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Sample records for cellular mutational assays

  1. Prospects for cellular mutational assays in human populations

    International Nuclear Information System (INIS)

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

  2. Prospects for cellular mutational assays in human populations

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1984-06-29

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

  3. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.

    2005-01-01

    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  4. Cellular effects of LRRK2 mutations

    OpenAIRE

    Cookson, Mark R.

    2012-01-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) are a relatively common cause of inherited Parkinson's disease (PD) but the mechanism(s) by which mutations lead to disease are poorly understood. Here, I will discuss what is known about LRRK2 in cellular models, focusing on specifically on assays that have been used to tease apart the effects of LRRK2 mutations on cellular phenotypes. LRRK2 expression has been suggested to cause loss of neuronal viability, although because it also has a stro...

  5. Cellular toxicity (High-Throughput Cellular Assays for Modeling Toxicity in the Fish Reproductive System)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The purpose of this project is to adapt cellular in vitro assay systems of the rainbow trout pituitary, liver and ovary for high-throughput screening (HTS) of...

  6. 40 CFR 79.68 - Salmonella typhimurium reverse mutation assay.

    Science.gov (United States)

    2010-07-01

    ... recommendations as specified under 40 CFR 79.60, the following specific information shall be reported: (i... consulted. (1) 40 CFR 798.5265, The Salmonella typhimurium reverse mutation asay. (2) Ames, B.N., McCann, J... mutation assay. 79.68 Section 79.68 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...

  7. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    International Nuclear Information System (INIS)

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

  8. A novel fluorescence-based cellular permeability assay.

    Science.gov (United States)

    Chandra, Ankur; Barillas, Samuel; Suliman, Ahmed; Angle, Niren

    2007-04-10

    Vascular permeability is a pathologic process in many disease states ranging from metastatic progression of malignancies to ischemia-reperfusion injury. In order to more precisely study tissue, and more specifically cell layer permeability, our goal was to create a fluorescence-based assay which could quantify permeability without radioactivity or electrical impedance measurements. Human aortic endothelial cells were grown in monolayer culture on Costar-Transwell clear polyester membrane 6-well cell culture inserts. After monolayer integrity was confirmed, vascular endothelial growth factor (VEGF(165)) at varying concentrations with a fixed concentration of yellow-green fluorescent 0.04 microm carboxylate-modified FluoSpheres microspheres were placed in the luminal chamber and incubated for 24 h. When stimulated with VEGF(165) at 20, 40, 80, and 100 ng/ml, this assay system was able to detect increases in trans-layer flux of 8.2+/-2.4%, 16.0+/-3.7%, 41.5+/-4.9%, and 58.6+/-10.1% for each concentration, respectively. This represents the first fluorescence-based permeability assay with the sensitivity to detect changes in the permeability of a cell layer to fluid flux independent of protein flux; as well as being simpler and safer than previous radioactive-and impedance-based permeability assays. With the application of this in vitro assay to a variety of pathologic conditions, both the dynamics and physiology relating to cellular permeability can be more fully investigated. PMID:16962665

  9. Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

    OpenAIRE

    Milbury, Coren A.; Qun Zhong; Jesse Lin; Miguel Williams; Jeff Olson; Link, Darren R.; Brian Hutchison

    2014-01-01

    Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were exp...

  10. [KRAS mutation assay on EUS-FNA specimens from pacients with pancreatic mass].

    Science.gov (United States)

    Bunganič, Bohuš; Hálková, Tereza; Benešová, Lucie; Belšánová, Barbora; Laclav, Martin; Hrůzová, Martina; Traboulsi, Eva; Frič, Přemysl; Suchánek, Štěpán; Minárik, Marek; Zavoral, Miroslav

    2016-01-01

    Differential diagnosis of solid pancreatic masses using EUS FNA is in 1015 % of cases still challenging. Promising method, which helps to distinguish between chronic pancreatitis and cancer, is point mutations of the proto-oncogene KRAS test. This method is not established in routine clinical practice yet.Objectives were the determination of the sensitivity of the KRAS assay using various kinds of samples of patients with pancreatic mass and testing the effect of the presence of KRAS mutations on the prognosis of survival. 147 patients underwent EUS-FNA examination of pancreatic mass, accompanied by blood sampling with subsequent separation of plasma for the detection of circulating tumor DNA. Part of biopsy sample was left native in a stabilizing solution and part as cytological smear. Samples (native aspirates, cytological smears, plasma) were examined for the presence of KRAS mutation by heteroduplex analysis, denaturing capillary electrophoresis.Among 147 patients with pancreatic masses, 118 were diagnosed as a cancer, 26 chronic pancreatitis, 3 neuroendocrine tumor. In total 147 native aspirates, 118 cytological smears and 94 plasma samples were examined. The highest sensitivity of KRAS mutation was reached in the group of pancreatic cancer patients using cytology, in which 90 % of KRAS mutation was detected (106/118 of the samples). When using the native cellular aspirates, mutation was detected in 78 % (92/118 samples), and examination of plasma was positive in 27 % (24/90 samples). In four patients with chronic pancreatitis KRAS mutations was detected, although none has been cytologically confirmed as a cancer. Two of these four patients were confirmed in the course of the disease as a cancer, one patient died because of alcoholic delirium and the last one was indicated for surgery recently.Examination of KRAS mutations can be performed in all patients undergoing EUS-FNA, with the cytology being the most reliable type of sample for genetic tests. KRAS

  11. A sentinel protein assay for simultaneously quantifying cellular processes

    Czech Academy of Sciences Publication Activity Database

    Soste, M.; Hrabáková, Rita; Wanka, S.; Melnik, A.; Boersema, P.; Maiolica, A.; Wernas, T.; Tognetti, M.; von Mering, Ch.; Picotti, P.

    2014-01-01

    Roč. 11, č. 10 (2014), s. 1045-1048. ISSN 1548-7091 R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : targeted proteomics * selected reaction monitoring * cellular signaling Subject RIV: CE - Biochemistry Impact factor: 32.072, year: 2014

  12. 40 CFR 798.5265 - The salmonella typhimurium reverse mutation assay.

    Science.gov (United States)

    2010-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following... mutation assay. 798.5265 Section 798.5265 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5265 The salmonella typhimurium reverse mutation assay. (a) Purpose. The Salmonella...

  13. A Rapid, Sensitive Assay to Detect EGFR Mutation in Small Biopsy Specimens from Lung Cancer

    OpenAIRE

    Yatabe, Yasushi; Hida, Toyoaki; Horio, Yoshitsugu; Kosaka, Takayuki; Takahashi, Takashi; Mitsudomi, Tetsuya

    2006-01-01

    It has been demonstrated that lung cancers, specifically a subset of pulmonary adenocarcinomas, with epidermal growth factor receptor (EGFR) mutation are highly sensitive to EGFR-targeted drugs. Therefore, a rapid, sensitive assay for mutation detection using routine pathological specimens is demanded in clinical practice to predict the response. We therefore developed a new assay for detecting EGFR mutation using only a paraffin section of a small biopsy specimen. The method was very sensiti...

  14. The relationship between cellular radiosensitivity and radiation-induced DNA damage measured by the comet assay

    International Nuclear Information System (INIS)

    The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by γ-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assay. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition of the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells. (author)

  15. The glycophorin A assay for somatic cell mutations in humans

    International Nuclear Information System (INIS)

    In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs

  16. Identification of new quinic acid derivatives as histone deacetylase inhibitors by fluorescence-based cellular assay.

    Science.gov (United States)

    Son, Dohyun; Kim, Chung Sub; Lee, Kang Ro; Park, Hyun-Ju

    2016-05-01

    A fluorescence-based cellular assay system was established to identify potential epigenetic modulator ligands. This assay method is to detect the de-repression of an EGFP reporter in cancer cells by the treatment of HDAC (histone deacetylase) or DNMT (DNA methyltransferase) inhibitor. Using this system, we conducted a preliminary screening of in-house natural product library containing extracts and pure compounds, and identified several active compounds. Among them, novel quinic acid derivatives were recognized as excellent HDAC inhibitors by both enzymatic and cell-based HDAC assays. PMID:26996372

  17. Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens

    OpenAIRE

    Bhalli, Javed A.; Shaddock, Joseph G.; Pearce, Mason G.; Dobrovolsky, Vasily N.

    2013-01-01

    Clastogens are potential human carcinogens whose detection by genotoxicity assays is important for safety assessment. Although some endogenous genes are sensitive to the mutagenicity of clastogens, many genes that are used as reporters for in vivo mutation (e.g. transgenes) are not. In this study, we have compared responses in the erythrocyte Pig-a gene mutation assay with responses in a gene mutation assay that is relatively sensitive to clastogens, the lymphocyte Hprt assay, and in the reti...

  18. Studying the genotoxicity of vincristine on human lymphocytes using comet assay, micronucleus assay and TCR gene mutation test in vitro

    International Nuclear Information System (INIS)

    The results of our previous investigation for workers occupationally exposed to vincristine (VCR) indicated that the genetic damage was detectable with comet assay, cytokinesis-block micronucleus (CBMN) assay and housekeeping gene mutation tests. In order to determine the results of above investigation and to inquire further the characteristics of genotoxicity of VCR, the cytogenetic effects of VCR on human lymphocytes were assessed with comet assay, CBMN assay and T-cell receptor (TCR) gene mutation test in vitro. The lymphocytes from two healthy donors were incubated for 24 h at doses of 0.00, 0.01, 0.02, 0.04, and 0.08 μg ml-1 VCR. The results of the present experiment showed that VCR not only could induce DNA damage, increase significantly micronucleus frequencies and the apoptotic cell ratios and decrease the nuclear division index (NDI) with dose-response relationship, but also could produce nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions and nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes. Moreover, VCR could enhance TCR gene mutation frequency (Mf-TCR) of human lymphocytes. There was good correlation between the parameters (mean tail length, mean tail moment, micronucleus frequency, micronucleated frequency and Mf-TCR). The results of present study supported the results of our previous investigation for workers occupationally exposed to VCR, and the genotoxicity of VCR was determined at the different genetic end-points in vitro

  19. A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin.

    Science.gov (United States)

    Li, Li; Du, Yuhong; Chen, Wei; Fu, Haian; Harrison, David G

    2011-09-01

    Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels. PMID:21693765

  20. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.

    2016-04-01

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  1. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    International Nuclear Information System (INIS)

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with 125I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  2. Cellular and deafness mechanisms underlying connexin mutation induced hearing loss – A common hereditary deafness

    Directory of Open Access Journals (Sweden)

    Hong-Bo Zhao

    2015-05-01

    Full Text Available Hearing loss due to mutations in the connexin gene family which encodes gap junctional proteins is a common form of hereditary deafness. In particular, connexin 26 (Cx26, GJB2 mutations are responsible for ~50% of nonsyndromic hearing loss, which is the highest incidence of genetic disease. In the clinic, Cx26 mutations cause various auditory phenotypes ranging from profound congenital deafness at birth to mild, progressive hearing loss in late childhood. Recent experiments demonstrate that congenital deafness mainly results from cochlear developmental disorders rather than hair cell degeneration and endocochlear potential (EP reduction, while late-onset hearing loss results from reduction of active cochlear amplification, even though cochlear hair cells have no connexin expression. Moreover, new experiments further demonstrate that the hypothesized K+-recycling disruption is not a principal deafness mechanism for connexin deficiency induced hearing loss. Additionally, there is no clear relationship between specific changes in connexin (channel functions and the phenotypes of mutation-induced hearing loss. Cx30, Cx29, Cx31, and Cx43 mutations can also cause hearing loss with distinct pathological changes in the cochlea. These new studies provide invaluable information about deafness mechanisms underlying connexin mutation induced hearing loss and also provide important information for developing new protective and therapeutic strategies for this common deafness. However, the detailed cellular mechanisms underlying these pathological changes and pathogeneses of specific-mutation induced hearing loss remain unclear. Finally, little information is available for humans. Further studies to address these deficiencies are urgently required.

  3. An improved assay for antibody dependent cellular cytotoxicity based on time resolved fluorometry.

    Science.gov (United States)

    Patel, A K; Boyd, P N

    1995-07-17

    A new and faster assay for antibody dependent cellular cytotoxicity based on release of europium from target cells is described. This has a number of important advantages over the traditional assays based on release of chromium-51 (51Cr). The new method involves labelling of Wein 133 target cells (B cell non-Hodgkin's lymphoma cells) which express the antigen, CDw52, with the chelate europium diethylenetriaminopentaacetic acid (EuDTPA) according to the method of Blomberg et al. (1986). Labelled cells are sensitised (coated) with the anti-lymphocytic monoclonal antibody, Campath-1H. Human peripheral blood mononuclear cells are added to mediate lysis of EuDTPA labelled Wein 133 cells by ADCC. Release of EuDTPA from lysed cells is determined by mixing supernatants with enhancement solution containing 2-naphthoyl trifluoroacetone, 2-NTA, to form a highly fluorescent chelate which is measured using time resolved fluorometry. Results obtained with the new EuDPTA release assays were comparable to traditional assays based on the release of the radioisotope 51Cr. It is anticipated that this assay will have a widespread application among laboratories performing ADCC assays. The method is non-hazardous and has been used routinely for over 2 years to monitor production and purification of Campath-1H. PMID:7622867

  4. SNaPshot assay for the detection of the most common CFTR mutations in infertile men.

    Directory of Open Access Journals (Sweden)

    Predrag Noveski

    Full Text Available Congenital bilateral absence of vas deferens (CBAVD is the most common CFTR-related disorder (CFTR-RD that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF and CFTR-related disorder (CFTR-RD mutations are found in a high percentage (22.7% of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men.

  5. Mismatch oxidation assay: detection of DNA mutations using a standard UV/Vis microplate reader.

    Science.gov (United States)

    Tabone, Tania; Sallmann, Georgina; Cotton, Richard G H

    2009-01-01

    Simple, low-cost mutation detection assays that are suitable for low-throughput analysis are essential for diagnostic applications where the causative mutation may be different in every family. The mismatch oxidation assay is a simple optical absorbance assay to detect nucleotide substitutions, insertions, and deletions in heteroduplex DNA. The method relies on detecting the oxidative modification products of mismatched thymine and cytosine bases by potassium permanganate as it is reduced to manganese dioxide. This approach, unlike other methods commonly used to detect sequence variants, does not require costly labeled probes or primers, toxic chemicals, or a time-consuming electrophoretic separation step. The oxidation rate, and hence the presence of a sequence variant, is detected by measuring the formation of the potassium permanganate reduction product (hypomanganate diester), which absorbs at the 420-nm visible wavelength, using a standard UV/vis microplate reader. PMID:19768598

  6. Performance of a novel KRAS mutation assay for formalin-fixed paraffin embedded tissues of colorectal cancer

    OpenAIRE

    Sakai, Kazuko; Yoneshige, Azusa; Ito, Akihiko; UEDA Yoji; Kondo, Satoshi; Nobumasa, Hitoshi; Fujita, Yoshihiko; TOGASHI, YOSUKE; Terashima, Masato; Velasco, Marco A.; Tomida, Shuta; Nishio, Kazuto

    2015-01-01

    We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independ...

  7. Generation and precise control of dynamic biochemical gradients for cellular assays

    CERN Document Server

    Saka, Yasushi; Giuraniuc, Claudiu V

    2016-01-01

    Spatial gradients of diffusible signalling molecules play crucial roles in controlling diverse cellular behaviour such as cell differentiation, tissue patterning and chemotaxis. Here we present a microfluidic platform for cellular assays that can generate and control diffusion-based gradients dynamically. A unique design of the device eliminates cross-flow between the source and sink channels, thereby stabilising gradients by passive diffusion. The platform also enables quick and flexible control of chemical concentration that makes highly dynamic gradients in diffusion chambers. Budding yeast cells cultured in a gradient of a chemical inducer expressed a reporter fluorescence protein in a concentration-dependent manner. This microfluidic platform serves as a versatile prototype applicable to a broad range of biomedical investigations.

  8. Platelet hexosaminidase a enzyme assay effectively detects carriers missed by targeted DNA mutation analysis.

    Science.gov (United States)

    Nakagawa, Sachiko; Zhan, Jie; Sun, Wei; Ferreira, Jose Carlos; Keiles, Steven; Hambuch, Tina; Kammesheidt, Anja; Mark, Brian L; Schneider, Adele; Gross, Susan; Schreiber-Agus, Nicole

    2012-01-01

    Biochemical testing of hexosaminidase A (HexA) enzyme activity has been available for decades and has the ability to detect almost all Tay-Sachs disease (TSD) carriers, irrespective of ethnic background. This is increasingly important, as the gene pool of those who identify as Ashkenazi Jewish is diversifying. Here we describe the analysis of a cohort of 4,325 individuals arising from large carrier screening programs and tested by the serum and/or platelet HexA enzyme assays and by targeted DNA mutation analysis. Our results continue to support the platelet assay as a highly effective method for TSD carrier screening, with a low inconclusive rate and the ability to detect possible disease-causing mutation carriers that would have been missed by targeted DNA mutation analysis. Sequence analysis performed on one such platelet assay carrier, who had one non-Ashkenazi Jewish parent, identified the amino acid change Thr259Ala (A775G). Based on crystallographic modeling, this change is predicted to be deleterious, as threonine 259 is positioned proximal to the HexA alpha subunit active site and helps to stabilize key residues therein. Accordingly, if individuals are screened for TSD in broad-based programs by targeted molecular testing alone, they must be made aware that there is a more sensitive and inexpensive test available that can identify additional carriers. Alternatively, the enzyme assays can be offered as a first tier test, especially when screening individuals of mixed or non-Jewish ancestry. PMID:23430931

  9. SNaPshot Assay for the Detection of the Most Common CFTR Mutations in Infertile Men

    OpenAIRE

    Predrag Noveski; Svetlana Madjunkova; Marija Mircevska; Toso Plaseski; Vanja Filipovski; Dijana Plaseska-Karanfilska

    2014-01-01

    Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA an...

