, indicating an irregular distribution in olive tree xylem. Thus, our observations point out the primary role of the pathogen in olive vessel occlusions. Furthermore, our findings indicate that the KO 210 FISH probe is suitable for the specific detection of X. fastidiosa. PMID:29681910

Sampling the Denatured State of Polypeptides in Water, Urea, and Guanidine Chloride to Strict Equilibrium Conditions with the Help of Massively Parallel Computers.

Science.gov (United States)

Meloni, Roberto; Camilloni, Carlo; Tiana, Guido

2014-02-11

The denatured state of polypeptides and proteins, stabilized by chemical denaturants like urea and guanidine chloride, displays residual secondary structure when studied by nuclear-magnetic-resonance spectroscopy. However, these experimental techniques are weakly sensitive, and thus molecular-dynamics simulations can be useful to complement the experimental findings. To sample the denatured state, we made use of massively-parallel computers and of a variant of the replica exchange algorithm, in which the different branches, connected with unbiased replicas, favor the formation and disruption of local secondary structure. The algorithm is applied to the second hairpin of GB1 in water, in urea, and in guanidine chloride. We show with the help of different criteria that the simulations converge to equilibrium. It results that urea and guanidine chloride, besides inducing some polyproline-II structure, have different effect on the hairpin. Urea disrupts completely the native region and stabilizes a state which resembles a random coil, while guanidine chloride has a milder effect.

Heavy ion-induced chromosomal aberrations analyzed by fluorescence in situ hybridization

International Nuclear Information System (INIS)

Durante, M.; Gialanella, G.; Grossi, G.; Pugliese, M.; Cella, L.; Greco, O.; George, K.; Yang, T.C.

1997-01-01

We have investigated the effectiveness of heavy ions in the induction of chromosomal aberrations in mammalian cells by the recent technique of fluorescence in situ hybridization (FISH) with whole-chromosome probes. FISH-painting was used both in metaphase and interphase (prematurely condensed) chromosomes. The purpose of our experiments was to address the following problems: (a) the ratio of different types of aberrations as a function of radiation quality (search for biomarkers); (b) the ratio between aberrations scored in interphase and metaphase as a function of radiation quality (role of apoptosis); (c) differences between cytogenetic effects produced by different ions at the same LET (role of track structure). (orig./MG)

Heavy ion-induced chromosomal aberrations analyzed by fluorescence in situ hybridization

Energy Technology Data Exchange (ETDEWEB)

Durante, M; Gialanella, G; Grossi, G; Pugliese, M [Univ. ` ` Federico II` ` , Naples (Italy). Dept. of Physics; [INFN, Naples (Italy); Cella, L; Greco, O [Univ. ` ` Federico II` ` , Naples (Italy). Dept. of Physics; Furusawa, Y [NIRS, Chiba (Japan); George, K; Yang, T C [NASA Lyndon B. Johnson Space Center, Houston, TX (United States)

1997-09-01

We have investigated the effectiveness of heavy ions in the induction of chromosomal aberrations in mammalian cells by the recent technique of fluorescence in situ hybridization (FISH) with whole-chromosome probes. FISH-painting was used both in metaphase and interphase (prematurely condensed) chromosomes. The purpose of our experiments was to address the following problems: (a) the ratio of different types of aberrations as a function of radiation quality (search for biomarkers); (b) the ratio between aberrations scored in interphase and metaphase as a function of radiation quality (role of apoptosis); (c) differences between cytogenetic effects produced by different ions at the same LET (role of track structure). (orig./MG)

On-site detection of Phytophthora spp.—single-stranded target DNA as the limiting factor to improve on-chip hybridization

International Nuclear Information System (INIS)

Schwenkbier, Lydia; Pollok, Sibyll; Popp, Jürgen; Weber, Karina; König, Stephan; Wagner, Stefan; Werres, Sabine; Weber, Jörg; Hentschel, Martin

2014-01-01

We report on a lab-on-a-chip approach for on-site detection of Phytophthora species that allows visual signal readout. The results demonstrate the significance of single-stranded DNA (ssDNA) generation in terms of improving the intensity of the hybridization signal and to improve the reliability of the method. Conventional PCR with subsequent heat denaturation, sodium hydroxide-based denaturation, lambda exonuclease digestion and two asymmetric PCR methods were investigated for the species P. fragariae, P. kernoviae, and P. ramorum. The positioning of the capture probe within the amplified yeast GTP-binding protein (YPT1) target DNA was also of interest because it significantly influences the intensity of the signal. Statistical tests were used to validate the impact of the ssDNA generation methods and the capture-target probe position. The single-stranded target DNA generated by Linear-After-The-Exponential PCR (LATE-PCR) was found to produce signal intensities comparable to post-PCR exonuclease treatment. The LATE-PCR is the best method for the on-site detection of Phytophthora because the enzymatic digestion after PCR is more laborious and time-consuming. (author)

ZyFISH: a simple, rapid and reliable zygosity assay for transgenic mice.

Directory of Open Access Journals (Sweden)

Donal McHugh

Full Text Available Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH. The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.

Denatured states of yeast cytochrome c induced by heat and guanidinium chloride are structurally and thermodynamically different.

Science.gov (United States)

Zaidi, Sobia; Haque, Md Anzarul; Ubaid-Ullah, Shah; Prakash, Amresh; Hassan, Md Imtaiyaz; Islam, Asimul; Batra, Janendra K; Ahmad, Faizan

2017-05-01

A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.

Development of Thinopyrum ponticum-specific molecular markers and FISH probes based on SLAF-seq technology.

Science.gov (United States)

Liu, Liqin; Luo, Qiaoling; Teng, Wan; Li, Bin; Li, Hongwei; Li, Yiwen; Li, Zhensheng; Zheng, Qi

2018-05-01

Based on SLAF-seq, 67 Thinopyrum ponticum-specific markers and eight Th. ponticum-specific FISH probes were developed, and these markers and probes could be used for detection of alien chromatin in a wheat background. Decaploid Thinopyrum ponticum (2n = 10x = 70) is a valuable gene reservoir for wheat improvement. Identification of Th. ponticum introgression would facilitate its transfer into diverse wheat genetic backgrounds and its practical utilization in wheat improvement. Based on specific-locus-amplified fragment sequencing (SLAF-seq) technology, 67 new Th. ponticum-specific molecular markers and eight Th. ponticum-specific fluorescence in situ hybridization (FISH) probes have been developed from a tiny wheat-Th. ponticum translocation line. These newly developed molecular markers allowed the detection of Th. ponticum DNA in a variety of materials specifically and steadily at high throughput. According to the hybridization signal pattern, the eight Th. ponticum-specific probes could be divided into two groups. The first group including five dispersed repetitive sequence probes could identify Th. ponticum chromatin more sensitively and accurately than genomic in situ hybridization (GISH). Whereas the second group having three tandem repetitive sequence probes enabled the discrimination of Th. ponticum chromosomes together with another clone pAs1 in wheat-Th. ponticum partial amphiploid Xiaoyan 68.

Bioinspired Smart Actuator Based on Graphene Oxide-Polymer Hybrid Hydrogels.

Science.gov (United States)

Wang, Tao; Huang, Jiahe; Yang, Yiqing; Zhang, Enzhong; Sun, Weixiang; Tong, Zhen

2015-10-28

Rapid response and strong mechanical properties are desired for smart materials used in soft actuators. A bioinspired hybrid hydrogel actuator was designed and prepared by series combination of three trunks of tough polymer-clay hydrogels to accomplish the comprehensive actuation of "extension-grasp-retraction" like a fishing rod. The hydrogels with thermo-creep and thermo-shrinking features were successively irradiated by near-infrared (NIR) to execute extension and retraction, respectively. The GO in the hydrogels absorbed the NIR energy and transformed it into thermo-energy rapidly and effectively. The hydrogel with adhesion or magnetic force was adopted as the "hook" of the hybrid hydrogel actuator for grasping object. The hook of the hybrid hydrogel actuator was replaceable according to applications, even with functional materials other than hydrogels. This study provides an innovative concept to explore new soft actuators through combining response hydrogels and programming the same stimulus.