  10. A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs

    OpenAIRE

    Alghafri, Rashed; Goodwin, Will; Ralf, Arwin; Kayser, Manfred; Hadi, Sibte

    2015-01-01

    textabstractAbstract A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other applications to improve differentiating unrelated males and allow separating related males. The 13 RM Y-STR markers included in the multiplex are: DYF387S1, DYF399S1, DYF403S1ab, DYF404S1, DYS449, DYS518...

  11. Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

    Science.gov (United States)

    Hagihara, Kenta; Tsukagoshi, Kazuhiko; Nakajima, Chinami; Esaki, Shinsuke; Hashimoto, Masahiko

    2016-01-01

    We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent. PMID:26960620

  12. Determining the functional significance of mismatch repair gene missense variants using biochemical and cellular assays

    DEFF Research Database (Denmark)

    Heinen, Christopher D; Juel Rasmussen, Lene

    2012-01-01

    provided an important experimental tool for studying the functional consequences of VUS. However, beyond this repair assay, a number of other experimental methods have been developed that allow us to test the effect of a VUS on discrete biochemical steps or other aspects of MMR function. Here, we describe......ABSTRACT: With the discovery that the hereditary cancer susceptibility disease Lynch syndrome (LS) is caused by deleterious germline mutations in the DNA mismatch repair (MMR) genes nearly 20 years ago, genetic testing can now be used to diagnose this disorder in patients. A definitive diagnosis of...... LS can direct how clinicians manage the disease as well as prevent future cancers for the patient and their families. A challenge emerges, however, when a germline missense variant is identified in a MMR gene in a suspected LS patient. The significance of a single amino acid change in these large...

  13. Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

    Science.gov (United States)

    Zeyer, Karina A; Reinhardt, Dieter P

    2015-01-01

    Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1. PMID:26281765

  14. Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test

    International Nuclear Information System (INIS)

    To evaluate the genotoxic effects of lead (Pb) exposure, 25 workers in a workplace producing storage battery were monitored for three genetic end-points using micronucleus (MN) assay, comet assay and TCR gene mutation test. Twenty-five controls were matched with workers according to age, gender and smoking. The air Pb concentration in the workplace was 1.26 mg/m3. All subjects were measured for Pb concentration of blood by atom absorption spectrophotometry. The mean Pb concentration of blood in workers (0.32 mg/l) was significantly higher than that in controls (0.02 mg/l). The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in workers were 9.04 ± 1.51 per mille and 7.76 ± 1.23 per mille , respectively, which were significantly higher than those (2.36 ± 0.42 per mille and 1.92 ± 0.31 per mille ) in controls (P -4 and 1.74 ± 0.17 x 10-4, respectively, there was no significant difference between workers and controls (P > 0.05). The results of our study indicated that the genetic damage was detectable in 25 workers occupationally exposed to lead

  15. 75Se-release: A short and long term assay system for cellular cytotoxicity

    International Nuclear Information System (INIS)

    The γ-emitting aminoacid 75Se-selenomethionine (75SeM) was examined as a target cell label in cytotoxic assays. It was efficiently taken up by activated, intensely metabolizing cells of various types but hardly at all by resting or low-metabolizing cells. Culturing activated cells in methionine-deficient medium with 3-5μCi75SeM/ml for 18-22 h usually resulted in an uptake of 3-20 cpm/cell which was 3-200 times that of 51Cr marked cells. 75SeM-labelled cells kept in medium at ambient temperature or at 370C maintained a high radioactivity per cell and a viability above 85% for at least 72 h without significant increase in spontaneous isotope release or loss in sensitivity in subsequent cytotoxic tests. 75Se-labelled material released from target cells was not reutilized by unlabelled lymphoid cells. Provided the cells were carefully washed after labelling and kept in optimal culture conditions, the reasonably low baseline release in the medium control allowed performance of long-term assays of up to 54 h. However, strong cytotoxic reactions could cause over 50% specific 75Se-release within 5 h. With constant amounts of effector cells, identical or even higher specific releases were obtained on 6 x 102 targets as compared to 1 x 104 targets/well. Thus, the 75Se-release assay offers a single monitoring system suitable for short and long term cytotoxic reactions on a microscale, using 1 x 103 or less targets/well. Its sensitivity permits evaluation of strong and weak reactions as well as early and delayed onser cytotoxicity. In addition with a γ-spectrometer the radioactivity of 75Se can easily be distinguished from that of 51Cr. Due to this, and an improved method for 51Cr labelling of cells, a double γ-labelling of cellular proteins is available which provides new possibilities for monitoring cellular interactions in short and long term tests. (orig./MG)

  16. Screening for novel LRRK2 inhibitors using a high-throughput TR-FRET cellular assay for LRRK2 Ser935 phosphorylation.

    Directory of Open Access Journals (Sweden)

    Spencer B Hermanson

    Full Text Available BACKGROUND: Mutations in the leucine-rich repeat kinase-2 (LRRK2 have been linked to Parkinson's disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973. CONCLUSIONS/SIGNIFICANCE: We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756

  17. Comprehensive TP53-denaturing gradient gel electrophoresis mutation detection assay also applicable to archival paraffin-embedded tissue

    NARCIS (Netherlands)

    Hayes, V M; Bleeker, W; Verlind, E; Timmer, T; Karrenbeld, A; Plukker, J T; Marx, M P; Hofstra, R M; Buys, C H

    1999-01-01

    A comprehensive mutation detection assay is described for the entire coding region and all splice site junctions of TP53. The assay is based on denaturing gradient gel electrophoresis, which follows either multiplex polymerase chain reaction (PCR) applied to DNA extracted from fresh or frozen tissue

  18. Serological detection of H-Y antigen in humans with a cellular radioimmunobinding assay and monoclonal antibody

    International Nuclear Information System (INIS)

    A sensitive and reliable serological assay for the detection of H-Y antigen is described which uses monoclonal H-Y antibody and a cellular radioimmunobinding assay on cultured human fibroblasts. Cell lines from 2 normal human females, 2 normal human males and one XX human male were tested in 3 separate assays. Cells from XY and XX males were found to contain H-Y antigen; however, the reaction against the XX male cells was found to be intermediate between the XY male and normal XX female cells. (Auth.)

  19. Cellular replication limits in the Luria-Delbrück mutation model

    Science.gov (United States)

    Rodriguez-Brenes, Ignacio A.; Wodarz, Dominik; Komarova, Natalia L.

    2016-08-01

    Originally developed to elucidate the mechanisms of natural selection in bacteria, the Luria-Delbrück model assumed that cells are intrinsically capable of dividing an unlimited number of times. This assumption however, is not true for human somatic cells which undergo replicative senescence. Replicative senescence is thought to act as a mechanism to protect against cancer and the escape from it is a rate-limiting step in cancer progression. Here we introduce a Luria-Delbrück model that explicitly takes into account cellular replication limits in the wild type cell population and models the emergence of mutants that escape replicative senescence. We present results on the mean, variance, distribution, and asymptotic behavior of the mutant population in terms of three classical formulations of the problem. More broadly the paper introduces the concept of incorporating replicative limits as part of the Luria-Delbrück mutational framework. Guidelines to extend the theory to include other types of mutations and possible applications to the modeling of telomere crisis and fluctuation analysis are also discussed.

  20. Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo

    Institute of Scientific and Technical Information of China (English)

    GUO Dong-xing; LI Jing-yun; LI Han-ping; LI Lin; ZHUANG Dao-min; JIAO Li-yan; WANG Zheng; BAO Zuo-yi; LIU Si-yang; LIU Yong-jian

    2010-01-01

    Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.Methods We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140clinical samples using this method.Results The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M1841, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Yemerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.

  1. Potency assay development for cellular therapy products: an ISCT review of the requirements and experiences in the industry.

    Science.gov (United States)

    Bravery, Christopher A; Carmen, Jessica; Fong, Timothy; Oprea, Wanda; Hoogendoorn, Karin H; Woda, Juliana; Burger, Scott R; Rowley, Jon A; Bonyhadi, Mark L; Van't Hof, Wouter

    2013-01-01

    The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development. PMID:23260082

  2. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

    OpenAIRE

    Zarbock Ralf; Woischnik Markus; Sparr Christiane; Thurm Tobias; Kern Sunčana; Kaltenborn Eva; Hector Andreas; Hartl Dominik; Liebisch Gerhard; Schmitz Gerd; Griese Matthias

    2012-01-01

    Abstract Background Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We al...

  3. Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems

    Directory of Open Access Journals (Sweden)

    Fukumori Nobutaka

    2009-09-01

    Full Text Available Abstract Background Recently, manufactured nano/microparticles such as fullerenes (C60, carbon black (CB and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles. Results In in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations. Conclusion Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

  4. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    Directory of Open Access Journals (Sweden)

    Jiali Xing

    Full Text Available Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS, reducing power (RP, and inhibition of linoleic acid peroxidation (ILAP. Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.

  5. Use of amplification refractory mutation system PCR assay as a simple and effective tool to detect HIV-1 drug resistance mutations.

    Science.gov (United States)

    Nanfack, Aubin J; Agyingi, Lucy; Noubiap, Jean Jacques N; Ngai, Johnson N; Colizzi, Vittorio; Nyambi, Phillipe N

    2015-05-01

    Access to genotyping assays to determine successful antiretroviral treatment (ART) is limited in resource-constrained settings by high cost, suggesting the need for a cost-effective and simplified method to identify HIV-1 drug resistance (HIVDR) mutations. In this study, an amplification refractory mutation system (ARMS)-PCR assay was developed and used to investigate the most frequent HIVDR mutations affecting first-line ART in settings where WHO ART guidelines are applied. Seventy-five HIV-positive (HIV(+)) samples from Cameroon were used to assess the performance of this assay. Sequencing of HIV-1 reverse transcriptase was simultaneously performed for comparison, and discordant samples were tested with a Trugene HIV-1 genotyping kit. The ARMS-PCR assay was able to detect M184V, T215Y/F, K103N, and Y181C mutations with sensitivities of 96.8%, 85.7%, 91.3%, and 70%, respectively, and specificities of 90.6%, 95%, 100%, 96.9%, respectively, compared with data on sequencing. The results indicated the highest positive predictive value for K103N (100%) and the highest negative predictive value for M184V (97.5%). ARMS-PCR's limits of detection for mutations M184V, T215Y/F, K103N, and Y181C were HIV-1 clades (CRF02_AG and non-CRF02_AG). In addition, this approach was more cost-effective than other genotyping assays. The high throughput, the cost-effectiveness, and the simplicity of the ARMS-PCR assay make it a suitable tool to monitor HIVDR patterns in resource-constrained settings with broad HIV-1 genetic diversity. PMID:25788547

  6. Responses of the L5178Y mouse lymphoma cell forward mutation assay. V: 27 coded chemicals

    Energy Technology Data Exchange (ETDEWEB)

    McGregor, D.B.; Brown, A.G.; Howgate, S.; McBride, D.; Riach, C. (Inveresk Research International Limited, Musselburgh (Scotland)); Caspary, W.J. (National Inst. of Health, Research Triangle Park, NC (United States))

    1991-01-01

    Twenty-seven chemicals were tested for their mutagenic potential in the L5178Y tk{sup +}/tk{sup {minus}} mouse lymphoma cell forward mutation assay. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 {mu}g/ml. The chemicals were tested at least twice. Statistically significant responses were obtained with acid orange 10, aniline, benzaldehyde o-chloroaniline, chlorodibromomethane, cytembena, 1,2-dibromo-4-(1,2-dibromomethyl) cyclohexane, dieldrin, lithocholic acid, oxytetracycline, phenazopyridine HCl, 1phenyl-3-methyl-5-pyrazolone, sodium diethyldithiocarbamate, solvent yellow 14, tetraethylthiuram disulfide (disulfiram), 2,4-toluene diisocyanate, and 2,6-toluene diisocyanate. Apart from phenazopyridine HCl, acid orange 10, and solvent yellow 14, rat liver S9 mix was not a requirement for the mutagenic activity of these compounds.

  7. Evaluation of the safety of the dietary antioxidant ergothioneine using the bacterial reverse mutation assay

    International Nuclear Information System (INIS)

    The dietary antioxidant L-(+)-ergothioneine was tested for its potential mutagenic activity using the bacterial reverse mutation assay. The experiments were carried out using histidine-requiring auxotrophic strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotrophic strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post-mitochondrial supernatant (S9) prepared from livers of phenobarbital/β-naphthoflavone-induced rats. The revertant colony numbers of vehicle control plates with and without S9 Mix were within the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biologically relevant increases in induced revertant colonies in all experimental phases in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with L-(+)-ergothioneine at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. On the basis of the data reported, it can be concluded that L-(+)-ergothioneine did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Thus L-(+)-ergothioneine has no mutagenic activity on the applied bacteria tester strains under the test conditions used in this study. Research is continuing to define the role of L-(+)-ergothioneine in disease pathophysiology. Further studies on its safety are suggested.

  8. Systematic Cellular Disease Models Reveal Synergistic Interaction of Trisomy 21 and GATA1 Mutations in Hematopoietic Abnormalities.

    Science.gov (United States)

    Banno, Kimihiko; Omori, Sayaka; Hirata, Katsuya; Nawa, Nobutoshi; Nakagawa, Natsuki; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Sakuma, Tetsushi; Yamamoto, Takashi; Toki, Tsutomu; Ito, Etsuro; Yamamoto, Toshiyuki; Kokubu, Chikara; Takeda, Junji; Taniguchi, Hidetoshi; Arahori, Hitomi; Wada, Kazuko; Kitabatake, Yasuji; Ozono, Keiichi

    2016-05-10

    Chromosomal aneuploidy and specific gene mutations are recognized early hallmarks of many oncogenic processes. However, the net effect of these abnormalities has generally not been explored. We focused on transient myeloproliferative disorder (TMD) in Down syndrome, which is characteristically associated with somatic mutations in GATA1. To better understand functional interplay between trisomy 21 and GATA1 mutations in hematopoiesis, we constructed cellular disease models using human induced pluripotent stem cells (iPSCs) and genome-editing technologies. Comparative analysis of these engineered iPSCs demonstrated that trisomy 21 perturbed hematopoietic development through the enhanced production of early hematopoietic progenitors and the upregulation of mutated GATA1, resulting in the accelerated production of aberrantly differentiated cells. These effects were mediated by dosage alterations of RUNX1, ETS2, and ERG, which are located in a critical 4-Mb region of chromosome 21. Our study provides insight into the genetic synergy that contributes to multi-step leukemogenesis. PMID:27134169

  9. Cytotoxicity and genotoxicity of Agaricus blazei methanolic extract fractions assessed using gene and chromosomal mutation assays

    Directory of Open Access Journals (Sweden)

    Marilanda Ferreira Bellini

    2008-01-01

    Full Text Available Functional food investigations have demonstrated the presence of substances that could be beneficial to human health when consumed. However, the toxic effects of some substances contained in foods have been determined. Reported medicinal and nutritive properties have led to the extensive commercialization of the basidiomycete fungi Agaricus blazei Murrill (sensu Heinemann, also known as Agaricus brasiliensis Wasser et al., Agaricus subrufescens Peck or the Brazilian medical mushroom (BMM. Different methanolic extract fractions (ME of this mushroom were submitted to the cytokinesis-block micronucleus (CBMN clastogenic assay and the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT assay for gene mutation, both using Chinese hamster ovary cells clone K1 (CHO-K1. The results suggest that all the fractions tested possess cytotoxic and mutagenic potential but no clastogenic effects. Further information is needed on the biochemical components of the A. blazei methanol fractions to identify any substances with cytotoxic and/or mutagenicity potential. These findings indicate that A. blazei methanolic extract should not be used due to their genotoxicity and care should be taken in the use of A. blazei by the general population until further biochemical characterization of this fungi is completed.

  10. Increased Chromosomal Radiosensitivity in Women Carrying BRCA1/BRCA2 Mutations Assessed With the G2 Assay

    International Nuclear Information System (INIS)

    Purpose: Several in vitro studies suggest that BRCA1 and BRCA2 mutation carriers present increased sensitivity to ionizing radiation. Different assays for the assessment of deoxyribonucleic acid double-strand break repair capacity have been used, but results are rather inconsistent. Given the concerns about the possible risks of breast screening with mammography in mutation carrier women and the potentially damaging effects of radiotherapy, the purpose of this study was to further investigate the radiosensitivity of this population. Methods and Materials: The G2 chromosomal radiosensitivity assay was used to assess chromosomal breaks in lymphocyte cultures after exposure to 1 Gy. A group of familiar breast cancer patients carrying a mutation in the BRCA1 or BRCA2 gene (n = 15) and a group of healthy mutation carriers (n = 5) were investigated and compared with a reference group of healthy women carrying no mutation (n = 21). Results: BRCA1 and BRCA2 mutation carriers had a significantly higher number of mean chromatid breaks per cell (p = 0.006) and a higher maximum number of breaks (p = 0.0001) as compared with their matched controls. Both healthy carriers and carriers with a cancer history were more radiosensitive than controls (p = 0.002 and p = 0.025, respectively). Age was not associated with increased radiosensitivity (p = 0.868). Conclusions: Our results indicate that BRCA1 and BRCA2 mutation carriers show enhanced radiosensitivity, presumably because of the involvement of the BRCA genes in deoxyribonucleic acid repair and cell cycle control mechanisms.

  11. Molecular heterogeneity for bovine alpha-mannosidosis: PCR based assays for detection of breed-specific mutations.

    Science.gov (United States)

    Berg, T; Healy, P J; Tollersrud, O K; Nilssen, O

    1997-01-01

    DNA tests, based on the polymerase chain reaction (PCR), were developed for the detection of two breed-specific mutations responsible for the autosomal recessive disorder bovine alpha-mannosidosis. The tests involve separate amplification of two exons of the lysosomal alpha-mannosidase gene followed by restriction enzyme digestion of the amplicons. We demonstrate that one of the mutations, the 662G-->A transition, is responsible for alpha-mannosidosis in Galloway cattle. The other mutation, the 961T-->C transition, is uniquely associated with alpha-mannosidosis in Angus, Murray Grey and Brangus cattle from Australia. The 961T-->C mutation was also detected in Red Angus cattle exported from Canada to Australia as embryos. All 39 animals classified as heterozygotes on the basis of biochemical assays were heterozygous for one of the two mutations. None of 102 animals classified as homozygous-normal on the basis of biochemical assays possessed the mutations. Our results indicate that the two breed-specific mutations may have arisen in Scotland and by the export of animals and germplasm disseminated to America, New Zealand and Australia. PMID:9491457

  12. Characterisation of an in vivo Pig-a gene mutation assay for use in regulatory toxicology studies.

    Science.gov (United States)

    Godin-Ethier, Jessica; Leroux, Francis; Wang, Nan; Thébaud, Sybil; Merah, Farida; Nelson, Andrew

    2015-05-01

    Integration of in vivo genotoxicity testing into standard toxicology studies presents multiple advantages as it reduces animal use and costs, accelerates data generation and provides concurrent data that are useful for interpreting results. The in vivo Pig-a assay is a mammalian gene mutation assay that utilises peripheral blood and thus has a high integration potential. Although inter-laboratory reproducibility has been well demonstrated, further characterisation is required for this assay. In this study, we evaluated intra-laboratory reproducibility of the in vivo Pig-a gene mutation assay (MutaFlow® kit) in rats through the conduct of an assay characterisation prior to subsequent use in Good Laboratory Practices (GLP) toxicology studies. To evaluate intra-laboratory reproducibility, intra-assay and inter-assay variability, ruggedness, robustness and blood storage stability were assessed. These assessments were performed using blood obtained from male Sprague-Dawley rats exposed to 0, 20 or 40mg/kg/day N-ethyl-N-nitrosourea via oral gavage for three consecutive days. Blood was collected from these rats on multiple occasions from Day 29 to Day 71 and samples were analysed for Pig-a mutation using the rat MutaFlow kit. Frequencies of reticulocytes (RET), mutant phenotype erythrocytes (RBC(CD59-)) and mutant phenotype RET (RET(CD59-)) were determined. Overall, the proportion of RET and frequencies of RBC(CD59-) and of RET(CD59-) were consistent throughout the different assessments. The assay demonstrated acceptable intra-run and inter-run variability with coefficients of variation of ≤4.8 and 20.6%, respectively. The method was shown to be independent of the analyst performing the assay and unaffected by small changes in assay conditions. Comparable results were obtained from freshly collected samples and samples refrigerated for up to 4 days. Although technically challenging, the rat Pig-a gene mutation assay using a high-throughput automated method was shown to

  13. Cellular apoptosis susceptibility (CAS) is overexpressed in thyroid carcinoma and maintains tumor cell growth: A potential link to the BRAFV600E mutation.

    Science.gov (United States)

    Holzer, Kerstin; Drucker, Elisabeth; Oliver, Scott; Winkler, Juliane; Eiteneuer, Eva; Herpel, Esther; Breuhahn, Kai; Singer, Stephan

    2016-04-01

    Thyroid carcinoma is among the most common malignant endocrine neoplasms with a rising incidence. Genetic alterations occurring in thyroid cancer frequently affect the RAS/RAF/MEK/ERK-pathway such as the oncogenic, kinase-activating BRAF(V600E) mutation. Nuclear transport receptors including importins and exportins represent an important part of the nuclear transport machinery providing nucleo-cytoplasmic exchange of macromolecules. The role of nuclear transport receptors in the development and progression of thyroid carcinomas is largely unknown. Here, we studied the expression and function of the exportin cellular apoptosis susceptibility (CAS) in thyroid carcinogenesis and its link to the BRAF(V600E) mutation. By using immunohistochemistry (IHC) we found significantly increased IHC scores of CAS in primary papillary (PTC) and medullary (MTC), but not in follicular (FTC) thyroid carcinoma compared to non-tumorous (NT) thyroid tissue. Interestingly, metastases of the aforementioned subtypes including FTC showed a strong CAS positivity. Among PTCs we observed that CAS immunoreactivity was significantly higher in the tumors harboring the BRAF(V600E) mutation. Furthermore, depletion of CAS by RNAi in the BRAF(V600E)-positive PTC cell line B-CPAP led to reduced tumor cell growth measured by crystal violet assays. This phenotype could be attributed to reduced proliferation and increased cell death as assayed by BrdU ELISAs and immunoblotting for PARP-cleavage, respectively. Finally, we found additive effects of CAS siRNA and vemurafenib treatment in B-CPAP cells. Collectively, these data suggest that CAS overexpression in thyroid carcinoma depends on the subtype and the disease stage. Our findings also indicate that CAS maintains PTC cell proliferation and survival. Targeting CAS could represent a potential therapeutic approach particularly in combination with BRAF inhibitors such as vemurafenib in BRAF(V600E)-positive tumors. PMID:26892809

  14. Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

    DEFF Research Database (Denmark)

    Russell, Rodney S; Meunier, Jean-Christophe; Takikawa, Shingo; Faulk, Kristina; Engle, Ronald E; Bukh, Jens; Purcell, Robert H; Emerson, Suzanne U

    2008-01-01

    The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five......, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast......, both the p7 and NS2 mutations independently increased the amount of virus released....

  15. Responses of the L5178Y mouse Lymphoma cell forward mutation assay. V: 27 coded chemicals.

    Science.gov (United States)

    McGregor, D B; Brown, A G; Howgate, S; McBride, D; Riach, C; Caspary, W J

    1991-01-01

    Twenty-seven chemicals were tested for their mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay using procedures based upon those described by McGregor et al. (McGregor DB, Martin R, Cattanach P, Edwards I, McBride D, Caspary WJ (1987): Environ Mol Mutagen 9:143-160). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Statistically significant responses were obtained with acid orange 10, aniline, benzaldehyde, o-chloroaniline, chlorodibromomethane, cytembena, 1,2-dibromo-4-(1,2-dibromomethyl) cyclohexane, dieldrin, lithocholic acid, oxytetracycline, phenazopyridine HCl, 1-phenyl-3-methyl-5-pyrazolone, sodium diethyldithiocarbamate, solvent yellow 14, tetraethylthiuram disulfide (disulfiram), 2,4-toluene diisocyanate, and 2,6-toluene diisocyanate. Apart from phenazopyridine HCl, acid orange 10, and solvent yellow 14, rat liver S9 mix was not a requirement for the mutagenic activity of these compounds. Chemical not identified as mutagens were N-4-acetylaminofluorene, chlorpheniramine maleate, chloropropamide, 1,4-dioxane, endrin, ethylene glycol, iron dextran, methapyrilene, sodium(2-ethylhexyl)alcohol PMID:1902415

  16. Cellular and molecular analyses of hprt mutation in human hepatocyte L02 cells after exposure to carbon ions

    Science.gov (United States)

    Li, Qiang; He, Jing; Jin, Xiao-Dong; Gong, Li; Li, Sha

    Mutations play an important role in carcinogenesis. The quantitative evaluation of mutation induction by heavy charged particles helps us to delineate the risks of space radiation on astronauts, as well as the risks of heavy ions on patients during tumor therapy. Hprt mutation assay, which has been used as a biological dosimeter, is an ideal gene mutation test in mammalian cells in vitro. In order to provide basic data and evidence for the risk assessment of heavy ions, the relationships between hprt mutation induction and radiation dose in human hepatocyte L02 cells were investigated for highand low-LET carbon ions and X-rays. Moreover, the carbon ion induced hprt mutation spectrum was analyzed. In our study, human hepatocyte L02 cells were irradiated with carbon ions with LET of 30keV/µm and X-rays (0.2keV/µm), respectively. The survival fraction of L02 cells was measured by means of colony-forming assay. The mutation frequency was detected by measuring 6-thioguanine-resistant clones after 10 days of incubation at the presence of 15mg/L 6-TG. To obtain the mutation spectrum, 9 10 mutant cell clones at each dose were randomly selected from the 6-TG containing medium, and were further cultured and analyzed. The deletion patterns of the 9 exons of hprt gene were analyzed with multiplex polymerase chain reactions (multiplex PCR). Our results show that the number of mutants per 106 surviving cells increased with increasing the radiation dose for both the irradiations, and the mutation frequency increased up to 1Gy while reduced with increasing dose further. Partial deletion was the most dominant deletion pattern in the hprt mutant cells, and with the increase of dose, hprt genes tended to have more total deletions and less point deletions. It can be inferred that human hepatocyte L02 cells are more radiosensitive to high-LET carbon ions than to low-LET X-rays, and carbon ions are more effective in inducing hprt mutation in L02 cells. It has been also found that the

  17. Cellular and Deafness Mechanisms Underlying Connexin Mutation-Induced Hearing Loss – A Common Hereditary Deafness

    OpenAIRE

    Wingard, Jeffrey C.; Zhao, Hong-Bo

    2015-01-01

    Hearing loss due to mutations in the connexin gene family, which encodes gap junctional proteins, is a common form of hereditary deafness. In particular, connexin 26 (Cx26, GJB2) mutations are responsible for ~50% of non-syndromic hearing loss, which is the highest incidence of genetic disease. In the clinic, Cx26 mutations cause various auditory phenotypes ranging from profound congenital deafness at birth to mild, progressive hearing loss in late childhood. Recent experiments demonstrate th...

  18. Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

    Directory of Open Access Journals (Sweden)

    Hideki Hayashi

    Full Text Available Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

  19. Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qPCR Assay.

    Science.gov (United States)

    Harb, Wael; Fan, Andrea; Tran, Tony; Danila, Daniel C; Keys, David; Schwartz, Michael; Ionescu-Zanetti, Cristian

    2013-01-01

    Circulating tumor cells (CTCs) provide a readily accessible source of tumor material from patients with cancer. Molecular profiling of these rare cells can lead to insight on disease progression and therapeutic strategies. A critical need exists to isolate CTCs with sufficient quantity and sample integrity to adapt to conventional analytical techniques. We present a microfluidic platform (IsoFlux) that uses flow control and immunomagnetic capture to enhance CTC isolation. A novel cell retrieval mechanism ensures complete transfer of CTCs into the molecular assay. Improved sensitivity to the capture antigen was demonstrated by spike-in experiments for three cell lines of varying levels of antigen expression. We obtained spike-in recovery rates of 74%, 75%, and 85% for MDA-MB-231 (low), PC3 (middle), and SKBR3 (high) cell lines. Recovery using matched enumeration protocols and matched samples (PC3) yielded 90% and 40% recovery for the IsoFlux and CellSearch systems, respectively. In matched prostate cancer samples (N = 22), patients presenting more than four CTCs per blood draw were 95% and 36% using IsoFlux and CellSearch, respectively. An assay for detecting KRAS mutations was described along with data from patients with colorectal cancer, of which 87% presented CTCs above the assay's limit of detection (four CTCs). The CTC KRAS mutant rate was 50%, with 46% of patients displaying a CTC KRAS mutational status that differed from the previously acquired tissue biopsy data. The microfluidic system and mutation assay presented here provide a complete workflow to track oncogene mutational changes longitudinally with high success rates. PMID:24151533

  20. Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qPCR Assay12

    Science.gov (United States)

    Harb, Wael; Fan, Andrea; Tran, Tony; Danila, Daniel C; Keys, David; Schwartz, Michael; Ionescu-Zanetti, Cristian

    2013-01-01

    Circulating tumor cells (CTCs) provide a readily accessible source of tumor material from patients with cancer. Molecular profiling of these rare cells can lead to insight on disease progression and therapeutic strategies. A critical need exists to isolate CTCs with sufficient quantity and sample integrity to adapt to conventional analytical techniques. We present a microfluidic platform (IsoFlux) that uses flow control and immunomagnetic capture to enhance CTC isolation. A novel cell retrieval mechanism ensures complete transfer of CTCs into the molecular assay. Improved sensitivity to the capture antigen was demonstrated by spike-in experiments for three cell lines of varying levels of antigen expression. We obtained spike-in recovery rates of 74%, 75%, and 85% for MDA-MB-231 (low), PC3 (middle), and SKBR3 (high) cell lines. Recovery using matched enumeration protocols and matched samples (PC3) yielded 90% and 40% recovery for the IsoFlux and CellSearch systems, respectively. In matched prostate cancer samples (N = 22), patients presenting more than four CTCs per blood draw were 95% and 36% using IsoFlux and CellSearch, respectively. An assay for detecting KRAS mutations was described along with data from patients with colorectal cancer, of which 87% presented CTCs above the assay's limit of detection (four CTCs). The CTC KRAS mutant rate was 50%, with 46% of patients displaying a CTC KRAS mutational status that differed from the previously acquired tissue biopsy data. The microfluidic system and mutation assay presented here provide a complete workflow to track oncogene mutational changes longitudinally with high success rates. PMID:24151533

  1. Bringing the light to high throughput screening: use of optogenetic tools for the development of recombinant cellular assays

    Science.gov (United States)

    Agus, Viviana; Di Silvio, Alberto; Rolland, Jean Francois; Mondini, Anna; Tremolada, Sara; Montag, Katharina; Scarabottolo, Lia; Redaelli, Loredana; Lohmer, Stefan

    2015-03-01

    The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called "optogenetic" technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

  2. Immune Responses to AAV in Canine Muscle Monitored by Cellular Assays and Noninvasive Imaging

    OpenAIRE

    Wang, Zejing; Storb, Rainer; Lee, Donghoon; Kushmerick, Martin J.; Chu, Baocheng; Berger, Carolina; Arnett, Andrea; Allen, James; Chamberlain, Jeffrey S.; Riddell, Stanley R.; Tapscott, Stephen J.

    2009-01-01

    We previously demonstrated that direct intramuscular injection of rAAV2 or rAAV6 in wild-type dogs resulted in robust T-cell responses to viral capsid proteins, and others have shown that cellular immunity to adeno-associated virus (AAV) capsid proteins coincided with liver toxicity and elimination of transgene expression in a human trial of hemophilia B. Here, we show that the heparin-binding ability of a given AAV serotype does not determine the induction of T-cell responses following intra...

  3. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

    Directory of Open Access Journals (Sweden)

    Zarbock Ralf

    2012-03-01

    Full Text Available Abstract Background Surfactant protein C (SP-C is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. Methods SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. Results Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. Conclusions We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy

  4. KRAS mutation analysis in ovarian samples using a high sensitivity biochip assay

    Directory of Open Access Journals (Sweden)

    Reinthaller Alexander

    2009-04-01

    Full Text Available Abstract Background Mutations in the KRAS gene are one of the most frequent genetic abnormalities in ovarian carcinoma. They are of renewed interest as new epidermal growth factor receptor (EGFR-targeted therapies are being investigated for use in ovarian carcinoma. As KRAS mutations are associated with poor response and resistance to EGFR-targeting drugs, this study was conducted to obtain more information on the spectrum of KRAS mutations in ovarian carcinoma. Methods The presence of KRAS mutations in codon 12 and 13 was analyzed in frozen and formalin-fixed paraffin-embedded (FFPE tissue with a low density biochip platform. 381 malignant (29 borderline malignancy, 270 primary carcinomas, and 82 recurrent carcinomas and 22 benign tissue samples from a total of 394 patients were examined. KRAS mutational status of each sample was correlated with dignity, FIGO stage, grade, histology, and survival. Results KRAS mutations were found in 60 (15% samples with 58 samples deriving from malignant tissue and 2 samples deriving from benign tissue. In 55 (92% samples codon 12 was found to be mutated. Frozen and FFPE samples concurred with respect to KRAS mutation status. Conclusion KRAS mutation is a common event in ovarian cancer primarily in carcinomas of lower grade, lower FIGO stage, and mucinous histotype. The KRAS mutational status is no prognostic factor for patients treated with standard therapy. However, in line with experience from colorectal cancer and non-small-cell-lung cancer (NSCLC, it may be important for prediction of response to EGFR-targeted therapies.