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization

International Nuclear Information System (INIS)

Lawrence, J.B.; Marselle, L.M.; Byron, K.S.; Johnson, C.V.; Sullivan, J.L.; Singer, R.H.

1990-01-01

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell

Microbiological spoilage of fish and fish products

DEFF Research Database (Denmark)

Gram, Lone; Huss, Hans Henrik

1996-01-01

Spoilage of fresh and lightly preserved fish products is caused by microbial action. This paper reviews the current knowledge in terms of the microbiology of fish and fish products with particular emphasis on identification of specific spoilage bacteria and the qualitative and quantitative...... biochemical indicators of spoilage. Shewanzella putrefaciens and Pseudomonas spp. are the specific spoilage bacteria of iced fresh fish regardless of the origin of the fish. Modified atmosphere stored marine fish from temperate waters are spoiled by the CO2 resistant Photobacterium phosphoreum whereas Gram......- positive bacteria are likely spoilers of CO2 packed fish from fresh or tropical waters. Fish products with high salt contents may spoil due to growth of halophilic bacteria (salted fish) or growth of anaerobic bacteria and yeasts (barrel salted fish). Whilst the spoilage of fresh and highly salted fish...

Multifocal peritoneal splenosis in Tc-99m-labeled heat-denatured red blood cell scintigraphy

Energy Technology Data Exchange (ETDEWEB)

Yoon, Min Ki; Hwang, Kyung Hoon; Choe, Won Sick [Gachon University Gil Medical Center, Incheon (Korea, Republic of)

2006-06-15

A 44-year-old man with a past medical history of splenectomy came to hospital because of epigastric pain abdominopelvic computed tomography(CT) showed a soft tissue mass and multifocal variable-sized nodules as well as finding suggestive of cholecystitis. Subsequently, he underwent Tc-99m-labeled heat- denatured red blood cell(RBC) scintigraphy to evaluate the mass and nodules. The scintigraphy confirmed multifocal peritoneal splenosis in the abdominopelvic cavity.

Resolving colocalization of bacteria and metal(loid)s on plant root surfaces by combining fluorescence in situ hybridization (FISH) with multiple-energy micro-focused X-ray fluorescence (ME μXRF).

Science.gov (United States)

Honeker, Linnea K; Root, Robert A; Chorover, Jon; Maier, Raina M

2016-12-01

Metal(loid)-contamination of the environment due to anthropogenic activities is a global problem. Understanding the fate of contaminants requires elucidation of biotic and abiotic factors that influence metal(loid) speciation from molecular to field scales. Improved methods are needed to assess micro-scale processes, such as those occurring at biogeochemical interfaces between plant tissues, microbial cells, and metal(loid)s. Here we present an advanced method that combines fluorescence in situ hybridization (FISH) with synchrotron-based multiple-energy micro-focused X-ray fluorescence microprobe imaging (ME μXRF) to examine colocalization of bacteria and metal(loid)s on root surfaces of plants used to phytostabilize metalliferous mine tailings. Bacteria were visualized on a small root section using SytoBC nucleic acid stain and FISH probes targeting the domain Bacteria and a specific group (Alphaproteobacteria, Gammaproteobacteria, or Actinobacteria). The same root region was then analyzed for elemental distribution and metal(loid) speciation of As and Fe using ME μXRF. The FISH and ME μXRF images were aligned using ImageJ software to correlate microbiological and geochemical results. Results from quantitative analysis of colocalization show a significantly higher fraction of As colocalized with Fe-oxide plaques on the root surfaces (fraction of overlap 0.49±0.19) than to bacteria (0.072±0.052) (proots, metal(loid)s and microbes, information that should lead to improved mechanistic models of metal(loid) speciation and fate. Copyright © 2016 Elsevier B.V. All rights reserved.

Repetitive sequences: the hidden diversity of heterochromatin in prochilodontid fish

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Maria L. Terencio

2015-08-01

Full Text Available The structure and organization of repetitive elements in fish genomes are still relatively poorly understood, although most of these elements are believed to be located in heterochromatic regions. Repetitive elements are considered essential in evolutionary processes as hotspots for mutations and chromosomal rearrangements, among other functions – thus providing new genomic alternatives and regulatory sites for gene expression. The present study sought to characterize repetitive DNA sequences in the genomes of Semaprochilodus insignis (Jardine & Schomburgk, 1841 and Semaprochilodus taeniurus (Valenciennes, 1817 and identify regions of conserved syntenic blocks in this genome fraction of three species of Prochilodontidae (S. insignis, S. taeniurus, and Prochilodus lineatus (Valenciennes, 1836 by cross-FISH using Cot-1 DNA (renaturation kinetics probes. We found that the repetitive fractions of the genomes of S. insignis and S. taeniurus have significant amounts of conserved syntenic blocks in hybridization sites, but with low degrees of similarity between them and the genome of P. lineatus, especially in relation to B chromosomes. The cloning and sequencing of the repetitive genomic elements of S. insignis and S. taeniurus using Cot-1 DNA identified 48 fragments that displayed high similarity with repetitive sequences deposited in public DNA databases and classified as microsatellites, transposons, and retrotransposons. The repetitive fractions of the S. insignis and S. taeniurus genomes exhibited high degrees of conserved syntenic blocks in terms of both the structures and locations of hybridization sites, but a low degree of similarity with the syntenic blocks of the P. lineatus genome. Future comparative analyses of other prochilodontidae species will be needed to advance our understanding of the organization and evolution of the genomes in this group of fish.

Microbiological spoilage of fish and fish products.

Science.gov (United States)

Gram, L; Huss, H H

1996-11-01

Spoilage of fresh and lightly preserved fish products is caused by microbial action. This paper reviews the current knowledge in terms of the microbiology of fish and fish products with particular emphasis on identification of specific spoilage bacteria and the qualitative and quantitative biochemical indicators of spoilage. Shewanella putrefaciens and Pseudomonas spp. are the specific spoilage bacteria of iced fresh fish regardless of the origin of the fish. Modified atmosphere stored marine fish from temperate waters are spoiled by the CO2 resistant Photobacterium phosphoreum whereas Gram-positive bacteria are likely spoilers of CO2 packed fish from fresh or tropical waters. Fish products with high salt contents may spoil due to growth of halophilic bacteria (salted fish) or growth of anaerobic bacteria and yeasts (barrel salted fish). Whilst the spoilage of fresh and highly salted fish is well understood, much less is known about spoilage of lightly preserved fish products. It is concluded that the spoilage is probably caused by lactic acid bacteria, certain psychotrophic Enterobacteriaceae and/or Photobacterium phosphoreum. However, more work is needed in this area.

Invasive hybridization in a threatened species is accelerated by climate change

Science.gov (United States)

Muhlfeld, Clint C.; Kovach, Ryan P.; Jones, Leslie A.; Al-Chokhachy, Robert K.; Boyer, Matthew C.; Leary, Robb F.; Lowe, Winsor H.; Luikart, Gordon; Allendorf, Fred W.

2014-01-01

Climate change will decrease worldwide biodiversity through a number of potential pathways, including invasive hybridization (cross-breeding between invasive and native species). How climate warming influences the spread of hybridization and loss of native genomes poses difficult ecological and evolutionary questions with little empirical information to guide conservation management decisions. Here we combine long-term genetic monitoring data with high-resolution climate and stream temperature predictions to evaluate how recent climate warming has influenced the spatio-temporal spread of human-mediated hybridization between threatened native westslope cutthroat trout (Oncorhynchus clarkii lewisi) and non-native rainbow trout (Oncorhynchus mykiss), the world’s most widely introduced invasive fish. Despite widespread release of millions of rainbow trout over the past century within the Flathead River system, a large relatively pristine watershed in western North America, historical samples revealed that hybridization was prevalent only in one (source) population. During a subsequent 30-year period of accelerated warming, hybridization spread rapidly and was strongly linked to interactions between climatic drivers—precipitation and temperature—and distance to the source population. Specifically, decreases in spring precipitation and increases in summer stream temperature probably promoted upstream expansion of hybridization throughout the system. This study shows that rapid climate warming can exacerbate interactions between native and non-native species through invasive hybridization, which could spell genomic extinction for many species.