  5. Cellular and molecular studies of mutation induction by low energy heavy ions

    Institute of Scientific and Technical Information of China (English)

    TomKHei; DavidJChen; 等

    1997-01-01

    Mutation induction by low energy heavy ions was scored at the hypoxanthine guanine phosphoribosyl transferase(HGPRT) locus using both normal human fibroblasts and the human-hamster hybrid AL cells.In addition,the mutation yield at a non-essential chromosome was also examined by using the S1 marker gene locating on human chromosome 11 in AL cells,Mutagenicity induced by low energy heavy ions was dose and LET dependent.THe induced mutant fractions at the S1 locus were consistently higher than those for HGPRT.Using a mutation system that can detect multilocus changes,it can be shown by either Southern blotting or multiplex PCR techniques that radiation can induce chromosomal deletions in the millions of basepairs.

  6. Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

    Energy Technology Data Exchange (ETDEWEB)

    French, C. Tenley [Cytomation GTX Inc., Fort Collins, CO (United States); Ross, Carley D. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Keysar, Stephen B. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Joshi, Dhanashree D. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Lim, Chang-Uk [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Fox, Michael H. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States) and Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523-1618 (United States)]. E-mail: mfox@colostate.edu

    2006-12-01

    Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A{sub L}). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59{sup -} and CD59{sup +} populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.

  7. The cytotoxicity of polycationic iron oxide nanoparticles: Common endpoint assays and alternative approaches for improved understanding of cellular response mechanism

    Directory of Open Access Journals (Sweden)

    Hoskins Clare

    2012-04-01

    Full Text Available Abstract Background Iron oxide magnetic nanoparticles (MNP's have an increasing number of biomedical applications. As such in vitro characterisation is essential to ensure the bio-safety of these particles. Little is known on the cellular interaction or effect on membrane integrity upon exposure to these MNPs. Here we synthesised Fe3O4 and surface coated with poly(ethylenimine (PEI and poly(ethylene glycol (PEG to achieve particles of varying surface positive charges and used them as model MNP's to evaluate the relative utility and limitations of cellular assays commonly applied for nanotoxicity assessment. An alternative approach, atomic force microscopy (AFM, was explored for the analysis of membrane structure and cell morphology upon interacting with the MNPs. The particles were tested in vitro on human SH-SY5Y, MCF-7 and U937 cell lines for reactive oxygen species (ROS production and lipid peroxidation (LPO, LDH leakage and their overall cytotoxic effect. These results were compared with AFM topography imaging carried out on fixed cell lines. Results Successful particle synthesis and coating were characterised using FTIR, PCS, TEM and ICP. The particle size from TEM was 30 nm (−16.9 mV which increased to 40 nm (+55.6 mV upon coating with PEI and subsequently 50 nm (+31.2 mV with PEG coating. Both particles showed excellent stability not only at neutral pH but also in acidic environment of pH 4.6 in the presence of sodium citrate. The higher surface charge MNP-PEI resulted in increased cytotoxic effect and ROS production on all cell lines compared with the MNP-PEI-PEG. In general the effect on the cell membrane integrity was observed only in SH-SY5Y and MCF-7 cells by MNP-PEI determined by LDH leakage and LPO production. AFM topography images showed consistently that both the highly charged MNP-PEI and the less charged MNP-PEI-PEG caused cell morphology changes possibly due to membrane disruption and cytoskeleton remodelling. Conclusions

  8. Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay

    Directory of Open Access Journals (Sweden)

    C Thakur

    2015-01-01

    Full Text Available Context: Tuberculosis (TB is a major public health problem in India and a principal cause of death in adults, especially among the economically productive age group. India accounts for one-fifth of the global burden of TB. It is estimated that about 40% of Indian population is infected with TB bacillus. The GenoType® MTBDRplus molecular method allows rapid diagnosis of the clinical samples and detection of the most common mutations in the genes associated with rifampicin (R and isoniazid (H resistance. Aims: To study the drug resistance and mutational patterns in multidrug-resistant (MDR suspects clinical strains using GenoType® MTBDRplus assay. Subjects and Methods: A total of 770 sputum samples of the MDR-TB suspects were included in this study, which were received at Intermediate Reference Laboratory, Government TB Sanatorium, Dharampur, Solan, Himachal Pradesh from the Designated Microscopy Centres of Himachal Pradesh for the culture and susceptibility testing. All the 521 Mycobacterium tuberculosis complex (MTBC strains were subjected to GenoType® MTBDRplus (HAIN Lifescience assay to detect molecular resistance pattern to first line anti-tubercular drugs (isoniazid and rifampicin. Results: Of 770 samples, 556 (72.20% were from male and 214 (27.80% were from female. Among the 521 MTBC strains, 19.76% were found to be MDR and mono-resistance to isoniazid and rifampicin was detected in 8.63% and 6.14% strains respectively. About 74.81%, 76.35% and 5.40% strains harboured known mutation in rpoB, katG and inhA genes respectively. Conclusions: In rpoB gene, the most common mutation is associated with S531 L region. The GenoType® MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In our study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.

  9. Biodosimetry of Chernobyl cleanup workers from Estonia and Latvia using the glycophorin A in vivo somatic cell mutation assay

    International Nuclear Information System (INIS)

    The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method. 25 refs., 2 figs., 3 tabs

  10. Use of an Anaerobic Chamber Environment for the Assay of Endogenous Cellular Protein-Tyrosine Phosphatase Activities

    Directory of Open Access Journals (Sweden)

    Zhu Li

    2002-01-01

    Full Text Available Protein-tyrosine phosphatases (PTPases have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible fraction of the endogenous PTPase pool.

  11. Effects of p35 Mutations Associated with Mental Retardation on the Cellular Function of p35-CDK5.

    Directory of Open Access Journals (Sweden)

    Shunsuke Takada

    Full Text Available p35 is an activation subunit of the cyclin-dependent kinase 5 (CDK5, which is a Ser/Thr kinase that is expressed predominantly in neurons. Disruption of the CDK5 or p35 (CDK5R1 genes induces abnormal neuronal layering in various regions of the mouse brain via impaired neuronal migration, which may be relevant for mental retardation in humans. Accordingly, mutations in the p35 gene were reported in patients with nonsyndromic mental retardation; however, their effect on the biochemical function of p35 has not been examined. Here, we studied the biochemical effect of mutant p35 on its known properties, i.e., stability, CDK5 activation, and cellular localization, using heterologous expression in cultured cells. We also examined the effect of the mutations on axon elongation in cultured primary neurons and migration of newborn neurons in embryonic brains. However, we did not detect any significant differences in the effects of the mutant forms of p35 compared with wild-type p35. Therefore, we conclude that these p35 mutations are unlikely to cause mental retardation.

  12. New Real-Time PCR Assay Using Locked Nucleic Acid Probes To Assess Prevalence of ParC Mutations in Fluoroquinolone-Susceptible Streptococcus pneumoniae Isolates from France

    OpenAIRE

    Decousser, Jean-Winoc; Methlouthi, Imen; Pina, Patrick; Collignon, Anne; Allouch, Pierre

    2006-01-01

    A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys.

  13. New real-time PCR assay using locked nucleic acid probes to assess prevalence of ParC mutations in fluoroquinolone-susceptible Streptococcus pneumoniae isolates from France.

    Science.gov (United States)

    Decousser, Jean-Winoc; Methlouthi, Imen; Pina, Patrick; Collignon, Anne; Allouch, Pierre

    2006-04-01

    A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys. PMID:16569894

  14. Endocrine, teratogenic and neurotoxic effects of cyanobacteria detected by cellular in vitro and zebrafish embryos assays.

    Science.gov (United States)

    Jonas, Adam; Scholz, Stefan; Fetter, Eva; Sychrova, Eliska; Novakova, Katerina; Ortmann, Julia; Benisek, Martin; Adamovsky, Ondrej; Giesy, John P; Hilscherova, Klara

    2015-02-01

    Cyanobacteria contain various types of bioactive compounds, which could cause adverse effects on organisms. They are released into surface waters during cyanobacterial blooms, but there is little information on their potential relevance for effects in vivo. In this study presence of bioactive compounds was characterized in cyanobacteria Microcystis aeruginosa (Chroococcales), Planktothrix agardhii (Oscillatoriales) and Aphanizomenon gracile (Nostocales) with selected in vitro assays. The in vivo relevance of detected bioactivities was analysed using transgenic zebrafish embryos tg(cyp19a1b-GFP). Teratogenic potency was assessed by analysis of developmental disorders and effects on functions of the neuromuscular system by video tracking of locomotion. Estrogenicity in vitro corresponded to 0.95-54.6 ng estradiol equivalent(g dry weight (dw))(-1). In zebrafish embryos, estrogenic effects could not be detected potentially because they were masked by high toxicity. There was no detectable (anti)androgenic/glucocorticoid activity in any sample. Retinoid-like activity was determined at 1-1.3 μg all-trans-retinoic acid equivalent(g dw)(-1). Corresponding to the retinoid-like activity A. gracile extract also caused teratogenic effects in zebrafish embryos. Furthermore, exposure to biomass extracts at 0.3 gd wL(-1) caused increase of body length in embryos. There were minor effects on locomotion caused by 0.3 gd wL(-1)M. aeruginosa and P. agardhii extracts. The traditionally measured cyanotoxins microcystins did not seem to play significant role in observed effects. This indicates importance of other cyanobacterial compounds at least towards some species or their developmental phases. More attention should be paid to activity of retinoids, estrogens and other bioactive substances in phytoplankton using in vitro and in vivo bioassays. PMID:25170595

  15. Detection of BRAF mutation in Chinese tumor patients using a highly sensitive antibody immunohistochemistry assay

    Science.gov (United States)

    Qiu, Tian; Lu, Haizhen; Guo, Lei; Huang, Wenting; Ling, Yun; Shan, Ling; Li, Wenbin; Ying, Jianming; Lv, Ning

    2015-03-01

    BRAF mutations can be found in various solid tumors. But accurate and reliable screening for BRAF mutation that is compatible for clinical application is not yet available. In this study, we used an automated immunohistochemistry (IHC) staining coupled with mouse monoclonal anti-BRAF V600E (VE1) primary antibody to screen the BRAF V600E mutation in 779 tumor cases, including 611 colorectal carcinomas (CRC), 127 papillary thyroid carcinomas (PTC) and 41 malignant melanomas. Among the 779 cases, 150 cases were positive for BRAF (V600E) staining, including 38 (of 611, 6%) CRCs, 102 (of 127, 80%) PTCs and 10 (of 41, 24%) malignant melanomas. Sanger sequencing and real-time PCR confirmed the sensitivity and specificity of IHC staining for the V600E mutation are 100% and 99%, respectively. Therefore, our study demonstrates that the fully automated IHC is a reliable tool to determine BRAF mutation status in CRC, PTC and melanoma and can be used for routine clinical screen.

  16. In vivo method for the detection of somatic mutations at the cellular level in mice

    Energy Technology Data Exchange (ETDEWEB)

    Searle, A.G.; Stephenson, D.A. (Medical Research Council, Harwell (UK). Radiobiology Unit)

    1982-01-01

    This new method is based on the fact that recessive mutations at the dilute and leaden loci change the shape of pigment-cells in the hair follicles from their normal highly dendritic appearance to a rounded-up almost non-dendritic form. Gene action is autonomous and individual mutant melanocytes can be recognised in whole mounts of dorsal skin made after the post-natal onset of melanogenesis. Foetal heterozygotes for d and In were treated with X-rays (0-200 rad) or procarbazine (0-300 mg/kg body mass by maternal i.p. injection) 17 after detection of a vaginal plug; whole mounts of dorsal skin (fixed 3 after birth) were microscopically scanned for mutant melanocytes in the hair follicles. Mutant cells were mainly in the form of small intrafollicular clones; the few mounts containing large interfollicular clones were excluded from the analysis. For both treatments, dose-response relationships showed good fits to both a quadratic and a power-law curve. However, neither of these differed significantly from a square-law relationship. The estimated mutation rate at the 100 rad X-ray level of 4.7x10/sup -7//rad/locus was in good agreement with the literature. Possible ways in which the present method might be improved and extended are discussed.

  17. An in vivo method for the detection of somatic mutations at the cellular level in mice

    International Nuclear Information System (INIS)

    This new method is based on the fact that recessive mutations at the dilute and leaden loci change the shape of pigment-cells in the hair follicles from their normal highly dendritic appearance to a rounded-up almost non-dendritic form. Gene action is autonomous and individual mutant melanocytes can be recognised in whole mounts of dorsal skin made after the post-natal onset of melanogenesis. Foetal heterozygotes for d and In were treated with X-rays (0-200 rad) or procarbazine (0-300 mg/kg body mass by maternal i.p. injection) 17 after detection of a vaginal plug; whole mounts of dorsal skin (fixed 3 after birth) were microscopically scanned for mutant melanocytes in the hair follicles. Mutant cells were mainly in the form of small intrafollicular clones; the few mounts containing large interfollicular clones were excluded from the analysis. For both treatments, dose-response relationships showed good fits to both a quadratic and a power-law curve. However, neither of these differed significantly from a square-law relationship. The estimated mutation rate at the 100 rad X-ray level of 4.7x10-7/rad/locus was in good agreement with the literature. Possible ways in which the present method might be improved and extended are discussed. (orig./MG)

  18. KSR2 Mutations Are Associated with Obesity, Insulin Resistance, and Impaired Cellular Fuel Oxidation

    OpenAIRE

    Pearce, Laura R.; Atanassova, Neli; Banton, Matthew C.; Bottomley, Bill; van der Klaauw, Agatha A.; Revelli, Jean-Pierre; Hendricks, Audrey; Keogh, Julia M.; Henning, Elana; Doree, Deon; Jeter-Jones, Sabrina; Garg, Sumedha; Bochukova, Elena G.; Bounds, Rebecca; Ashford, Sofie

    2013-01-01

    Summary Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxida...

  19. A balanced salt solution that prevents agglomeration of nano iron oxo-hydroxides in serum-free cellular assays

    Science.gov (United States)

    Pereira, Dora I. A.; Lederer, Bianca; Powell, Jonathan J.

    2015-01-01

    Nano iron oxo-hydroxides have numerous and increasing applications in biology and medicine. Assessment of their uptake and toxicity often requires cell culture but maintaining iron oxides uniformly nano-dispersed in such conditions can be challenging. We describe a balanced salt solution (BSS) compatible with cellular assays for use in such investigations. We determined hydrodynamic particle size and dispersibility of nano iron in BSS. The BSS, containing 130 mM NaCl, 10 mM KCl, 1 mM MgSO4, 5 mM glucose and 1.8 mM CaCl2 in 10 mM PIPES buffer (pH 7.4), was capable of maintaining nanoparticulate tartrate-modified Fe(III) oxo-hydroxide (i.e. nano Fe) mono-disperse (≥95% nanoparticulate) with a mean hydrodynamic particle diameter of 6.1 ± 0.3 nm. This size was similar to the native form of the nano Fe material (i.e. as synthesized) with a mean hydrodynamic particle diameter of 4.3 ± 0.1 nm in water. Furthermore, we show that BSS also adequately maintains nano Fe dispersion when supplemented with inhibitors of particle uptake or lysosomal acidification, namely chloropromazine and monensin, and when used at pHs 6.5 or 5.8. In conclusion, we provide a method for nanodispersion of iron oxo-hydroxides that is suitable for short term (1-3 h) cellular exposure investigations.

  20. A balanced salt solution that prevents agglomeration of nano iron oxo-hydroxides in serum-free cellular assays

    International Nuclear Information System (INIS)

    Nano iron oxo-hydroxides have numerous and increasing applications in biology and medicine. Assessment of their uptake and toxicity often requires cell culture but maintaining iron oxides uniformly nano-dispersed in such conditions can be challenging. We describe a balanced salt solution (BSS) compatible with cellular assays for use in such investigations. We determined hydrodynamic particle size and dispersibility of nano iron in BSS. The BSS, containing 130 mM NaCl, 10 mM KCl, 1 mM MgSO4, 5 mM glucose and 1.8 mM CaCl2 in 10 mM PIPES buffer (pH 7.4), was capable of maintaining nanoparticulate tartrate-modified Fe(III) oxo-hydroxide (i.e. nano Fe) mono-disperse (≥95% nanoparticulate) with a mean hydrodynamic particle diameter of 6.1 ± 0.3 nm. This size was similar to the native form of the nano Fe material (i.e. as synthesized) with a mean hydrodynamic particle diameter of 4.3 ± 0.1 nm in water. Furthermore, we show that BSS also adequately maintains nano Fe dispersion when supplemented with inhibitors of particle uptake or lysosomal acidification, namely chloropromazine and monensin, and when used at pHs 6.5 or 5.8. In conclusion, we provide a method for nanodispersion of iron oxo-hydroxides that is suitable for short term (1–3 h) cellular exposure investigations. (paper)

  1. Microsatellite instability, KRAS mutations and cellular distribution of TRAIL-receptors in early stage colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Lydia Kriegl

    Full Text Available BACKGROUND: The fact that the receptors for the TNF-related apoptosis inducing ligand (TRAIL are almost invariably expressed in colorectal cancer (CRC represents the rationale for the employment of TRAIL-receptors targeting compounds for the therapy of patients affected by this tumor. Yet, first reports on the use of these bioactive agents provided disappointing results. We therefore hypothesized that loss of membrane-bound TRAIL-R might be a feature of some CRC and that the evaluation of membrane staining rather than that of the overall expression of TRAIL-R might predict the response to TRAIL-R targeting compounds in this tumor. AIM AND METHODS: Thus, we evaluated the immunofluorescence pattern of TRAIL-receptors and E-cadherin to assess the fraction of membrane-bound TRAIL-receptors in 231 selected patients with early-stage CRC undergoing surgical treatment only. Moreover, we investigated whether membrane staining for TRAIL-receptors as well as the presence of KRAS mutations or of microsatellite instability (MSI had an effect on survival and thus a prognostic effect. RESULTS: As expected, almost all CRC samples stained positive for TRAIL-R1 and 2. Instead, membrane staining for these receptors was positive in only 71% and 16% of samples respectively. No correlation between KRAS mutation status or MSI-phenotype and prognosis could be detected. TRAIL-R1 staining intensity correlated with survival in univariate analysis, but only membranous staining of TRAIL-R1 and TRAIL-R2 on cell membranes was an independent predictor of survival (cox multivariate analysis: TRAIL-R1: p = 0.019, RR 2.06[1.12-3.77]; TRAIL-R2: p = 0.033, RR 3.63[1.11-11.84]. CONCLUSIONS: In contrast to the current assumptions, loss of membrane staining for TRAIL-receptors is a common feature of early stage CRC which supersedes the prognostic significance of their staining intensity. Failure to achieve therapeutic effects in recent clinical trials using TRAIL-receptors targeting

  2. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

    Institute of Scientific and Technical Information of China (English)

    Jian-yong WANG; Yang-shun GU; Jing WANG; Yi TONG; Ying WANG; Jun-bing SHAO; Ming QI

    2008-01-01

    Objective:Leber's hereditary optic neuropathY (LHON)is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA(mtDNA).Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing.This study aims to develop a minor groove binder(MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction(PCR).Methods:Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation,with 20 normal individuals as a control group at the same time.A real-time PCR involving two MGB probes was used to detect the mtDNA 11778 mutation and heteroplasmy.A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones.Results:All 48 LHON patients and their matemal relatives were positive for mtDNA 11778 mutation in our assay,27 heteroplasmic and 21 homoplasmic.Eighteen cases did not show an occurrence of the disease,while 9 developed the disease among the 27 heteroplasmic mutation cases.Eleven did not show an occurrence of the disease,while 10 cases developed the disease among 21 homoplasmic mutation cases.There was a significant difierence in the incidence between the heteroplasmic and the homoplasmic mutation types.The time needed for running a real-time PCR assay was only 80 min.Conclusion:This real-time PCR assay is a rapid,reliable method for mtDNA mutation detection as well as heteroplasmy quantification.Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

  3. An extended set of yeast-based functional assays accurately identifies human disease mutations

    OpenAIRE

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L.; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E.; Vidal, Marc

    2016-01-01

    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (...

  4. A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice

    International Nuclear Information System (INIS)

    Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as ''mean ratio'') was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group)

  5. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    DEFF Research Database (Denmark)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik; Wu, Lin; Wei, Wen; Tsai, Julie; Khalil, Azza; Nexo, Ebba; Sorensen, Boe S

    2014-01-01

    mutations in exons 18-21 of the EGFR gene, employing the cobas(®) EGFR Tissue Test and cobas(®) EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA). RESULTS: Test results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were......BACKGROUND: Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma...... samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence of...

  6. NGS-Based Assay for the Identification of Individuals Carrying Recessive Genetic Mutations in Reproductive Medicine.

    Science.gov (United States)

    Abulí, Anna; Boada, Montserrat; Rodríguez-Santiago, Benjamín; Coroleu, Buenaventura; Veiga, Anna; Armengol, Lluís; Barri, Pedro N; Pérez-Jurado, Luis A; Estivill, Xavier

    2016-06-01

    Next-generation sequencing (NGS) has the capacity of carrier screening in gamete donation (GD) programs. We have developed and validated an NGS carrier-screening test (qCarrier test) that includes 200 genes associated with 368 disorders (277 autosomal recessive and 37 X-linked). Carrier screening is performed on oocyte donation candidates and the male partner of oocyte recipient. Carriers of X-linked conditions are excluded from the GD program, whereas donors are chosen who do not carry mutations for the same gene/disease as the recipients. The validation phase showed a high sensitivity (>99% sensitivity) detecting all single-nucleotide variants, 13 indels, and 25 copy-number variants included in the validation set. A total of 1,301 individuals were analysed with the qCarrier test, including 483 candidate oocyte donors and 635 receptor couples, 105 females receiving sperm donation, and 39 couples seeking pregnancy. We identified 56% of individuals who are carriers for at least one genetic condition and 1.7% of female donors who were excluded from the program due to a carrier state of X-linked conditions. Globally, 3% of a priori assigned donations had a high reproductive risk that could be minimized after testing. Genetic counselling at different stages is essential for helping to facilitate a successful and healthy pregnancy. PMID:26990548

  7. Antioxidant activity of proanthocyanidins-rich fractions from Choerospondias axillaris peels using a combination of chemical-based methods and cellular-based assay.

    Science.gov (United States)

    Li, Qian; Wang, Xieyi; Chen, Jun; Liu, Chengmei; Li, Ti; McClements, David Julian; Dai, Taotao; Liu, Jiyan

    2016-10-01

    An extract isolated from Choerospondias axillaris peels was separated into five fractions using size-exclusion chromatography. The structural composition and mean degree of polymerization (mDP) of these fractions were then characterized by acid-catalysis followed by HPLC analysis. The antioxidant activity of each fraction was determined using a combination of chemical-based methods (DPPH, ABTS(+) radical scavenging activity, ferric-reducing antioxidant power, and phosphomolybdate assay) and a cellular-based assay. All fractions tested were found to have high total phenolics contents and were rich in proanthocyanidins. The mDP of fractions (F1-F5) ranged from 1.92 to 9.25. When tested by the chemical-based assays, the antioxidant activity of the fractions did not depend on molecular weight of the phenolics. Conversely, when tested by the cellular-based assay the antioxidant activity actually decreased with increasing molecular weight of the proanthocyanidins. These experiments highlight the limitations of using chemical-based assays to establish the antioxidant activity of proanthocyanidins within biological systems. PMID:27132855

  8. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    International Nuclear Information System (INIS)

    Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma samples with allele-specific PCR assays. Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence of mutations in exons 18–21 of the EGFR gene, employing the cobas® EGFR Tissue Test and cobas® EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA). Test results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue was 179/196 (91%) (kappa value: 0.621). Mutational analysis of the EGFR gene in plasma samples is feasible with allele-specific PCR assays and represents a non-invasive supplement to biopsy analysis. M-20080012 from March 10, 2008 and reported to ClinicalTrials.gov: http://www.clinicaltrials.gov/ct2/show/NCT00815971

  9. Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

    Directory of Open Access Journals (Sweden)

    R Singhal

    2015-01-01

    Full Text Available Purpose: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT. GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF and katG and inhA genes for isoniazid (INH were analyzed. Results: A total of 366 (16.8% MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0% and for INH was S315T1 (88.3%. Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

  10. Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO).

    Science.gov (United States)

    Winkler, Dirk; Beconi, Maria; Toledo-Sherman, Leticia M; Prime, Michael; Ebneth, Andreas; Dominguez, Celia; Muñoz-Sanjuan, Ignacio

    2013-09-01

    Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells. PMID:23690293

  11. Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

    Science.gov (United States)

    Kessler, Jan H; Mommaas, Bregje; Mutis, Tuna; Huijbers, Ivo; Vissers, Debby; Benckhuijsen, Willemien E; Schreuder, Geziena M Th; Offringa, Rienk; Goulmy, Els; Melief, Cornelis J M; van der Burg, Sjoerd H; Drijfhout, Jan W

    2003-02-01

    We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules. PMID:12559627

  12. Germ Line Transmission of the Cdk4R24C Mutation Facilitates Tumorigenesis and Escape from Cellular Senescence

    OpenAIRE

    Rane, Sushil G; Cosenza, Stephen C.; Mettus, Richard V.; Reddy, E. Premkumar

    2002-01-01

    Mutations in CDK4 and its key kinase inhibitor p16INK4a have been implicated in the genesis and progression of familial human melanoma. The importance of the CDK4 locus in human cancer first became evident following the identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16INK4a. To determine the role of the Cdk4R24C germ line mutation in the genesis of other cancer types, we introduced the R24C mutation in the Cdk4 locus of mice by usi...

  13. Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

    OpenAIRE

    R Singhal; Myneedu, V. P.; J Arora; Singh, N; Bhalla, M; Verma, A.; Sarin, R

    2015-01-01

    Purpose: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosi...

  14. Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies▿ †

    OpenAIRE

    Lalonde, Matthew S.; Troyer, Ryan M.; Syed, Aslam R.; Bulime, Stanley; Demers, Korey; Bajunirwe, Francis; Arts, Eric J

    2007-01-01

    This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVP-resistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for e...

  15. Expression of cellular FLICE-inhibitory protein and its association with p53 mutation in colon cancer

    Institute of Scientific and Technical Information of China (English)

    Xiao-Dong Zhou; Jie-Ping Yu; Hong-Xia Chen; Hong-Gang Yu; He-Sheng Luo

    2005-01-01

    AIM: To investigate the expression of cellular FLICE (Fas associated death domain-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) and its association with p53mutation in colon cancer.METHODS: Immunohistochemical staining of c-FLIP and mutant p53 by using specific antibodies was performed by the standard streptavidin-peroxidase technique for 45 colon cancer tissue samples with matched normal tissues. Semi-quantitative reverse transcriptional (RT)-PCR was used to measure c-FLIP mRNA levels. t-test statistical method was used in data analyses.RESULTS: c-FLIP mRNA was expressed in all colon cancer tissues and its level (0.63±0.12) was significantly higher than that in normal tissues (0.38±0.10, P<0.01). Immunohistochemically, c-FLIP protein was also expressed in all colon cancers (45/45) and 71.1% (32/45) showed an intense immunostaining, in contrast, 93.3% (42/45) of normal colonic mucosa showed positive staining and none of them immunostained intensely. The quantity of c-FLIP protein was significantly higher in cancer tissues than in normal mucosa (7.04±1.20 vs 5.21±0.86, P<0.01).Positive staining of mutant p53 protein was found in 60%(27/45) colon cancers. c-FLIP mRNA level was decreased in p53 positive group compared with p53 negative cancer tissues (0.59±0.13 vs 0.69±0.14, P<0.01), but c-FLIP protein had a significantly higher level in p53 positive cancer tissues than in negative ones (7.57±1.30 vs 6.25±1.27,P<0.01).CONCLUSION: c-FLIP is specially overexpressed in colon cancers and it might contribute to carcinogenesis of normal colonic mucosa. p53 may exert transcriptional upregulation effects on c-FLIP gene and more potent effects on promoting the degradation of c-FLIP protein.

  16. Prognostic value of IDH1 mutations identified with PCR-RFLP assay in acute myeloid leukemia patients

    International Nuclear Information System (INIS)

    Background: Somatic mutations in isocitrate dehydrogenase 1 (1DH1) gene occur frequently in primary brain tumors. Recently theses mutations were demonstrated in acute myeloid leukemia (AML). So far, assessment of these mutations relied on the DNA sequencing technique. Aim of the work: The aim of this study was to detect somatic mutations in IDH1 gene using mismatched primers suitable for endonuclease based detection, without the need for DNA sequencing, and to estimate its prognostic value, on patients with de novo AML. Methods: Residual DNA extracted from pretreatment bone marrow (BM) samples of 100 patients with de novo AML was used. The polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was adapted to IDHl gene, codon 132 mutations screening. Results: The frequency of IDH1 mutations was 13%. In the non-acute promyelocytic leukemia group (non-APL), IDH1 mutations were significantly associated with FLT3-ITD negative patients (p = 0.03). Patients with 1DH1 mutations did not achieve complete remission (CR). There was a trend for shorter overall survival (OS) in patients with IDH1 mutation compared to those with wild type (p = 0.08). Conclusion: IDH1 mutations are recurring genetic alterations in AML and they may have unfavorable impact on clinical outcome in adult AML. The PCR-RFLP method allows for a fast, inexpensive, and sensitive method for the detection of IDF11 mutations in AML.

  17. A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in Salmonella enterica serovars Typhi and Paratyphi A.

    LENUS (Irish Health Repository)

    Song, Yajun

    2010-08-01

    OBJECTIVES: Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers. METHODS: By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (Nal(R)) and\\/or decreased susceptibility to fluoroquinolones. RESULTS: This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (Nal(R) = 223 and Nal(S) = 69) and 106 isolates of Salmonella Paratyphi A (Nal(R) = 24 and Nal(S) = 82). All of the 247 Nal(R) Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143\\/223 for Salmonella Typhi and 18\\/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight Nal(S) Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies. CONCLUSIONS: The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes.

  18. International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials.

    Science.gov (United States)

    Galipeau, Jacques; Krampera, Mauro; Barrett, John; Dazzi, Francesco; Deans, Robert J; DeBruijn, Joost; Dominici, Massimo; Fibbe, Willem E; Gee, Adrian P; Gimble, Jeffery M; Hematti, Peiman; Koh, Mickey B C; LeBlanc, Katarina; Martin, Ivan; McNiece, Ian K; Mendicino, Michael; Oh, Steve; Ortiz, Luis; Phinney, Donald G; Planat, Valerie; Shi, Yufang; Stroncek, David F; Viswanathan, Sowmya; Weiss, Daniel J; Sensebe, Luc

    2016-02-01

    Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an "open-access" manner, such as through publication or database collection. PMID:26724220

  19. Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay

    OpenAIRE

    Kovvali, Gopala K.; Mehta, Bena; Epstein, Charles B.; Lutzker, Stuart G.

    2001-01-01

    Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that ‘hot-spot’ p53 mutants (which comprise ∼30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have m...

  20. Dissection of the Critical Binding Determinants of Cellular Retinoic Acid Binding Protein II by Mutagenesis and Fluorescence Binding Assay

    OpenAIRE

    Vasileiou, Chrysoula; Lee, Kin Sing Stephen; Crist, Rachael M.; Vaezeslami, Soheila; Goins, Sarah M.; Geiger, James H.; Borhan, Babak

    2009-01-01

    The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by ...

  1. Malignant monoblasts can function as effector cells in natural killer cell and antibody-dependent cellular cytotoxicity assays

    DEFF Research Database (Denmark)

    Hokland, P; Hokland, M; Ellegaard, J

    1981-01-01

    This is the first report describing natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) of malignant monoblasts. Pure acute monoblastic leukemia was diagnosed in bone marrow aspirations from two patients by use of conventional cytochemical methods as well as multiple immunologic...... modulation was seen in ADCC. These findings are discussed in the light of our present knowledge of lymphoid NK cells. Udgivelsesdato: 1981-May...

  2. Mutation Analysis of the Common Deafness Genes in Patients with Nonsyndromic Hearing Loss in Linyi by SNPscan Assay

    Science.gov (United States)

    Zhang, Fengguo; Xu, Lei; Zhang, Xue; Zhang, Guodong; Li, Jianfeng; Lv, Huaiqing; Bai, Xiaohui; Wang, Haibo

    2016-01-01

    Hearing loss is a common sensory disorder, and at least 50% of cases are due to a genetic etiology. Although hundreds of genes have been reported to be associated with nonsyndromic hearing loss, GJB2, SLC26A4, and mtDNA12SrRNA are the major contributors. However, the mutation spectrum of these common deafness genes varies among different ethnic groups. The present work summarized mutations in these three genes and their prevalence in 339 patients with nonsyndromic hearing loss at three different special education schools and one children's hospital in Linyi, China. A new multiplex genetic screening system “SNPscan assay” was employed to detect a total of 115 mutations of the above three genes. Finally, 48.67% of the patients were identified with hereditary hearing loss caused by mutations in GJB2, SLC26A4, and mtDNA12SrRNA. The carrying rate of mutations in the three genes was 37.76%, 19.75%, and 4.72%, respectively. This mutation profile in our study is distinct from other parts of China, with high mutation rate of GJB2 suggesting a unique mutation spectrum in this area. PMID:27247933

  3. A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

    KAUST Repository

    Aouida, Mustapha

    2013-06-07

    Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides\\' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to

  4. A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

    Directory of Open Access Journals (Sweden)

    Mustapha Aouida

    Full Text Available Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p. The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA signaling and AMP activated protein kinase (AMPK phosphorylation in S. cerevisiae, which are

  5. Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes.