Invasive hybridization in a threatened species is accelerated by climate change

Science.gov (United States)

Muhlfeld, Clint C.; Kovach, Ryan P.; Jones, Leslie A.; Al-Chokhachy, Robert; Boyer, Matthew C.; Leary, Robb F.; Lowe, Winsor H.; Luikart, Gordon; Allendorf, Fred W.

2014-07-01

Climate change will decrease worldwide biodiversity through a number of potential pathways, including invasive hybridization (cross-breeding between invasive and native species). How climate warming influences the spread of hybridization and loss of native genomes poses difficult ecological and evolutionary questions with little empirical information to guide conservation management decisions. Here we combine long-term genetic monitoring data with high-resolution climate and stream temperature predictions to evaluate how recent climate warming has influenced the spatio-temporal spread of human-mediated hybridization between threatened native westslope cutthroat trout (Oncorhynchus clarkii lewisi) and non-native rainbow trout (Oncorhynchus mykiss), the world's most widely introduced invasive fish. Despite widespread release of millions of rainbow trout over the past century within the Flathead River system, a large relatively pristine watershed in western North America, historical samples revealed that hybridization was prevalent only in one (source) population. During a subsequent 30-year period of accelerated warming, hybridization spread rapidly and was strongly linked to interactions between climatic drivers--precipitation and temperature--and distance to the source population. Specifically, decreases in spring precipitation and increases in summer stream temperature probably promoted upstream expansion of hybridization throughout the system. This study shows that rapid climate warming can exacerbate interactions between native and non-native species through invasive hybridization, which could spell genomic extinction for many species.

Combination of microautoradiography and fluorescence in situ hybridization for identification of microorganisms degrading xenobiotic contaminants.

Science.gov (United States)

Yang, Yanru; Zarda, Annatina; Zeyer, Josef

2003-12-01

One of the central topics in environmental bioremediation research is to identify microorganisms that are capable of degrading the contaminants of interest. Here we report application of combined microautoradiography (MAR) and fluorescence in situ hybridization (FISH). The method has previously been used in a number of systems; however, here we demonstrate its feasibility in studying the degradation of xenobiotic compounds. With a model system (coculture of Pseudomonas putida B2 and Sphingomonas stygia incubated with [14C] o-nitrophenol), combination of MAR and FISH was shown to be able to successfully identify the microorganisms degrading o-nitrophenol. Compared with the conventional techniques, MAR-FISH allows fast and accurate identification of the microorganisms involved in environmental contaminant degradation.

Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology

Directory of Open Access Journals (Sweden)

Hovhannisyan Galina G

2010-09-01

Full Text Available Abstract Comet assay and micronucleus (MN test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH techniques. FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology.

TA3 - Dosimetry and instrumentation supply of the M-Fish technique to the Fish-3 painting technique for analysing translocations: A radiotherapy-treated patient study

International Nuclear Information System (INIS)

Pouzoulet, F.; Roch-Lefevre, S.; Giraudet, A.L.; Vaurijoux, A.; Voisin, P.A.; Buard, V.; Delbos, M.; Voisin, Ph.; Roy, L.; Bourhis, J.

2006-01-01

Purpose: Currently, the chromosome translocation study is the best method to estimate the dose of an old radiation exposure. Fluorescent In Situ Hybridization (F.I.S.H.) technique allows an easy detection of this kind of aberrations. However, as only a few number of chromosomes is usually painted, some bias could skew the result. To evaluate the advantage of using full genome staining (M-F.I.S.H. technique) compared with three chromosomes labelling (F.I.S.H.-3 painting), we compared translocation yields in radiotherapy treated patients. Methods: Chromosome aberration analyses were performed on peripheral blood lymphocyte cultures of two patients treated for a throat cancer by radiotherapy. Blood samples were obtained, before, along the treatment and six or four months later. For each sample, a dicentrics analysis was performed together with translocation analysis either with F.I.S.H.-3 painting or M-F.I.S.H.. Results: By confronting results from the F.I.S.H.-3 painting technique and the M-F.I.S.H. technique, significant differences were revealed. The translocations yield seemed to be stable with the F.I.S.H.-3 painting technique whereas it is not the case with the M-F.I.S.H. technique. This difference in results was explained by the bias induced by F.I.S.H.-3 Painting technique in the visualisation of complex aberrations. Furthermore, we found the presence of a clone bearing a translocation involving a painted chromosome. Conclusions: According to the potential bias of F.I.S.H.-3 painting on translocations study, the M-F.I.S.H. technique should provide more precise and reproducible results. Because of its more difficult implement, it seems hardly applicable to retrospective dosimetry instead of F.I.S.H.-3 painting technique. (authors)

TA3 - Dosimetry and instrumentation supply of the M-Fish technique to the Fish-3 painting technique for analysing translocations: A radiotherapy-treated patient study

Energy Technology Data Exchange (ETDEWEB)

Pouzoulet, F.; Roch-Lefevre, S.; Giraudet, A.L.; Vaurijoux, A.; Voisin, P.A.; Buard, V.; Delbos, M.; Voisin, Ph.; Roy, L. [Institut de Radioprotection et de Surete Nucleaire, Lab. de Dosimetrie Biologique, 92 - Fontenay aux Roses (France); Bourhis, J. [Laboratoire UPRES EA 27-10, Radiosensibilite des Tumeurs et Tissus sains, PR1, 94 - Villejuif (France)

2006-07-01

Purpose: Currently, the chromosome translocation study is the best method to estimate the dose of an old radiation exposure. Fluorescent In Situ Hybridization (F.I.S.H.) technique allows an easy detection of this kind of aberrations. However, as only a few number of chromosomes is usually painted, some bias could skew the result. To evaluate the advantage of using full genome staining (M-F.I.S.H. technique) compared with three chromosomes labelling (F.I.S.H.-3 painting), we compared translocation yields in radiotherapy treated patients. Methods: Chromosome aberration analyses were performed on peripheral blood lymphocyte cultures of two patients treated for a throat cancer by radiotherapy. Blood samples were obtained, before, along the treatment and six or four months later. For each sample, a dicentrics analysis was performed together with translocation analysis either with F.I.S.H.-3 painting or M-F.I.S.H.. Results: By confronting results from the F.I.S.H.-3 painting technique and the M-F.I.S.H. technique, significant differences were revealed. The translocations yield seemed to be stable with the F.I.S.H.-3 painting technique whereas it is not the case with the M-F.I.S.H. technique. This difference in results was explained by the bias induced by F.I.S.H.-3 Painting technique in the visualisation of complex aberrations. Furthermore, we found the presence of a clone bearing a translocation involving a painted chromosome. Conclusions: According to the potential bias of F.I.S.H.-3 painting on translocations study, the M-F.I.S.H. technique should provide more precise and reproducible results. Because of its more difficult implement, it seems hardly applicable to retrospective dosimetry instead of F.I.S.H.-3 painting technique. (authors)

Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter.

Science.gov (United States)

He, Weiguo; Qin, Qinbo; Liu, Shaojun; Li, Tangluo; Wang, Jing; Xiao, Jun; Xie, Lihua; Zhang, Chun; Liu, Yun

2012-01-01

Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I-N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.

Fluorescence in situ hybridization with reference to biodosimetry: a review

International Nuclear Information System (INIS)

Venkatachalam, P.; Paul, S.F.D.; Jeevanram, R.K.

1996-01-01

Many advances have taken place in the field of radiation biodosimetry in the recent past. Measurement of dicentric chromosome aberrations, was first developed and followed by micronuclei scoring. These, however, are unstable type aberrations and the cells carrying such aberrations are eliminated from the body in few years. They are therefore of use primarily in case of accidental exposures. The challenge is to measure the cumulative radiation exposure resulting from normal operations by measuring stable chromosome aberrations. Banding technique can measure stable chromosome aberration but require long time to analyse the banding pattern to study translocations. On the other hand fluorescence in situ hybridization (FISH) technique is sensitive, fast and easy to identify the translocations as the chromosomes involved in translocation are painted with different colours. This review brings out the requirements of various materials, their preparations, method of detection of fluorescence etc. for carrying out FISH. The experience of various laboratories using FISH in the monitoring of radiation absorbed dose is discussed. (author)

Luciferase genes cloned from the unculturable luminous bacteroid symbiont of the Caribbean flashlight fish, Kryptophanaron alfredi.