    Science.gov (United States)

    Twigg, Stephen R F; Babbs, Christian; van den Elzen, Marijke E P; Goriely, Anne; Taylor, Stephen; McGowan, Simon J; Giannoulatou, Eleni; Lonie, Lorne; Ragoussis, Jiannis; Sadighi Akha, Elham; Knight, Samantha J L; Zechi-Ceide, Roseli M; Hoogeboom, Jeannette A M; Pober, Barbara R; Toriello, Helga V; Wall, Steven A; Rita Passos-Bueno, M; Brunner, Han G; Mathijssen, Irene M J; Wilkie, Andrew O M

    2013-04-15

    Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females. PMID:23335590

  6. Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

    Science.gov (United States)

    Twigg, Stephen R.F.; Babbs, Christian; van den Elzen, Marijke E.P.; Goriely, Anne; Taylor, Stephen; McGowan, Simon J.; Giannoulatou, Eleni; Lonie, Lorne; Ragoussis, Jiannis; Akha, Elham Sadighi; Knight, Samantha J.L.; Zechi-Ceide, Roseli M.; Hoogeboom, Jeannette A.M.; Pober, Barbara R.; Toriello, Helga V.; Wall, Steven A.; Rita Passos-Bueno, M.; Brunner, Han G.; Mathijssen, Irene M.J.; Wilkie, Andrew O.M.

    2013-01-01

    Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries—a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5′ untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5′ UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females. PMID:23335590

  7. Detection of knockdown resistance (kdr mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    Directory of Open Access Journals (Sweden)

    Ball Amanda

    2007-08-01

    Full Text Available Abstract Background Knockdown resistance (kdr is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1 TaqMan probes and 2 high resolution melt (HRM analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR, Heated Oligonucleotide Ligation Assay (HOLA, Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost, and safety (requirement for hazardous chemicals. Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions and the most specific (with the lowest number of incorrect scores. Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS

  8. Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

    OpenAIRE

    Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

    2013-01-01

    We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutati...

  9. "Sickle Cell Anemia: Tracking down a Mutation": An Interactive Learning Laboratory That Communicates Basic Principles of Genetics and Cellular Biology

    Science.gov (United States)

    Jarrett, Kevin; Williams, Mary; Horn, Spencer; Radford, David; Wyss, J. Michael

    2016-01-01

    "Sickle cell anemia: tracking down a mutation" is a full-day, inquiry-based, biology experience for high school students enrolled in genetics or advanced biology courses. In the experience, students use restriction endonuclease digestion, cellulose acetate gel electrophoresis, and microscopy to discover which of three putative patients…

  10. Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay

    Science.gov (United States)

    Chi, Kai-Hua; Pillay, Allan; Nachamkin, Eli; Su, John R.; Ballard, Ronald C.

    2013-01-01

    Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum. PMID:23284026

  11. Development of a novel PTT assay for mutation detection in PALB2 large exons and PALB2 screening in medullary breast cancer.

    Science.gov (United States)

    Poumpouridou, Nikoleta; Goutas, Nikolaos; Tsionou, Christina; Dimas, Kleanthi; Lianidou, Evi; Kroupis, Christos

    2016-04-01

    Beyond BRCA1 and BRCA2 genes, PALB2 (Partner and localizer of BRCA2) emerges as the third breast cancer susceptibility gene due to its role in the same DNA repair pathway: homologous recombination. In most populations studied so far, PALB2 mutations are detected in 1-2% of BRCA negative female patients. PALB2 gene contains 13 exons; exons 4 and 5 consist 65% of the coding area. We developed a protein truncation test (PTT) for quick screening of truncating pathogenic mutations of these two large exons. Specific primers were de novo, in silico designed and the PTT-PCR products were translated in the presence of biotinylated lysine and detected colorimetrically. The assay was initially tested in 30 patients with hereditary breast cancer, negative for BRCA mutations and then, in 17 patients with the rare medullary breast cancer subtype. Small PALB2 exons were screened with high-resolution melting curve analysis (HRMA) and the large DNA rearrangements with multiplex ligation-dependent probe amplification (MLPA). Any alterations detected were verified by Sanger DNA Sequencing. The developed PTT methodology is highly specific for clinical significant mutations; positive control samples that produce truncated PALB2 peptides were correctly identified and the method was accurate when compared to DNA sequencing. We did not detect any deleterious PALB2 mutation in both groups of patients. HRMA and MLPA were also negative for all tested samples. However, our novel, fast and cost-effective PTT method for pathogenic mutation detection of the two large PALB2 exons can be applied in screening of a large number of breast cancer patients. PMID:26573693

  12. Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Mohamed-Yassine eAMAROUCH

    2015-02-01

    Full Text Available Voltage-gated sodium channels (Nav are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the voltage-gated sodium channels. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the voltage-gated sodium channels by shifting the voltage-dependence of steady state activation towards more negative potentials.

  13. Human reporter gene assays: Transcriptional activity of the androgen receptor is modulated by the cellular environment and promoter context

    International Nuclear Information System (INIS)

    The androgen receptor (AR) is a member of the nuclear receptor superfamily and mediates the physiological effects of androgens. Androgens are essential for male development and disruption of androgen signaling may cause androgen-dependent developmental defects and/or tumors. Here we present a comparative analysis of various model systems for the investigation of endocrine active compounds in human cell lines. We generated reporter plasmids containing androgen response elements derived from the human secretory component or the rat probasin genes as well as the glucocorticoid consensus response element and compared their activities to that of the mouse mammary tumor virus promotor. Additionally, we generated an expression plasmid containing the AR cDNA derived from LNCaP cells. In 22Rv1 cells transiently transfected with human AR, all reporters displayed a dose-dependent, high activity when treated with androgens. Interestingly, the potency of testosterone and its metabolite dihydrotestosterone was very low in HepG2 but not in 22Rv1 cells, independent of the reporter used. The efficacies of the androgens tested were comparable in both cell lines but highly dependent on the reporter used. Based on these results, 22Rv1 cells provide a highly sensitive in vitro test system to analyze endocrine activities of xenobiotics. Furthermore, this study highlights the need to investigate the (anti-) androgenic activity of compounds in dependence of the cellular and promoter context

  14. Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection

    International Nuclear Information System (INIS)

    The molecular characterization of tumors using next generation sequencing (NGS) is an emerging diagnostic tool that is quickly becoming an integral part of clinical decision making. Cancer genomic profiling involves significant challenges including DNA quality and quantity, tumor heterogeneity, and the need to detect a wide variety of complex genetic mutations. Most available comprehensive diagnostic tests rely on primer based amplification or probe based capture methods coupled with NGS to detect hotspot mutation sites or whole regions implicated in disease. These tumor panels utilize highly customized bioinformatics pipelines to perform the difficult task of accurately calling cancer relevant alterations such as single nucleotide variations, small indels or large genomic alterations from the NGS data. In this review, we will discuss the challenges of solid tumor assay design/analysis and report a case study that highlights the need to include complementary technologies (i.e., arrays) and germline analysis in tumor testing to reliably identify copy number alterations and actionable variants

  15. Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Phillip N., E-mail: pgray@ambrygen.com; Dunlop, Charles L.M.; Elliott, Aaron M. [Ambry Genetics, 15 Argonaut, Aliso Viejo, CA 92656 (United States)

    2015-07-17

    The molecular characterization of tumors using next generation sequencing (NGS) is an emerging diagnostic tool that is quickly becoming an integral part of clinical decision making. Cancer genomic profiling involves significant challenges including DNA quality and quantity, tumor heterogeneity, and the need to detect a wide variety of complex genetic mutations. Most available comprehensive diagnostic tests rely on primer based amplification or probe based capture methods coupled with NGS to detect hotspot mutation sites or whole regions implicated in disease. These tumor panels utilize highly customized bioinformatics pipelines to perform the difficult task of accurately calling cancer relevant alterations such as single nucleotide variations, small indels or large genomic alterations from the NGS data. In this review, we will discuss the challenges of solid tumor assay design/analysis and report a case study that highlights the need to include complementary technologies (i.e., arrays) and germline analysis in tumor testing to reliably identify copy number alterations and actionable variants.

  16. Mutation of the Sensor Kinase chvG in Rhizobium leguminosarum Negatively Impacts Cellular Metabolism, Outer Membrane Stability, and Symbiosis

    OpenAIRE

    Vanderlinde, Elizabeth M.; Yost, Christopher K.

    2012-01-01

    Two-component signal transduction systems (TCS) are a main strategy used by bacteria to sense and adapt to changes in their environment. In the legume symbiont Rhizobium leguminosarum biovar viciae VF39, mutation of chvG, a histidine kinase, caused a number of pleiotropic phenotypes. ChvG mutants are unable to grow on proline, glutamate, histidine, or arginine as the sole carbon source. The chvG mutant secreted smaller amounts of acidic and neutral surface polysaccharides and accumulated abno...

  17. Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori ▿ †

    OpenAIRE

    Burucoa, Christophe; Garnier, Martine; Silvain, Christine; Fauchère, Jean-Louis

    2008-01-01

    We developed a single-vessel multiplex real-time PCR assay that detects Helicobacter pylori infection and identified the four existing alleles of the 23S rRNA genes of H. pylori—the wild-type sequence and the three mutations conferring clarithromycin resistance—using allele-specific Scorpion primers directly on biopsy specimens. The Scorpion primers combine a primer and a probe in a single molecule and are able to distinguish single-nucleotide polymorphism. Fluorescent signals, produced when ...

  18. Detection of single nucleotide polymorphisms in p53 mutation hotspots and expression of mutant p53 in human cell lines using an enzyme-linked electrochemical assay

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Šimková, Eva; Vychodilová, Zdenka; Brázdová, Marie; Fojta, Miroslav

    2009-01-01

    Roč. 21, č. 15 (2009), s. 1723-1729. ISSN 1040-0397 R&D Projects: GA ČR(CZ) GA203/07/1195; GA AV ČR(CZ) IAA400040901; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : enzyme-linked electrochemical assay * SNP typing * p53 mutation Subject RIV: AQ - Safety, Health Protection, Human - Machine Impact factor: 2.630, year: 2009

  19. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Sirirat Seekhuntod

    Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

  20. A novel quantitative hemolytic assay coupled with restriction fragment length polymorphisms analysis enabled early diagnosis of atypical hemolytic uremic syndrome and identified unique predisposing mutations in Japan.

    Directory of Open Access Journals (Sweden)

    Yoko Yoshida

    Full Text Available For thrombotic microangiopathies (TMAs, the diagnosis of atypical hemolytic uremic syndrome (aHUS is made by ruling out Shiga toxin-producing Escherichia coli (STEC-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP, often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45 had moderate-to-severe (≥50% hemolysis, whereas the remaining 76% (34/45 patients had mild or no hemolysis (<50%. The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34, which were identified by restriction fragment length polymorphism (RFLP analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45 of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients.

  1. Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis

    Science.gov (United States)

    Rahman, Arfatur; Sahrin, Mahfuza; Afrin, Sadia; Earley, Keith; Ahmed, Shahriar; Rahman, S. M. Mazidur; Banu, Sayera

    2016-01-01

    Background GeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding. Methods We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing. Results The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases. Conclusion Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis. PMID:27054344

  2. Two novel nonradioactive polymerase chain reaction-based assays of dried blood spots, genomic DNA, or whole cells for fast, reliable detection of Z and S mutations in the alpha 1-antitrypsin gene

    DEFF Research Database (Denmark)

    Andresen, B S; Knudsen, I; Jensen, P K;

    1992-01-01

    Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples...... from individuals who are normal, heterozygous, or homozygous for the mutations. We show that the two assays can be performed with purified genomic DNA as well as with boiled blood spots. The new assays were validated by parallel testing with a technique in which PCR is combined with allele......-specific oligonucleotide (ASO) probes. In all cases tested the results obtained by the different techniques were in accordance. The new assays can be used for prenatal diagnostics and can be performed directly with boiled tissue samples. Because the new assays are easy to perform and reliable, we conclude that they are...

  3. Mutations in the rpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Isolates in Spain and Their Rapid Detection by PCR–Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Garcia, Lucia; Alonso-Sanz, Mercedes; Rebollo, Maria J.; Tercero, Juan C.; Chaves, Fernando

    2001-01-01

    Genetic alterations in the rpoB gene were characterized in 50 rifampin-resistant (Rifr) clinical isolates of Mycobacterium tuberculosis complex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of rpoB mutations was evaluated with isolates of the M. tuberculosis complex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 different rpoB mutations among the Rifr isolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rifr allele within each strain, 10 of the 11 Rifr genotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of M. tuberculosis complex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rifr organisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods. PMID:11325996

  4. Mutations in human C2CD3 cause skeletal dysplasia and provide new insights into phenotypic and cellular consequences of altered C2CD3 function.

    Science.gov (United States)

    Cortés, Claudio R; McInerney-Leo, Aideen M; Vogel, Ida; Rondón Galeano, Maria C; Leo, Paul J; Harris, Jessica E; Anderson, Lisa K; Keith, Patricia A; Brown, Matthew A; Ramsing, Mette; Duncan, Emma L; Zankl, Andreas; Wicking, Carol

    2016-01-01

    Ciliopathies are a group of genetic disorders caused by defective assembly or dysfunction of the primary cilium, a microtubule-based cellular organelle that plays a key role in developmental signalling. Ciliopathies are clinically grouped in a large number of overlapping disorders, including the orofaciodigital syndromes (OFDS), the short rib polydactyly syndromes and Jeune asphyxiating thoracic dystrophy. Recently, mutations in the gene encoding the centriolar protein C2CD3 have been described in two families with a new sub-type of OFDS (OFD14), with microcephaly and cerebral malformations. Here we describe a third family with novel compound heterozygous C2CD3 mutations in two fetuses with a different clinical presentation, dominated by skeletal dysplasia with no microcephaly. Analysis of fibroblast cultures derived from one of these fetuses revealed a reduced ability to form cilia, consistent with previous studies in C2cd3-mutant mouse and chicken cells. More detailed analyses support a role for C2CD3 in basal body maturation; but in contrast to previous mouse studies the normal recruitment of the distal appendage protein CEP164 suggests that this protein is not sufficient for efficient basal body maturation and subsequent axonemal extension in a C2CD3-defective background. PMID:27094867

  5. WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations

    Directory of Open Access Journals (Sweden)

    Assawamakin Anunchai

    2007-08-01

    Full Text Available Abstract Background Allele-specific (AS Polymerase Chain Reaction is a convenient and inexpensive method for genotyping Single Nucleotide Polymorphisms (SNPs and mutations. It is applied in many recent studies including population genetics, molecular genetics and pharmacogenomics. Using known AS primer design tools to create primers leads to cumbersome process to inexperience users since information about SNP/mutation must be acquired from public databases prior to the design. Furthermore, most of these tools do not offer the mismatch enhancement to designed primers. The available web applications do not provide user-friendly graphical input interface and intuitive visualization of their primer results. Results This work presents a web-based AS primer design application called WASP. This tool can efficiently design AS primers for human SNPs as well as mutations. To assist scientists with collecting necessary information about target polymorphisms, this tool provides a local SNP database containing over 10 million SNPs of various populations from public domain databases, namely NCBI dbSNP, HapMap and JSNP respectively. This database is tightly integrated with the tool so that users can perform the design for existing SNPs without going off the site. To guarantee specificity of AS primers, the proposed system incorporates a primer specificity enhancement technique widely used in experiment protocol. In particular, WASP makes use of different destabilizing effects by introducing one deliberate 'mismatch' at the penultimate (second to last of the 3'-end base of AS primers to improve the resulting AS primers. Furthermore, WASP offers graphical user interface through scalable vector graphic (SVG draw that allow users to select SNPs and graphically visualize designed primers and their conditions. Conclusion WASP offers a tool for designing AS primers for both SNPs and mutations. By integrating the database for known SNPs (using gene ID or rs number

  6. Responses of the L5178Y tk/sup +//tk/sup -/ mouse lymphoma cell forward mutation assay. II. 18 coded chemicals

    Energy Technology Data Exchange (ETDEWEB)

    McGregor, D.B.; Brown, A.; Cattanach, P.; Edwards, I.; McBride, D.; Caspary, W.J.

    1988-01-01

    Eighteen chemicals were tested for their mutagenic potential in the L5178Y tk/sup +///sup -/ mouse lymphoma cell forward mutation assay by the use of procedures based upon those described previously. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 ..mu..g/ml. The chemicals were tested at least twice. Significant responses were obtained with benzofuran, benzyl chloride, bromodichloromethane, butylated hydroxytoluene, chlorendic acid, o-chlorobenzalmalonitrile, 1,2,3,4-diepoxybutane, dimethyl formamide, dimethyl hydrogen phosphite, furfural, glutaraldehyde, hydroquinone, 8-hydroxyquinoline, and resorcinol. Apart from bromodichloromethane, butylated hydroxytoluene and dimethyl hydrogen phosphite, rat liver S9 mix was not a requirement for the activity of any of these compounds. Chemicals not identified as mutagens were water, tert-butyl alcohol, pyridine, and witch hazel.