Science.gov (United States)

Haygood, M G; Cohn, D H

1986-01-01

Light organs of anomalopid (flashlight) fish contain luminous bacteroids that have never been cultured and, consequently, have been difficult to study. We have characterized the luciferase (lux) region of DNA extracted from light organs of the Caribbean flashlight fish Kryptophanaron alfredi by hybridization of cloned Vibrio harveyi lux genes to restriction-endonuclease-digested, light organ DNA. Comparison of the hybridization pattern of light organ DNA with that of DNA of a putative symbiotic isolate provides a method for identifying the authentic luminous symbiont regardless of its luminescence, and was used to reject one such isolate. Light organ DNA was further used to construct a cosmid clone bank and the luciferase genes were isolated. Unlike other bacterial luciferase genes, the genes were not expressed in Escherichia coli. When placed under the control of the E. coli trp promoter, the genes were transcribed but no luciferase was detected, suggesting a posttranscriptional block to expression.

INTERACTION OF IRON(II MIXED-LIGAND COMPLEXES WITH DNA: BASE-PAIR SPECIFICITY AND THERMAL DENATURATION STUDIES

Directory of Open Access Journals (Sweden)

Mudasir Mudasir

2010-06-01

Full Text Available A research about base-pair specificity of the DNA binding of [Fe(phen3]2+, [Fe(phen2(dip]2+ and [Fe(phen(dip2]2+ complexes and the effect of calf-thymus DNA (ct-DNA binding of these metal complexes on thermal denaturation of ct-DNA has been carried out. This research is intended to evaluate the preferential binding of the complexes to the sequence of DNA (A-T or G-C sequence and to investigate the binding strength and mode upon their interaction with DNA. Base-pair specificity of the DNA binding of the complexes was determined by comparing the equilibrium binding constant (Kb of each complex to polysynthetic DNA that contain only A-T or G-C sequence. The Kb value of the interaction was determined by spectrophotometric titration and thermal denaturation temperature (Tm was determined by monitoring the absorbance of the mixture solution of each complex and ct-DNA at λ =260 nm as temperature was elevated in the range of 25 - 100 oC. Results of the study show that in general all iron(II complexes studied exhibit a base-pair specificity in their DNA binding to prefer the relatively facile A-T sequence as compared to the G-C one. The thermal denaturation experiments have demonstrated that Fe(phen3]2+ and [Fe(phen2(dip]2+ interact weakly with double helical DNA via electrostatic interaction as indicated by insignificant changes in melting temperature, whereas [Fe(phen2(dip]2+  most probably binds to DNA in mixed modes of interaction, i.e.: intercalation and electrostatic interaction. This conclusion is based on the fact that the binding of [Fe(phen2(dip]2+ to ct-DNA moderately increase the Tm value of ct- DNA   Keywords: DNA Binding, mixed-ligand complexes

Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues.

Science.gov (United States)

Bulgari, Daniela; Casati, Paola; Faoro, Franco

2011-11-01

In the present study, we developed a rapid and efficient fluorescence in situ hybridization assay (FISH) in non-embedded tissues of the model plant Catharanthus roseus for co-localizing phytoplasmas and endophytic bacteria, opening new perspectives for studying the interaction between these microorganisms. Copyright © 2011 Elsevier B.V. All rights reserved.

Innovation in utilization of fish tanks for fish culture among fish ...

African Journals Online (AJOL)

This study investigated innovation in utilization of fish tanks for fish culture among fish farmers in Obio/Akpor Local Government Area of Rivers State, Nigeria. Data for this study was obtained through the administration of questionnaire and scheduled interview to 120 sampled fish farmers randomly selected from the study ...

Evaluation of several techniques to modify denatured muscle tissue to obtain a scaffold for peripheral nerve regeneration

NARCIS (Netherlands)

Meek, MF; den Dunnen, WFA; Schakenraad, JM; Robinson, PH

The aim of this study was to (1) evaluate the effect of several preparation techniques of denatured muscle tissue to obtain an open three-dimensional structure, and (2) test if this scaffold is suitable for peripheral nerve regeneration. Four samples (A-D) of muscle tissue specimens were evaluated

Fish health and fish quality

DEFF Research Database (Denmark)

Ingerslev, Hans-Christian

Aquaculture is an expanding worldwide industry producing an increasing amount of fish every year. The quality of the fish meat is dependent upon many biological and non-biological factors. Infectious diseases are known to cause bleedings and damage of the muscle tissue that may lead to scarring...... are poorly described in fish. The present work in this thesis focused on: 1) examination of potential changes in the quality regarding texture of the muscle tissue in rainbow trout (Oncorhynchus mykiss) after previous infection with the bacterial pathogens Yersinia ruckeri and Vibrio anguillarum; 2...... of these studies showed that previous infections by Yersinia ruckeri and Vibrio anguillarum gave rise to subsequent changes regarding textural quality parameters in fresh fish meat, while no differences were seen for cold-smoked meat from the same fish. The texture in previous infected fish was less flaky and less...

Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line, FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization

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Isayama Teruto

2010-11-01

Full Text Available Abstract Background Pleomorphic malignant fibrous histiocytoma (MFH is one of the most frequent malignant soft tissue tumors in adults. Despite the considerable amount of research on MFH cell lines, their characterization at a molecular cytogenetic level has not been extensively analyzed. Methods and results We established a new permanent human cell line, FU-MFH-2, from a metastatic pleomorphic MFH of a 72-year-old Japanese man, and applied multicolor fluorescence in situ hybridization (M-FISH, Urovysion™ FISH, and comparative genomic hybridization (CGH for the characterization of chromosomal aberrations. FU-MFH-2 cells were spindle or polygonal in shape with oval nuclei, and were successfully maintained in vitro for over 80 passages. The histological features of heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original tumor. Cytogenetic and M-FISH analyses displayed a hypotriploid karyotype with numerous structural aberrations. Urovysion™ FISH revealed a homozygous deletion of the p16INK4A locus on chromosome band 9p21. CGH analysis showed a high-level amplification of 9q31-q34, gains of 1p12-p34.3, 2p21, 2q11.2-q21, 3p, 4p, 6q22-qter, 8p11.2, 8q11.2-q21.1, 9q21-qter, 11q13, 12q24, 15q21-qter, 16p13, 17, 20, and X, and losses of 1q43-qter, 4q32-qter, 5q14-q23, 7q32-qter, 8p21-pter, 8q23, 9p21-pter, 10p11.2-p13, and 10q11.2-q22. Conclusion The FU-MFH-2 cell line will be a particularly useful model for studying molecular pathogenesis of human pleomorphic MFH.

Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

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Zonggao Shi

2012-01-01

Full Text Available The noncoding RNA designated as microRNA (miRNA is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH procedures to detect miR-146a with (a different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA incorporated DNA oligonucleotides; (b different reporters for the probes: biotin versus digoxigenin (DIG; (c different visualization: traditional versus tyramide signal amplification (TSA system; (d different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value.

Behavior of variable V3 region from 16S rDNA of lactic acid bacteria in denaturing gradient gel electrophoresis.

Science.gov (United States)

Ercolini, D; Moschetti, G; Blaiotta, G; Coppola, S

2001-03-01

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects.

Molecular cytogenetic of the Amoy croaker, Argyrosomus amoyensis (Teleostei, Sciaenidae)

Science.gov (United States)

Liao, Mengxiang; Zheng, Jiao; Wang, Zhiyong; Wang, Yilei; Zhang, Jing; Cai, Mingyi

2017-08-01

The family Sciaenidae is remarkable for its species richness and economic importance. However, the cytogenetic data available in this fish group are still limited, especially those obtained using fluorescence in situ hybridization (FISH). In the present study, the chromosome characteristics of a sciaenid species, Argyrosomus amoyensis, were examined with several cytogenetic methods, including dual-FISH with 18S and 5S rDNA probes, and a self-genomic in situ hybridization procedure (Self-GISH). The karyotype of A. amoyensis comprised 2n=48 acrocentric chromosomes. A single pair of nucleolar organizer regions (NORs) was located at the proximal position of chromosome 1, which was positive for silver nitrate impregnation (AgNO3) staining and denaturation-propidium iodide (DPI) staining but negative for Giemsa staining and 4',6-diamidino-2-phenylindole (DAPI) staining, and was confirmed by FISH with 18S rDNA probes. The 5S rDNA sites were located at the centromeric region of chromosome 3. Telomeric FISH signals were detected at all chromosome ends with different intensities, but internal telomeric sequences (ITSs) were not found. Self-GISH resulted in strong signals distributed at the centromeric regions of all chromosomes. C-banding revealed not only centromeric heterochromatin, but also heterochromatin that located on NORs, in interstitial and distal telomeric regions of specific chromosomes. These results suggest that the karyotype of Amoy croaker was relatively conserved and primitive. By comparison with the reported cytogenetic data of other sciaenids, it can be deduced that although the karyotypic macrostructure and chromosomal localization of 18S rDNA are conserved, the distribution of 5S rDNA varies dynamically among sciaenid species. Thus, the 5S rDNA sites may have different evolutionary dynamics in relation to other chromosomal regions, and have the potential to be effective cytotaxonomic markers in Sciaenidae.

[Influences of ration level and initial body weight on growth and size hierarchy of hybrid tilapia, Oreochromis mossambicus x O. niloticus].

Science.gov (United States)

Wang, Yan

2003-02-01

72 hybrid tilapia were distributed into twelve tanks, each stocked in six fish (A, B, C, D, E, F) with different body size. The initial body weight was 62.69 +/- 1.46 g for fish A, 56.48 +/- 1.30 g for B, 50.75 +/- 1.19 g for C, 35.56 +/- 1.18 g for D, 31.05 +/- 0.88 g for E, and 27.35 +/- 0.95 g for F (mean +/- SE). The fish were reared under four ration levels (deprived of feed, fed at 1.5% body weight per day (BW.d-1), fed at 3.0% BW.d-1, or fed excess) throughout four weeks, respectively. The specific growth rate (SGR) and feed efficiency (FE) of the fish increased with increasing ration level up to 3.0% BW.d-1, and then, SGR maintained at a constant level while FE dropped with continuing increase of ration. The variance coefficient of SGR and final body weight was relatively high in the fish that fed at 1.5% BW.d-1. For fish A, SGR was not significantly different, between 1.5% BW.d-1, 3.0% BW.d-1 and fed excess, while in fish F, relatively high variations were found among different ration treatments. This experiment revealed that the growth and size hierarchy of hybrid tilapia were related to their ration level and initial body size. Under ration levels more than 3.0% BW.d-1, SGR was usually high, and size hierarchy was relatively weak.

Fish oil quality of by-product (fish skin from swangi fish

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La Ode Huli

2015-01-01

Full Text Available The skin of swangi fish is a potential fish skin to be produced for fish oil. The objectives of this research were aimed to determine the yield and the best quality of fish oil and also to compare fatty acid profile of the fish according to different extraction methods. Fish oil extractions were used by wet rendering method with extraction temperatures of 60, 70, 80, 90, 100°C for 20, 30, and 40 minutes. Fish oil quality was determined by the chemical oil characteristics i.e. PV, FFA, AV, anisidin, and TOTOX. Fatty acid profile was analyzed using gas chromatography (Shimadzu. The results of the study showed that the highest fish oil yield in each treatment was obtained extraction temperature of 60°C for 30 minutes with percentage of 0.33, (70°C for 30 minutes 0.46, (80°C for 30 minutes 1.23, (90°C for 20 minutes 1.14 and (100°C for 20 minutes 0.84. These values were lower compare to Bligh & Dyer and Soxhlet methods. Then, the best fish oil quality was resulted on temperature extraction of 60°C for 30 minutes with PV, FFA, anisidin, AV, and TOTOX were 9.17 meq/kg, 6.92%, 13,77 mg KOH/g, 0.86 meq/kg and 19.19 meq/kg, respectively. FUFA fatty acid compositions of swangi skin fish oil especially EPA and DHA in wet rendering method were gained 0.73% and 2.53%, respectively. These results were lower than Bligh & Dyer method which was consisted of 3.66% (EPA, and 13.29% (DHA and also Soxhlet extraction method with value of EPA was 2.78% and DHA was 9.62%.Keywords: EPA, extraction temperature, DHA, fish oil quality, fish skin

Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method.

Science.gov (United States)

Gouveia, Juceli Gonzalez; Wolf, Ivan Rodrigo; de Moraes-Manécolo, Vivian Patrícia Oliveira; Bardella, Vanessa Belline; Ferracin, Lara Munique; Giuliano-Caetano, Lucia; da Rosa, Renata; Dias, Ana Lúcia

2016-12-01

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.

Kalispel Non-Native Fish Suppression Project 2007 Annual Report.

Energy Technology Data Exchange (ETDEWEB)

Wingert, Michele; Andersen, Todd [Kalispel Natural Resource Department

2008-11-18

Non-native salmonids are impacting native salmonid populations throughout the Pend Oreille Subbasin. Competition, hybridization, and predation by non-native fish have been identified as primary factors in the decline of some native bull trout (Salvelinus confluentus) and westslope cutthroat trout (Oncorhynchus clarki lewisi) populations. In 2007, the Kalispel Natural Resource Department (KNRD) initiated the Kalispel Nonnative Fish Suppression Project. The goal of this project is to implement actions to suppress or eradicate non-native fish in areas where native populations are declining or have been extirpated. These projects have previously been identified as critical to recovering native bull trout and westslope cutthroat trout (WCT). Lower Graham Creek was invaded by non-native rainbow (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis) after a small dam failed in 1991. By 2003, no genetically pure WCT remained in the lower 700 m of Graham Creek. Further invasion upstream is currently precluded by a relatively short section of steep, cascade-pool stepped channel section that will likely be breached in the near future. In 2008, a fish management structure (barrier) was constructed at the mouth of Graham Creek to preclude further invasion of non-native fish into Graham Creek. The construction of the barrier was preceded by intensive electrofishing in the lower 700 m to remove and relocate all captured fish. Westslope cutthroat trout have recently been extirpated in Cee Cee Ah Creek due to displacement by brook trout. We propose treating Cee Cee Ah Creek with a piscicide to eradicate brook trout. Once eradication is complete, cutthroat trout will be translocated from nearby watersheds. In 2004, the Washington Department of Fish and Wildlife (WDFW) proposed an antimycin treatment within the subbasin; the project encountered significant public opposition and was eventually abandoned. However, over the course of planning this 2004 project, little public

An intergeneric hybrid of a native minnow, the golden shiner, and an exotic minnow, the rudd

Science.gov (United States)

Burkhead, N.M.; Williams, J.D.

1991-01-01

The hybrid golden shiner Notemigonus crysoleucas × rudd Scardinius erythrophthalmus is the first known nonsalmonid, intergeneric hybrid of an exotic species and a North American native species. The cross is also the first valid record of a viable hybrid involving the native golden shiner. Meristic and mensural characters of 30 artificially produced hybrids of male golden shiners and female rudds were analyzed. Forty-seven percent of the meristic traits exhibited character states intermediate between those of parents. Twenty-seven percent of the meristic characters were supernumerary, suggesting developmental instability of the hybrid genome. Mensural hybrid characters were significantly skewed to the golden shiner phenotype. The skewed mensural inheritance and other skewed patterns of morphological inheritance also suggest problems in canalization of the hybrid phenome or atypical patterns of dominance. All hybrids were identifiable by intermediate squamation of the cultrate abdomen: the keel was mostly scaled but exhibited a small fleshy ridge posteriorly. This minnow hybrid allows general inferences to be made about the phylogenetic affinity of the golden shiner to other cultrate cyprinids of Eurasia. The hybrid cross has important management and conservation implications for fishes in North America. The hybrid is an example of how an exotic species may negatively affect a native species.