  7. Biological efficiency of the Brookhaven Medical Research Reactor mixed neutron beam estimated from gene mutations in Tradescantia stamen hair cells assay

    International Nuclear Information System (INIS)

    The relative biological effectiveness (RBE) of low energy neutrons for the induction of various abnormalities in Tradescantia stamen hair mutation (Trad-SH) assay was studied using two clones (T-4430 and T-02), heterozygous for flower color. Dose response relationship for gene mutations induced in somatic cells of Trad-SH were investigated after irradiation with a mixed neutron beam of the Brookhaven Medical Research Reactor (BMRR), currently used in a clinical trial of boron neutron capture therapy (BNCT) for glioblastoma. To establish the RBE of the BMRR beam in the induction of various biological end-points in Tradescantia, irradiation with various doses of γ-rays was also performed. After irradiation all plants were cultivated several days at Brookhaven National Laboratory (BNL), then transported to Poland for screening the biological end-points. Due to the post-exposure treatment, all plants showed high levels of lethal events and alteration of the cell cycle. Plants of clone 4430 were more reactive to post-treatment conditions, resulting in decreased blooming efficiency that affected the statistics. Slope coefficients estimated from the dose response curves for gene mutation frequencies allowed the evaluation of ranges for the maximal RBE values of the applied beam vs. γ rays as 6.0 and 5.4 for the cells of T-02 and T-4430, respectively. Estimated fraction of doses from neutrons and corresponding biological effects for the clones T-02 and T-4430 allowed to evaluate the RBE values for neutrons part in the beam as 32.3 and 45.4, respectively. (author)

  8. Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer

    International Nuclear Information System (INIS)

    Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. The assay’s limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test

  9. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma

    Science.gov (United States)

    Harlé, Alexandre; Salleron, Julia; Franczak, Claire; Dubois, Cindy; Filhine-Tressarieu, Pierre; Leroux, Agnès; Merlin, Jean-Louis

    2016-01-01

    Background Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies. Methods Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM) PCR, Real-time allele-specific amplification (RT-ASA) PCR, Next generation sequencing (NGS), immunohistochemistry (IHC) and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays. Results BRAF mutations were found in 28(47.5%), 29(49.2%), 31(52.5%), 29(49.2%) and 27(45.8%) samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2%) samples were found bearing a c.1799T>A (p.Val600Glu) mutation, three (9.4%) with a c.1798_1799delinsAA (p.Val600Lys) mutation and one with c.1789_1790delinsTC (p.Leu597Ser) mutation. Two samples were found bearing complex mutations. Conclusions HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays. PMID:27111917

  10. Chemical analysis and mutational assay of distilled oils from the H-coal direct liquefaction process: a status report

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, B.W.; Later, D.W.; Wright, C.W.; Stewart, D.L.

    1985-01-01

    Samples from the H-Coal process, a catalytic, single-stage, coal liquefaction technology, were chemically characterized and screened for microbial mutagenicity. For these investigations, a blend of light and heavy H-Coal process oils was fractionally distilled into 50/sup 0/F boiling point cuts. The chemical analyses and biological testing results presented in this status report deal primarily with the blended material and the distillate fractions boiling above 650/sup 0/F. Results from the microbial mutagenicity assays indicated that onset of biological activity in the crude materials occurred above 700/sup 0/F. Similar trends have been observed for Solvent Refined Coal (SRC) I, SRC II, Integrated Two-Stage Liquefaction (ITSL) and Exxon EDS process materials. After chemical class fractionation, the primary source of microbial mutagenicity of the crude boiling point cuts was the nitrogen-containing polycyclic aromatic compound (N-PAC) fractions. Amino polycyclic aromatic hydrocarbons (amino-PAH) were present at sufficient concentration levels in the N-PAC fractions to account for the observed mutagenic responses. In general, the chemical composition of the H-Coal materials studied was similar to that of other single-stage liquefaction materials. The degree of alkylation in these materials was determined to be greater than in the SRC and less than in the EDS process distillate cuts. 13 references, 8 figures, 11 tables.

  11. Cellular-signaling pathways unveil the carcinogenic potential of chemicals.

    Science.gov (United States)

    Hendriks, Giel; van de Water, Bob; Schoonen, Willem; Vrieling, Harry

    2013-06-01

    Most of the current in vitro carcinogenicity assays assess the potential carcinogenic properties of chemicals through the detection of inflicted DNA damage or subsequent chromosome damage and gene mutations. Unfortunately, these assays generally do not provide mechanistic insight into the reactive properties of a chemical. Upon chemical-induced damage of biomolecules, molecular sensors will activate general and damage-specific cellular response pathways that provide protection against the (geno)toxic and potential carcinogenic properties of chemicals. These cellular defense mechanisms include activation of cell-cycle checkpoints, DNA repair systems and induction of apoptosis or necrosis. Visualization of activated cellular-signaling pathways forms a powerful means to readily detect the genotoxic potential of chemical compounds and simultaneously gain insight into their reactive properties. Over the past years, various in vitro reporter assays have been developed that monitor activation of general and more specific cellular-signaling pathways, including the GreenScreen HC and ToxTracker assays. In this review we provide a perspective on how we can exploit activation of cellular signaling pathways to shed light on the mode of action of the chemical exposure and to develop sophisticated mechanism-based in vitro assays for cancer risk assessment. PMID:23339022

  12. Melanomas Associated With Blue Nevi or Mimicking Cellular Blue Nevi: Clinical, Pathologic, and Molecular Study of 11 Cases Displaying a High Frequency of GNA11 Mutations, BAP1 Expression Loss, and a Predilection for the Scalp.

    Science.gov (United States)

    Costa, Sebastian; Byrne, Michelle; Pissaloux, Daniel; Haddad, Veronique; Paindavoine, Sandrine; Thomas, Luc; Aubin, Francois; Lesimple, Thierry; Grange, Florent; Bonniaud, Bertille; Mortier, Laurent; Mateus, Christine; Dreno, Brigitte; Balme, Brigitte; Vergier, Beatrice; de la Fouchardiere, Arnaud

    2016-03-01

    Melanomas associated with blue nevi (MABN) or mimicking cellular blue nevi (MMCBN) represent exceptional variants of malignant cutaneous melanocytic tumors. Uveal and leptomeningeal melanomas frequently have somatic mutations of GNAQ or GNA11, which are believed to be early driver mutations. In uveal melanomas, monosomy 3, linked to the BAP1 gene, is an adverse prognostic factor. We have studied the clinical, histologic, BAP1 expression profile, and molecular data of 11 cases of MABN/MMCBN and 24 cellular blue nevi. Most of the cases of MABN/MMCBN occurred on the scalps of adult patients and presented as rapidly growing nodules, typically >1 cm, often arising at the site of a preexisting melanocytic lesion. The MABN/MMCBN were composed of dense nests of large dermal atypical melanocytes, in some cases lying adjacent to a blue nevus. Four patients developed metastatic disease, and 2 died from their disease. A GNA11 mutation was found in 8/11 cases and a GNAQ mutation in 1 case. Seven of 11 cases showed loss of nuclear BAP1 immunohistochemical (IHC) expression in the malignant component, sparing the adjacent nevus. Array comparative genomic hybridization revealed recurrent deletions of chromosomes 1p, 3p, 4q, 6q, 8p, 16q, and 17q and recurrent gains of chromosomes 6p, 8q, and 21q. The 24 cases of cellular blue nevi frequently occurred on the sacrum, had GNAQ mutations, and showed normal positive IHC staining for BAP1. These results underscore overlapping features in all blue-like malignant melanocytic tumors. Loss of BAP1 IHC expression was restricted to melanomas, including all metastatic cases. PMID:26645730

  13. Epidemiology of Rifampicin Resistant Tuberculosis and Common Mutations in rpoB Gene of Mycobacterium tuberculosis: A Retrospective Study from Six Districts of Punjab (India) Using Xpert MTB/RIF Assay

    Science.gov (United States)

    Kaur, Ramandeep; Jindal, Neerja; Arora, Shilpa; Kataria, Shajla

    2016-01-01

    Background: Xpert MTB/RIF assay has revolutionized the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (RIF), a surrogate marker for multidrug-resistant TB (MDR-TB) in tuberculosis (MTB). Materials and Methods: A total of 1612 specimens received between October 2013 and February 2015 were tested by Xpert MTB/RIF assay following manufacturer's instructions. The results thus obtained were analyzed using SPSS version 20.0.0 (SPSS Inc., Chicago, IL, USA) statistical software. Result: RIF resistance was statistically higher in previously treated patients in comparison to the new patients (P = 0.006) and in patients with acid fast-Bacilli (AFB) positive smears to AFB-negative smears (P = 0.048). RIF resistance mutations in 130 specimens revealed frequency of E 73/130 (56%), B 28/130 (21.5%), D 18/130 (13.8%), A 11/130 (8.4%), and C 1/130 (0.7%) while in one specimen, mutation combination, i.e., mutations associated with more than one probe (A and B both) was present. Conclusion: Xpert MTB/RIF assay is a user-friendly screening tool for detection of MTB and RIF resistance from suspected TB/MDR cases in a shorter period of time. It could also serve as a useful technique to have simultaneous preliminary information regarding the mutation pattern of RIF resistance in MTB isolates. PMID:27365918

  14. Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

    Directory of Open Access Journals (Sweden)

    Boerrigter Michaël

    2004-01-01

    Full Text Available Abstract Background Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. Methods Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP, 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls. The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. Results Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. Conclusion The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.

  15. Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Vestergaard, Hanne; Møller, Michael Boe

    2011-01-01

    The vast majority of patients with systemic mastocytosis (SM) carry the somatic D816V mutation in the KIT gene. The KIT D816V mutation is one of the minor criteria for a diagnosis of SM according to the 2008 World Health Organization classification of myeloproliferative neoplasms. In the present...

  16. A novel protein-RNA binding assay: functional interactions of the foot-and-mouth disease virus internal ribosome entry site with cellular proteins.

    OpenAIRE

    Stassinopoulos, I A; Belsham, G J

    2001-01-01

    Translation initiation on foot-and-mouth disease virus (FMDV) RNA occurs by a cap-independent mechanism directed by a highly structured element (approximately 435 nt) termed an internal ribosome entry site (IRES). A functional assay to identify proteins that bind to the FMDV IRES and are necessary for FMDV IRES-mediated translation initiation has been developed. In vitro-transcribed polyadenylated RNAs corresponding to the whole or part of the FMDV IRES were immobilized on oligo-dT Dynabeads ...

  17. Planar optical waveguide based sandwich assay sensors and processes for the detection of biological targets including protein markers, pathogens and cellular debris

    Science.gov (United States)

    Martinez, Jennifer S.; Swanson, Basil I.; Grace, Karen M.; Grace, Wynne K.; Shreve, Andrew P.

    2009-06-02

    An assay element is described including recognition ligands bound to a film on a single mode planar optical waveguide, the film from the group of a membrane, a polymerized bilayer membrane, and a self-assembled monolayer containing polyethylene glycol or polypropylene glycol groups therein and an assay process for detecting the presence of a biological target is described including injecting a biological target-containing sample into a sensor cell including the assay element, with the recognition ligands adapted for binding to selected biological targets, maintaining the sample within the sensor cell for time sufficient for binding to occur between selected biological targets within the sample and the recognition ligands, injecting a solution including a reporter ligand into the sensor cell; and, interrogating the sample within the sensor cell with excitation light from the waveguide, the excitation light provided by an evanescent field of the single mode penetrating into the biological target-containing sample to a distance of less than about 200 nanometers from the waveguide thereby exciting the fluorescent-label in any bound reporter ligand within a distance of less than about 200 nanometers from the waveguide and resulting in a detectable signal.

  18. Clinical and cellular consequences of the mutation m.12300G>A in the mitochondrial tRNA(Leu(CUN)) gene.

    Science.gov (United States)

    Martín-Jiménez, Rebeca; Martín-Hernández, Elena; Cabello, Ana; García-Silva, María Teresa; Arenas, Joaquín; Campos, Yolanda

    2012-03-01

    We report, for the first time, a patient with an overlap MERRF-NARP syndrome who carries the mutation m.12300G>A in the mitochondrial tRNA(Leu(CUN)) gene. The mutation was heteroplamic and more abundant in her muscle and fibroblast than in blood from her oligosymptomatic mother. Single muscle fiber analysis revealed that the proportion of mutant mtDNA in ragged red fibers was higher than that in normal fibers. Combined defects of mitochondrial respiratory chain complexes were detected in muscle, fibroblasts and transmitochondrial hybrid cells. Significant reduction of total ATP and mitochondrial membrane potential and an increased production of reactive oxygen species were observed. PMID:22094595

  19. Cellular and systemic effects of Parkinson’s disease-related LRRK2 mutations: An investigation of cytoskeletal function and the innate immune system in transgenic mice and human LRRK2 mutation carriers

    OpenAIRE

    Caesar, Mareike

    2016-01-01

    Parkinson’s disease (PD) is, after Alzheimer’s disease, the most common neurodegenerative disorder. Mutations in the leucine rich repeat kinase 2 (LRRK2) are the most common known cause of familial PD but also constitute about 3.5 % of all sporadic PD cases. This work focuses on the effects of LRRK2 mutations on cytoskeletal function and on the innate immune system. Findings from animal models were translated to human material to assess their relevance in human disease states. Changes in ...

  20. Positive newborn screen for methylmalonic aciduria identifies the first mutation in TCblR/CD320, the gene for cellular uptake of transcobalamin-bound vitamin B12.

    OpenAIRE

    Quadros,, Edward; Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira (Reisman), Pedro (Twins); Hannibal,, Luciana; Wang, Sihe; Jacobsen,, Donald; Fedosov, Sergey; Wright, Erica; Gallagher, Renata; Anastasio, Natascia; Watkins, David; Rosenblatt, David

    2010-01-01

    Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-dire...

  1. Functional Analysis of a Missense Mutation in the Serine Protease Inhibitor SPINT2 Associated with Congenital Sodium Diarrhea

    OpenAIRE

    Faller, Nicolas; Gautschi, Ivan; Schild, Laurent

    2014-01-01

    Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functio...

  2. Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

    OpenAIRE

    Faller N.; Gautschi I.; Schild L.

    2014-01-01

    Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functio...

  3. A p38 substrate-specific MK2-EGFP translocation assay for identification and validation of new p38 inhibitors in living cells: a comprising alternative for acquisition of cellular p38 inhibition data.

    Directory of Open Access Journals (Sweden)

    Roman Anton

    Full Text Available The fundamental role of p38 mitogen-activated protein kinases (MAPKs in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood.

  4. A p38 Substrate-Specific MK2-EGFP Translocation Assay for Identification and Validation of New p38 Inhibitors in Living Cells: A Comprising Alternative for Acquisition of Cellular p38 Inhibition Data

    Science.gov (United States)

    Anton, Roman; Bauer, Silke M.; Keck, Peter R. W. E. F.; Laufer, Stefan; Rothbauer, Ulrich

    2014-01-01

    The fundamental role of p38 mitogen-activated protein kinases (MAPKs) in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA) system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood. PMID:24743242

  5. A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity

    OpenAIRE

    Debrand, Emmanuel; Lykoudi, Alexandra; Bradshaw, Elizabeth; Allen, Stephanie K.

    2015-01-01

    Introduction Non-invasive prenatal diagnosis (NIPD) makes use of cell-free fetal DNA (cffDNA) in the mother’s bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5–1% risk of fetal loss. We describe a droplet digital PCR (ddPCR) assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in th...

  6. Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

    OpenAIRE

    Twigg, SR; Babbs, C.; van den Elzen, ME; Goriely, A.; Taylor, S.; McGowan, SJ; Giannoulatou, E; Lonie, L; Ragoussis, J; Sadighi Akha, E; Knight, SJ; Zechi-Ceide, RM; Hoogeboom, JA; Pober, BR; Toriello, HV

    2013-01-01

    Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with dif...

  7. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2003-01-01

    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  8. Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: Application of a differential interaction trap assay for examining protein-protein interactions

    Energy Technology Data Exchange (ETDEWEB)

    Inouye, C.; Dhillon, N.; Durfee, T. [Univ. of California, Berkeley, CA (United States)] [and others

    1997-10-01

    Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organized the components of the mitogen-activated protein kinase (MAKP) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein {beta} subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5{delta} cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer. 69 refs., 6 figs., 3 tabs.

  9. On-Chip Cellomics Assay Enabling Algebraic and Geometric Understanding of Epigenetic Information in Cellular Networks of Living Systems. 1. Temporal Aspects of Epigenetic Information in Bacteria

    Directory of Open Access Journals (Sweden)

    Kenji Yasuda

    2012-05-01

    Full Text Available A series of studies aimed at developing methods and systems of analyzing epigenetic information in cells and in cell networks, as well as that of genetic information, was examined to expand our understanding of how living systems are determined. Because cells are minimum units reflecting epigenetic information, which is considered to map the history of a parallel-processing recurrent network of biochemical reactions, their behaviors cannot be explained by considering only conventional DNA information-processing events. The role of epigenetic information on cells, which complements their genetic information, was inferred by comparing predictions from genetic information with cell behaviour observed under conditions chosen to reveal adaptation processes, population effects and community effects. A system of analyzing epigenetic information was developed starting from the twin complementary viewpoints of cell regulation as an “algebraic” system (emphasis on temporal aspects and as a “geometric” system (emphasis on spatial aspects. Exploiting the combination of latest microfabrication technology and measurement technologies, which we call on-chip cellomics assay, we can control and re-construct the environments and interaction of cells from “algebraic” and “geometric” viewpoints. In this review, temporal viewpoint of epigenetic information, a part of the series of single-cell-based “algebraic” and “geometric” studies of celluler systems in our research groups, are summerized and reported. The knowlege acquired from this study may lead to the use of cells that fully control practical applications like cell-based drug screening and the regeneration of organs.