Implementation of the Fluorescent in Situ Hybridization technique in the Faculty of Medicine, UdelaR

Directory of Open Access Journals (Sweden)

Andrea Cairus

2017-11-01

Full Text Available The Cytogenetic Laboratory of the Faculty of Medicine processes, on average, 300 annual samples of public and private healthcare centers by conventional cytogenetics. It is essential to implement new techniques to improve the quality of the service offered. The purpose of this work was to implement the Fluorescent in situ Hybridization technique (FISH. An observational, cross-sectional, analytical study was performed. Peripheral blood samples from patients with sex chromosomopathies diagnosed by conventional cytogenetics were analyzed. Fluorescent in situ hybridization technique was applied, comparing results with FISH and with conventional cytogenetics. The percentage of mosaicism detected by conventional cytogenetics and Fluorescent in situ Hybridization was studied: 24 samples were analyzed; 19 presented numerical alterations, 3 structural and 2 both. Numerical alterations were Turner syndrome, Klinefelter syndrome, XXX syndrome and XYY syndrome. Concordance in diagnoses was found for both techniques. For Turner syndrome, 8 of 12 samples corresponded to mosaicism, and there were no significant differences between conventional cytogenetics and the technique studied (p0.05. Klinefelter syndrome and XYY were both presented in a non-mosaic karyotype. For XXX syndrome, a normal line (46, XX was observed in three of the samples, in a percentage close to the cut off. From this research, it will be possible to implement Fluorescent in situ Hybridization in this service, to extend it to other pathologies and to enable the training of human resources; consolidating this laboratory as a national academic reference center.

Interfacial transduction of nucleic acid hybridization using immobilized quantum dots as donors in fluorescence resonance energy transfer.

Science.gov (United States)

Algar, W Russ; Krull, Ulrich J

2009-01-06

Fluorescence resonance energy transfer (FRET) using immobilized quantum dots (QDs) as energy donors was explored as a transduction method for the detection of nucleic acid hybridization at an interface. This research was motivated by the success of the QD-FRET-based transduction of nucleic acid hybridization in solution-phase assays. This new work represents a fundamental step toward the assembly of a biosensor, where immobilization of the selective chemistry on a surface is desired. After immobilizing QD-probe oligonucleotide conjugates on optical fibers, a demonstration of the retention of selectivity was achieved by the introduction of acceptor (Cy3)-labeled single-stranded target oligonucleotides. Hybridization generated the proximity required for FRET, and the resulting fluorescence spectra provided an analytical signal proportional to the amount of target. This research provides an important framework for the future development of nucleic acid biosensors based on QDs and FRET. The most important findings of this work are that (1) a QD-FRET solid-phase hybridization assay is viable and (2) a passivating layer of denatured bovine serum albumin alleviates nonspecific adsorption, ultimately resulting in (3) the potential for a reusable assay format and mismatch discrimination. In this, the first incarnation of a solid-phase QD-FRET hybridization assay, the limit of detection was found to be 5 nM, and the dynamic range was almost 2 orders of magnitude. Selective discrimination of the target was shown using a three-base-pairs mismatch from a fully complementary sequence. Despite a gradual loss of signal, reuse of the optical fibers over multiple cycles of hybridization and dehybridization was possible. Directions for further improvement of the analytical performance by optimizing the design of the QD-probe oligonucleotide interface are identified.

Gene Expression Profiling in Fish Toxicology: A Review.

Science.gov (United States)

Kumar, Girish; Denslow, Nancy D

In this review, we present an overview of transcriptomic responses to chemical exposures in a variety of fish species. We have discussed the use of several molecular approaches such as northern blotting, differential display reverse transcription-polymerase chain reaction (DDRT-PCR), suppression subtractive hybridization (SSH), real time quantitative PCR (RT-qPCR), microarrays, and next-generation sequencing (NGS) for measuring gene expression. These techniques have been mainly used to measure the toxic effects of single compounds or simple mixtures in laboratory conditions. In addition, only few studies have been conducted to examine the biological significance of differentially expressed gene sets following chemical exposure. Therefore, future studies should focus more under field conditions using a multidisciplinary approach (genomics, proteomics and metabolomics) to understand the synergetic effects of multiple environmental stressors and to determine the functional significance of differentially expressed genes. Nevertheless, recent developments in NGS technologies and decreasing costs of sequencing holds the promise to uncover the complexity of anthropogenic impacts and biological effects in wild fish populations.

Fishing down the largest coral reef fish species.

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Fenner, Douglas

2014-07-15

Studies on remote, uninhabited, near-pristine reefs have revealed surprisingly large populations of large reef fish. Locations such as the northwestern Hawaiian Islands, northern Marianas Islands, Line Islands, U.S. remote Pacific Islands, Cocos-Keeling Atoll and Chagos archipelago have much higher reef fish biomass than islands and reefs near people. Much of the high biomass of most remote reef fish communities lies in the largest species, such as sharks, bumphead parrots, giant trevally, and humphead wrasse. Some, such as sharks and giant trevally, are apex predators, but others such as bumphead parrots and humphead wrasse, are not. At many locations, decreases in large reef fish species have been attributed to fishing. Fishing is well known to remove the largest fish first, and a quantitative measure of vulnerability to fishing indicates that large reef fish species are much more vulnerable to fishing than small fish. The removal of large reef fish by fishing parallels the extinction of terrestrial megafauna by early humans. However large reef fish have great value for various ecological roles and for reef tourism. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biological function evaluation and effects of laser micro-pore burn-denatured acellular dermal matrix.

Science.gov (United States)

Zhang, Youlai; Zeng, Yuanlin; Xin, Guohua; Zou, Lijin; Ding, Yuewei; Duyin, Jiang

2018-03-01

In the field of burns repairs, many problems exist in the shortage of donor skin, the expense of allograft or xenograft skin, temporary substitution and unsatisfactory extremity function after wound healing. Previous studies showed that burn-denatured skin could return to normal dermis formation and function. This study investigates the application of laser micro-pore burn-denatured acellular dermis matrix (DADM) from an escharotomy in the repair of burn wounds and evaluates the biological properties and wound repair effects of DADM in implantation experiments in Kunming mice. Specific-pathogen-free (SPF) Kunming mice were used in this study. A deep II° burn wound was created on the dorsum of the mice by an electric heated water bath. The full-thickness wound tissue was harvested. The necrotic tissue and subcutaneous tissue were removed. The denatured dermis was preserved and treated with 0.25% trypsin, 0.5% Triton X-100. The DADM was drilled by laser micro-pore. The biological properties and grafting effects of laser micro-pore burn-DADM were evaluated by morphology, cytokine expression levels and subcutaneous implantation experiments in Kunming mice. We found statistical significance (Ppore burn-DADM (experimental group) compared to the control group (no laser micro-pore burn-DADM). Cytokine expression level was different in the dermal matrixes harvested at various time points after burn (24h, 48h, 72h and infected wound group). Comparing the dermal matrix from 24h burn tissue to infected wound tissue, the expression level of IL-6, MMP-24, VE-cadherin and VEGF were decreased. We found no inflammatory cells infiltration in the dermal matrix were observed in both experimental and control groups (24h burn group), while the obviously vascular infiltration and fiber fusion were observed in the experimental group after subcutaneous implantation experiments. There was better bio-performance, low immunogenicity and better dermal incorporation after treated by laser

RNA FISH for detecting expanded repeats in human diseases.

Science.gov (United States)

Urbanek, Martyna O; Krzyzosiak, Wlodzimierz J

2016-04-01

RNA fluorescence in situ hybridization (FISH) is a widely used technique for detecting transcripts in fixed cells and tissues. Many variants of RNA FISH have been proposed to increase signal strength, resolution and target specificity. The current variants of this technique facilitate the detection of the subcellular localization of transcripts at a single molecule level. Among the applications of RNA FISH are studies on nuclear RNA foci in diseases resulting from the expansion of tri-, tetra-, penta- and hexanucleotide repeats present in different single genes. The partial or complete retention of mutant transcripts forming RNA aggregates within the nucleoplasm has been shown in multiple cellular disease models and in the tissues of patients affected with these atypical mutations. Relevant diseases include, among others, myotonic dystrophy type 1 (DM1) with CUG repeats, Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3) with CAG repeats, fragile X-associated tremor/ataxia syndrome (FXTAS) with CGG repeats, myotonic dystrophy type 2 (DM2) with CCUG repeats, amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) with GGGGCC repeats and spinocerebellar ataxia type 32 (SCA32) with GGCCUG. In this article, we summarize the results obtained with FISH to examine RNA nuclear inclusions. We provide a detailed protocol for detecting RNAs containing expanded CAG and CUG repeats in different cellular models, including fibroblasts, lymphoblasts, induced pluripotent stem cells and murine and human neuronal progenitors. We also present the results of the first single-molecule FISH application in a cellular model of polyglutamine disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of genome in tetraploid StY species of Elymus (Triticeae: Poaceae) using sequential FISH and GISH.

Science.gov (United States)

Liu, Ruijuan; Wang, Richard R-C; Yu, Feng; Lu, Xingwang; Dou, Quanwen

2017-08-01

Genomes of ten species of Elymus, either presumed or known as tetraploid StY, were characterized using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH). These tetraploid species could be grouped into three categories. Type I included StY genome reported species-Roegneria pendulina, R. nutans, R. glaberrima, R. ciliaris, and Elymus nevskii, and StY genome presumed species-R. sinica, R. breviglumis, and R. dura, whose genome could be separated into two sets based on different GISH intensities. Type I genome constitution was deemed as putative StY. The St genome were mainly characterized with intense hybridization with pAs1, fewer AAG sites, and linked distribution of 5S rDNA and 18S-26S rDNA, while the Y genome with less intense hybridization with pAs1, more varied AAG sites, and isolated distribution of 5S rDNA and 18S-26S rDNA. Nevertheless, further genomic variations were detected among the different StY species. Type II included E. alashanicus, whose genome could be easily separated based on GISH pattern. FISH and GISH patterns suggested that E. alashanicus comprised a modified St genome and an unknown genome. Type III included E. longearistatus, whose genome could not be separated by GISH and was designated as St l Y l . Notably, a close relationship between S l and Y l genomes was observed.

A Proposed Mechanism for the Thermal Denaturation of a Recombinant Bacillus Halmapalus Alpha-amylase - the Effect of Calcium Ions

Science.gov (United States)

Nielsen, Anders D.; Pusey, Marc L.; Fuglsang, Claus C.; Westh, Peter

2003-01-01

The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where previous crystallographic studies have identified the existence of 3 calcium binding sites in the structure. Denaturation of BHA is irreversible with a Tm of approximately 89 C, and DSC thermograms can be described using a one-step irreversible model. A 5 C increase in T(sub m) in the presence of 10 fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40 fold excess calcium chelator (EDTA or EGTA) results in a large destabilization of BHA corresponding to about 40 C lower T(sub m), as determined by both CD and DSC. Ten fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which possibly originate from different populations of BHA:calcium complexes. The observations in the present study have, in combination with structural information of homologous alpha-amylases, provided the basis for the proposal of a simple denaturation mechanism of BHA. The proposed mechanism describes the irreversible thermal denaturation of different BHA:calcium complexes and the calcium binding equilibrium involved. Furthermore, the model accounts for a temperature induced reversible structural change associated with calcium binding.

Thermal and chemical denaturation of Bacillus circulans xylanase: A biophysical chemistry laboratory module.

Science.gov (United States)

Raabe, Richard; Gentile, Lisa

2008-11-01

A number of institutions have been, or are in the process of, modifying their biochemistry major to include some emphasis on the quantitative physical chemistry of biomolecules. Sometimes this is done as a replacement for part for the entire physical chemistry requirement, while at other institutions this is incorporated as a component into the traditional two-semester biochemistry series. The latter is the model used for biochemistry and molecular biology majors at the University of Richmond, whose second semester of biochemistry is a course entitled Proteins: Structure, Function, and Biophysics. What is described herein is a protein thermodynamics laboratory module, using the protein Bacillus circulans xylanase, which reinforces many lecture concepts, including: (i) the denatured (D) state ensemble of a protein can be different, depending on how it was populated; (ii) intermediate states may be detected by some spectroscopic techniques but not by others; (iii) the use and assumptions of the van't Hoff approach to calculate ΔH(o) , ΔS(o) , and ΔG(o) (T) for thermal protein unfolding transitions; and (iv) the use and assumptions of an approach that allows determination of the Gibb's free energy of a protein unfolding transition based on the linear dependence of ΔG(o) on the concentration of denaturant used. This module also requires students to design their own experimental protocols and spend time in the primary literature, both important parts of an upper division lab. Copyright © 2008 International Union of Biochemistry and Molecular Biology, Inc.

Detection of ROS1 Gene Rearrangement in Lung Adenocarcinoma: Comparison of IHC, FISH and Real-Time RT-PCR

OpenAIRE

Shan, Ling; Lian, Fang; Guo, Lei; Qiu, Tian; Ling, Yun; Ying, Jianming; Lin, Dongmei

2015-01-01

Aims To compare fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative real-time reverse transcription-PCR (qRT-PCR) assays for detection of ROS1 fusion in a large number of ROS1-positive lung adenocatcinoma (ADC) patients. Methods Using IHC analysis, sixty lung ADCs including 16 cases with ROS1 protein expression and 44 cases without ROS1 expression were selected for this study. The ROS1 fusion status was examined by FISH and qRT-PCR assay. Results Among 60 ca...

Denaturation of collagen structures and their transformation under the physical and chemical effects

Science.gov (United States)

Ivankin, A.; Boldirev, V.; Fadeev, G.; Baburina, M.; Kulikovskii, A.; Vostrikova, N.

2017-11-01

The process of denaturation of collagen structures under the influence of physical and chemical factors play an important role in the manufacture of food technology and the production of drugs for medicine and cosmetology. The paper discussed the problem of the combined effects of heat treatment, mechanical dispersion and ultrasonic action on the structural changes of the animal collagen in the presence of weak protonated organic acids. Algorithm combined effects of physical and chemical factors as a result of the formation of the technological properties of products containing collagen has been shown.

Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

DEFF Research Database (Denmark)

Christiansen, L; Ged, C; Hombrados, I

1999-01-01

to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

Laboratory Exercise for Studying the Morphology of Heat-Denatured and Amyloid Aggregates of Lysozyme by Atomic Force Microscopy

Science.gov (United States)

Gokalp, Sumeyra; Horton, William; Jónsdóttir-Lewis, Elfa B.; Foster, Michelle; Török, Marianna

2018-01-01

To facilitate learning advanced instrumental techniques, essential tools for visualizing biomaterials, a simple and versatile laboratory exercise demonstrating the use of Atomic Force Microscopy (AFM) in biomedical applications was developed. In this experiment, the morphology of heat-denatured and amyloid-type aggregates formed from a low-cost…

Evaluation of HER2 gene amplification in invasive breast cancer using a dual-color chromogenic in situ hybridization (dual CISH).

Science.gov (United States)

Kato, Nobuaki; Itoh, Hitoshi; Serizawa, Akihiko; Hatanaka, Yutaka; Umemura, Shinobu; Osamura, R Yoshiyuki

2010-07-01

Fluorescence in situ hybridization (FISH) assay is considered the 'gold standard' for evaluation of HER2/neu (HER2) gene status, however, it is difficult to recognize morphologic features of tumors using fluorescence microscopy. Thus, chromogenic in situ hybridization (CISH) has been proposed as an alternative method to evaluate HER2 gene amplification. Here, we examined the dual color CISH (dual CISH) method which provides information regarding the copy number of the HER2 gene and chromosome 17 centromere from a single slide. We examined 40 cases of invasive ductal carcinomas of the breast that were resected surgically. HER2 gene status was assessed with FISH (Abbott) and dual CISH (Dako). HER2 gene amplification status was classified according to the guidelines of the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP). Comparison of the cut-off values for HER2/chromosome 17 centromere copy number ratio obtained by dual CISH and FISH showed that there was almost perfect agreement between two methods (Kappa coefficient 0.96). The results of the two commercial products were almost consistent for evaluation of HER2 gene counts on the sections. The current study proved that dual CISH is comparable with FISH for evaluating HER2 gene status.

Lysis solution composition and non-linear dose-response to ionizing radiation in the non-denaturing DNA filter elution technique

International Nuclear Information System (INIS)

Radford, I.R.

1990-01-01

The suggestion by Okayasu and Iliakis (1989) that the non-linear dose-response curve, obtained with the non-denaturing filter elution technique for mammalian cells exposed to low-LET radiation, is the result of a technical artefact, was not confirmed. (author)

Imaging of Chromosome Dynamics in Mouse Testis Tissue by Immuno-FISH.

Science.gov (United States)

Scherthan, Harry

2017-01-01

The mouse (Mus musculus) represents the central mammalian genetic model system for biomedical and developmental research. Mutant mouse models have provided important insights into chromosome dynamics during the complex meiotic differentiation program that compensates for the genome doubling at fertilization. Homologous chromosomes (homologues) undergo dynamic pairing and recombine during first meiotic prophase before they become partitioned into four haploid sets by two consecutive meiotic divisions that lack an intervening S-phase. Fluorescence in situ hybridization (FISH) has been instrumental in the visualization and imaging of the dynamic reshaping of chromosome territories and mobility during prophase I, in which meiotic telomeres were found to act as pacemakers for the chromosome pairing dance. FISH combined with immunofluorescence (IF) co-staining of nuclear proteins has been instrumental for the visualization and imaging of mammalian meiotic chromosome behavior. This chapter describes FISH and IF methods for the analysis of chromosome dynamics in nuclei of paraffin-embedded mouse testes. The techniques have proven useful for fresh and archived paraffin testis material of several mammalian species.

Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

Science.gov (United States)

Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

2015-02-01

In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

Accuracy Assessment of Interphase Fluorescence In-Situ Hybridization on Uncultured Amniotic Fluid Cells

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Hamideh Karimi

2007-01-01

Full Text Available Background: Parental anxiety while waiting for the results of amniocentesis has been investigatedby many authors. It seems that the implementation of faster techniques such as fluorescence in-situhybridization (FISH will have some benefits in reducing this anxiety. Besides the patients' attitudesto choosing this method, gynecologists who are the persons responsible for treatment, must feelcomfortable about prescribing FISH techniques.Materials and Methods: This study, using a simple methodology, was undertaken to evaluate theresults of FISH tests on the amniotic fluid from 40 pregnant women undergoing cesarean surgery.Two sets of probes including X/Y cocktail and 13, 21 and 18 were applied on different slides.Results: The results of FISH tests were compared with the reports of the pediatrician about thehealth condition of the newborn. Complete conformity between the two sets of findings, haveconvinced our gynecologists of the benefit of prescribing this method to reduce the anxiety ofpatients at risk of having abnormal offspring due to chromosomal anuploidies.Conclusion: As has been documented by many authors, conventional chromosome analysis hasgreat advantages over fluorescence in situ hybridization of interphase amniocytes, but reducing theanxiety of parents is a good reason for employing the FISH technique.

X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids.

Directory of Open Access Journals (Sweden)

Tanmoy Bhattacharyya

2014-02-01

Full Text Available Hybrid sterility (HS belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2(Mmm allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes.

X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids.

Science.gov (United States)

Bhattacharyya, Tanmoy; Reifova, Radka; Gregorova, Sona; Simecek, Petr; Gergelits, Vaclav; Mistrik, Martin; Martincova, Iva; Pialek, Jaroslav; Forejt, Jiri

2014-02-01

Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2(Mmm) allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes.

X Chromosome Control of Meiotic Chromosome Synapsis in Mouse Inter-Subspecific Hybrids

Science.gov (United States)

Bhattacharyya, Tanmoy; Reifova, Radka; Gregorova, Sona; Simecek, Petr; Gergelits, Vaclav; Mistrik, Martin; Martincova, Iva; Pialek, Jaroslav; Forejt, Jiri

2014-01-01

Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2Mmm allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes. PMID:24516397

FISH DETECTION OF CAMPYLOBACTER AND ARCOBACTER ADHERED TO STAINLESS STEEL COUPONS

Directory of Open Access Journals (Sweden)

Lucie Šilhová

2015-02-01

Full Text Available This study focuses on detecting biofilm and planktonic bacterial cells of the genera Arcobacter and Campylobacter. This study is, to our knowledge, the first study deals with application of FISH procedure to detect biofilm formation on stainless steel coupons of Arcobacter-isolates from real-world environments. These bacteria can cause a lot of diseases. Especially, in the last decade, arcobacters have been increasingly isolated from feces of clinically healthy and ill animals, foods of animal origin and various types of water. Fluorescence in situ hybridization was used to detect biofilm and planktonic cells of selected microorganisms. This method was optimized and subsequently applied to biofilm samples prepared on stainless steel coupons. The study results indicate that fluorescence in situ hybridization is suitable for detecting biofilm and planktonic cells of the studied bacteria.

Dual-colour CISH is a reliable alternative to FISH for assessment of topoisomerase 2-alpha amplification in breast carcinomas.

Science.gov (United States)

García-Caballero, Tomás; Prieto, Olga; Vázquez-Boquete, Angel; Gude, Francisco; Viaño, Patricia; Otero, María; Curiel, Teresa; Fernández-Rodríguez, Beatriz; Parrado, Concepción; Fraga, Máximo; Antúnez, José R

2014-01-01

Anthracyclines are among the most powerful antineoplastic drugs available for breast cancer treatment. Although HER2 amplification has been postulated to predict anthracycline benefit, numerous reports have demonstrated that HER2/TOP2A co-amplification is the clinically useful predictive marker of response to anthracyclines. The standard technique to evaluate gene status for target therapy selection is fluorescence in situ hybridization (FISH), but this technique has some disadvantages. Dual-colour chromogenic in situ hybridization (CISH) is an extension of the FISH protocol that allows bright-field microscopy and thus represents a user-friendly alternative to FISH. In order to evaluate whether dual-colour CISH is a reliable alternative to FISH in determining TOP2A gene amplification and to determine the frequency with which TOP2A and HER2 were co-amplified, we analysed 100 invasive breast cancer specimens (70 consecutive and 30 HER2-amplified samples) using tissue microarrays. Thus, a 99 % agreement was found between TOP2A status determined by dual-colour CISH and FISH, as well as a high degree of correlation in TOP2A ratios using both techniques. TOP2A gene amplification was present in 8.6 % of the 70 consecutive samples studied, all of which were HER2-amplified. Co-amplification of TOP2A was observed in 46.5 % of the additional 30 HER2-amplified samples (no TOP2A amplification was seen in non-amplified HER2 samples). We conclude that dual-colour CISH represents an excellent alternative to FISH for determination of TOP2A gene status in invasive breast cancer. Our results showing TOP2A amplification only in HER2-amplified cases also add to the evidence that TOP2A determination should be restricted to those cases.

Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH

NARCIS (Netherlands)

Ruiz, Geraldine Avila; Opazo-Navarrete, Mauricio; Meurs, Marlon; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Stieger, Markus; Janssen, Anja E.M.

2016-01-01

The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60,

Fish welfare: Fish capacity to experience pain

Directory of Open Access Journals (Sweden)

Vučinić Marijana

2009-01-01

Full Text Available Teleost fish possess similar nociceptive processing systems to those found in terrestrial vertebrates. It means that they react to potential painful stimuli in a similar manner as mammals and birds. However, the welfare of fish has been the focus of less research than that of higher vertebrates. Humans may affect the welfare of fish through fisheries, aquaculture and a number of other activities. There is scientific evidence to support the assumption that fish have the capacity to experience pain because they possess functional nociceptors, endogenous opioids and opioid receptors, brain structures involved in pain processing and pathways leading from nociceptors to higher brain structures. Also, it is well documented that some anaesthetics and analgesics may reduce nociceptive responses in fish. Behavioural indicators in fish such as lip-rubbing and rocking behaviours are the best proof that fish react to potential painful stimuli. This paper is an overview of some scientific evidence on fish capacity to experience pain.