  10. Establishment of a non-radioactive cleavage assay to assess the DNA repair capacity towards oxidatively damaged DNA in subcellular and cellular systems and the impact of copper

    International Nuclear Information System (INIS)

    Oxidative stress is involved in many diseases, and the search for appropriate biomarkers is one major focus in molecular epidemiology. 8-Oxoguanine (8-oxoG), a potentially mutagenic DNA lesion, is considered to be a sensitive biomarker for oxidative stress. Another approach consists in assessing the repair capacity towards 8-oxoG, mediated predominantly by the human 8-oxoguanine DNA glycosylase 1 (hOGG1). With respect to the latter, during the last few years so-called cleavage assays have been described, investigating the incision of 32P-labelled and 8-oxoG damaged oligonucleotides by cell extracts. Within the present study, a sensitive non-radioactive test system based on a Cy5-labelled oligonucleotide has been established. Sources of incision activity are isolated proteins or extracts prepared from cultured cells and peripheral blood mononuclear cells (PBMC). After comparing different oligonucleotide structures, a hairpin-like structure was selected which was not degraded by cell extracts. Applying this test system the impact of copper on the activity of isolated hOGG1 and on hOGG activity in A549 cells was examined, showing a distinct inhibition of the isolated protein at low copper concentration as compared to a modest inhibition of hOGG activity in cells at beginning cytotoxic concentrations. For investigating PBMC, all reaction conditions, including the amounts of oligonucleotide and cell extract as well as the reaction time have been optimized. The incision activities of PBMC protein extracts obtained from different donors have been investigated, and inter-individual differences have been observed. In summary, the established method is as sensitive and even faster than the radioactive technique, and additionally, offers the advantage of reduced costs and low health risk.

  11. Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1-Transfected Human Embryonic Kidney 293 Cells.

    Science.gov (United States)

    Chien, Huan-Chieh; Zur, Arik A; Maurer, Tristan S; Yee, Sook Wah; Tolsma, John; Jasper, Paul; Scott, Dennis O; Giacomini, Kathleen M

    2016-03-01

    Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method to determine the intracellular unbound drug concentrations in cultured cells, and we apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected human embryonic kidney 293 (HEK293) cells. Intracellular space (ICS) was calculated by subtracting the [(3)H]-inulin distribution volume (extracellular space, ECS) from the [(14)C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean ± S.E.M.; μl/10(6) cells) of monolayers of HEK cells (HEK-empty vector [EV]) and cells overexpressing human organic cation transporter 1 (HEK-OCT1), 1.21± 0.07 and 1.25±0.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 µM metformin, the intracellular concentrations were 26.4 ± 7.8 μM and 268 ± 11.0 μM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K(+) buffer (140 mM KCl) compared with normal K(+) buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8 ± 3.8 μM and 198.1 ± 11.2 μM, respectively; P < 0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiologic values, the positively charged metformin accumulates to particularly high levels in endoplasmic reticulum and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells. PMID:26700958

  12. Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl Transferase Gene : New Assays to Increase the Sensitivity and Specificity of Newborn Screening for Galactosemia

    OpenAIRE

    Dobrowolski, Steven F.; Banas, Richard A.; Suzow, Joseph G.; Berkley, Michelle; Naylor, Edwin W.

    2003-01-01

    Classical galactosemia is a genetic disease caused by mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. Prospective newborn screening for galactosemia is routine and utilizes the universally collected newborn dried blood specimen on filter paper. Screening for galactosemia is achieved through analysis of total galactose (galactose and galactose-1-phosphate) and/or determining the activity of the GALT enzyme. While this approach is effective, en vironmental factors and the...

  13. Development and Evaluation of an Oligonucleotide Ligation Assay for Detection of Drug Resistance-Associated Mutations in the Human Immunodeficiency Virus Type 2 pol Gene▿

    OpenAIRE

    Jallow, Sabelle; Kaye, Steve; Schutten, Martin; Brandin, Eleonor; Albert, Jan; McConkey, Samuel J; Corrah, Tumani; Whittle, Hilton; Vanham, Guido; Rowland-Jones, Sarah; Janssens, Wouter

    2007-01-01

    Human immunodeficiency virus type 2 (HIV-2) is naturally resistant to several antiretroviral drugs, including all of the non-nucleoside reverse transcriptase inhibitors and the entry inhibitor T-20, and may have reduced susceptibility to some protease inhibitors. These resistance properties make treatment of HIV-2 patients difficult, with very limited treatment options. Therefore, early detection of resistance mutations is important for understanding treatment failures and guiding subsequent ...

  14. Development of Novel PCR Assays To Detect Azole Resistance-Mediating Mutations of the Aspergillus fumigatus cyp51A Gene in Primary Clinical Samples from Neutropenic Patients

    OpenAIRE

    Spiess, Birgit; Seifarth, Wolfgang; Merker, Natalia; Howard, Susan J.; Reinwald, Mark; Dietz, Anne; Hofmann, Wolf-Karsten; Buchheidt, Dieter

    2012-01-01

    The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establis...

  15. Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

    OpenAIRE

    Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida , Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei

    2006-01-01

    A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adj...

  16. A Non-Invasive Droplet Digital PCR (ddPCR Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity.

    Directory of Open Access Journals (Sweden)

    Emmanuel Debrand

    Full Text Available Non-invasive prenatal diagnosis (NIPD makes use of cell-free fetal DNA (cffDNA in the mother's bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5-1% risk of fetal loss. We describe a droplet digital PCR (ddPCR assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in the cffDNA, it is possible to predict whether the fetus will be an unaffected carrier (absence or whether further invasive testing is indicated (presence.We selected a family in which the parents were known to carry different mutated CFTR alleles as our test system. NIPD was performed for three of their pregnancies during the first trimester (at around 11-12 weeks of gestation. Taqman probes were designed against an amplicon in exon 11 of the CFTR gene, to quantify the proportion of mutant (ΔF508-MUT; FAM and normal (ΔF508-NOR; VIC alleles at position c.1521_1523 of the CFTR gene.The assay correctly and unambiguously recognized the ΔF508-MUT CFTR allele in the cffDNA of all three proband fetuses and none of the six unaffected control fetuses. In conclusion, the Bio-Rad QX100 was found to be a cost-effective and technically undemanding platform for designing bespoke NIPD assays.

  17. A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity

    Science.gov (United States)

    Debrand, Emmanuel; Lykoudi, Alexandra; Bradshaw, Elizabeth; Allen, Stephanie K.

    2015-01-01

    Introduction Non-invasive prenatal diagnosis (NIPD) makes use of cell-free fetal DNA (cffDNA) in the mother’s bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5–1% risk of fetal loss. We describe a droplet digital PCR (ddPCR) assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in the cffDNA, it is possible to predict whether the fetus will be an unaffected carrier (absence) or whether further invasive testing is indicated (presence). Methods We selected a family in which the parents were known to carry different mutated CFTR alleles as our test system. NIPD was performed for three of their pregnancies during the first trimester (at around 11–12 weeks of gestation). Taqman probes were designed against an amplicon in exon 11 of the CFTR gene, to quantify the proportion of mutant (ΔF508-MUT; FAM) and normal (ΔF508-NOR; VIC) alleles at position c.1521_1523 of the CFTR gene. Discussion The assay correctly and unambiguously recognized the ΔF508-MUT CFTR allele in the cffDNA of all three proband fetuses and none of the six unaffected control fetuses. In conclusion, the Bio-Rad QX100 was found to be a cost-effective and technically undemanding platform for designing bespoke NIPD assays. PMID:26561302

  18. In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

    Directory of Open Access Journals (Sweden)

    Sergio Luiz Montego Ferreira Junior

    2014-06-01

    Full Text Available Drug-resistant tuberculosis (TB threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR that is able to identify the most frequent mutations related to rifampicin (RMP and isoniazid (INH resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB. The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%, respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1, respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5 for RIF and 87.4% (84.3-96.2 for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

  19. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    OpenAIRE

    Slyskova, Jana; Langie, Sabine A. S.; Collins, Andrew R.; Vodicka, Pavel

    2014-01-01

    Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER) in peripheral blood cells based on modified comet assay protocols have been widely applied i...

  20. A novel autosomal recessive TERT T1129P mutation in a dyskeratosis congenita family leads to cellular senescence and loss of CD34+ hematopoietic stem cells not reversible by mTOR-inhibition.

    Science.gov (United States)

    Stockklausner, Clemens; Raffel, Simon; Klermund, Julia; Bandapalli, Obul Reddy; Beier, Fabian; Brümmendorf, Tim H; Bürger, Friederike; Sauer, Sven W; Hoffmann, Georg F; Lorenz, Holger; Tagliaferri, Laura; Nowak, Daniel; Hofmann, Wolf-Karsten; Buergermeister, Rebecca; Kerber, Carolin; Rausch, Tobias; Korbel, Jan O; Luke, Brian; Trumpp, Andreas; Kulozik, Andreas E

    2015-11-01

    The TERT gene encodes for the reverse transcriptase activity of the telomerase complex and mutations in TERT can lead to dysfunctional telomerase activity resulting in diseases such as dyskeratosis congenita (DKC). Here, we describe a novel TERT mutation at position T1129P leading to DKC with progressive bone marrow (BM) failure in homozygous members of a consanguineous family. BM hematopoietic stem cells (HSCs) of an affected family member were 300-fold reduced associated with a significantly impaired colony forming capacity in vitro and impaired repopulation activity in mouse xenografts. Recent data in yeast suggested improved cellular checkpoint controls by mTOR inhibition preventing cells with short telomeres or DNA damage from dividing. To evaluate a potential therapeutic option for the patient, we treated her primary skin fibroblasts and BM HSCs with the mTOR inhibitor rapamycin. This led to prolonged survival and decreased levels of senescence in T1129P mutant fibroblasts. In contrast, the impaired HSC function could not be improved by mTOR inhibition, as colony forming capacity and multilineage engraftment potential in xenotransplanted mice remained severely impaired. Thus, rapamycin treatment did not rescue the compromised stem cell function of TERTT1129P mutant patient HSCs and outlines limitations of a potential DKC therapy based on rapamycin. PMID:26546739

  1. γδ T cell-mediated antibody-dependent cellular cytotoxicity with CD19 antibodies assessed by an impedance-based label-free real-time cytotoxicity assay

    Directory of Open Access Journals (Sweden)

    Ursula Jördis Eva Seidel

    2014-12-01

    Full Text Available γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation (SCT strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC with monoclonal antibodies (mAbs. These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL. To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc optimized CD19 antibody (4G7SDIE and a bispecific antibody with the specificities CD19 and CD16 (N19-C16 was evaluated in CD107a degranulation assays and intracellular cytokine staining (ICS. CD107a, TNF-α and IFN-γ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits an promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and

  2. γδ T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay.

    Science.gov (United States)

    Seidel, Ursula Jördis Eva; Vogt, Fabian; Grosse-Hovest, Ludger; Jung, Gundram; Handgretinger, Rupert; Lang, Peter

    2014-01-01

    γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNγ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical

  3. A multi-scale approach to colorectal cancer: from a biochemical- interaction signaling-network level, to multi-cellular dynamics of malignant transformation. Interplay with mutations and onco-protein inhibitor drugs.

    Science.gov (United States)

    Tortolina, L; Castagnino, N; De Ambrosi, C; Moran, E; Patrone, F; Ballestrero, A; Parodi, S

    2012-05-01

    This review article is part of a special Current Cancer Drug Targets issue devoted to colorectal cancer and molecularly targeted treatments. In our paper we made an attempt to connect more basic aspects with preclinical, pharmacological / therapeutic and clinical aspects. Reconstruction of a Molecular Interaction Map (MIM) comprising an important part of the G0 - G1 - S cell cycle transition, was a major component of our review. Such a MIM serves also as a convenient / organized database of a large set of important molecular events. The frequency of mutated / altered signaling-proteins indicates the importance of this signaling-network region. We have considered problems at different scale levels. Our MIM works at a biochemical-interaction level. We have also touched the multi-cellular dynamics of normal and aberrant colon crypts. Until recently, dynamic simulations at a biochemical or multi-cellular scale level were considered as a sort of esoteric approach. We tried to convince the reader, also on the basis of a rapidly growing literature, mostly published in high quality journals, that suspicion towards simulations should dissipate, as the limitations and advantages of their application are better appreciated, opening the door to their permanent adoption in everyday research. What is really required is a more interdisciplinary mentality and an interdisciplinary approach. The prize is a level of understanding going beyond mere intuition. PMID:22385511

  4. Campylobacter jejuni CsrA complements an Escherichia coli csrA mutation for the regulation of biofilm formation, motility and cellular morphology but not glycogen accumulation

    Directory of Open Access Journals (Sweden)

    Fields Joshua A

    2012-10-01

    Full Text Available Abstract Background Although Campylobacter jejuni is consistently ranked as one of the leading causes of bacterial diarrhea worldwide, the mechanisms by which C. jejuni causes disease and how they are regulated have yet to be clearly defined. The global regulator, CsrA, has been well characterized in several bacterial genera and is known to regulate a number of independent pathways via a post transcriptional mechanism, but remains relatively uncharacterized in the genus Campylobacter. Previously, we reported data illustrating the requirement for CsrA in several virulence related phenotypes of C. jejuni strain 81–176, indicating that the Csr pathway is important for Campylobacter pathogenesis. Results We compared the Escherichia coli and C. jejuni orthologs of CsrA and characterized the ability of the C. jejuni CsrA protein to functionally complement an E. coli csrA mutant. Phylogenetic comparison of E. coli CsrA to orthologs from several pathogenic bacteria demonstrated variability in C. jejuni CsrA relative to the known RNA binding domains of E. coli CsrA and in several amino acids reported to be involved in E. coli CsrA-mediated gene regulation. When expressed in an E. coli csrA mutant, C. jejuni CsrA succeeded in recovering defects in motility, biofilm formation, and cellular morphology; however, it failed to return excess glycogen accumulation to wild type levels. Conclusions These findings suggest that C. jejuni CsrA is capable of efficiently binding some E. coli CsrA binding sites, but not others, and provide insight into the biochemistry of C. jejuni CsrA.

  5. Mismatch Amplification Mutation Assay Real-Time PCR Analysis of the Leptin Gene G2548A and A19G Polymorphisms and Serum Leptin in Infancy: A Preliminary Investigation.

    Science.gov (United States)

    Savino, Francesco; Rossi, Lorenza; Di Stasio, Liliana; Galliano, Ilaria; Montanari, Paola; Bergallo, Massimiliano

    2016-01-01

    Leptin is a hormone that regulates food intake and energy metabolism. Its coding gene (LEP) is one of the most promising candidates for obesity. Although some studies have detected associations of different single nucleotide polymorphisms (SNPs) in the LEP gene with serum leptin levels and obesity-related traits, the results are still conflicting. We investigated two SNPs to find relationships with leptin concentrations. Thirty healthy Caucasian infants younger than 6 months were genotyped for the SNPs G2548A and A19G with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system-mismatch amplification mutation assay (ARMS- MAMA) real-time PCR, and serum leptin concentrations were measured with a radioimmunoassay method. Considering the significant linkage disequilibrium observed between the two SNPs, we divided the sample according to the number of GG haplotypes and observed that individuals homozygous for the GG haplotype had higher serum leptin levels in early infancy than the others. Although these preliminary results are based on a limited sample, they suggest that the genetic background seems to play a role in modulating leptin levels in infancy, but changes in leptin levels over infancy and their correlation with obesity need to be further explored. We describe an ARMS-MAMA real-time PCR procedure which could be profitably applied in routine genetic screening. PMID:27008407

  6. Cellular automata

    CERN Document Server

    Codd, E F

    1968-01-01

    Cellular Automata presents the fundamental principles of homogeneous cellular systems. This book discusses the possibility of biochemical computers with self-reproducing capability.Organized into eight chapters, this book begins with an overview of some theorems dealing with conditions under which universal computation and construction can be exhibited in cellular spaces. This text then presents a design for a machine embedded in a cellular space or a machine that can compute all computable functions and construct a replica of itself in any accessible and sufficiently large region of t

  7. Bacterial mutagenicity assays: test methods.

    Science.gov (United States)

    Gatehouse, David

    2012-01-01

    The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems. PMID:22147566

  8. Genome Destabilizing Mutator Alleles Drive Specific Mutational Trajectories in Saccharomyces cerevisiae

    Science.gov (United States)

    Stirling, Peter C.; Shen, Yaoqing; Corbett, Richard; Jones, Steven J. M.; Hieter, Philip

    2014-01-01

    In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13–Stn1–Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes. PMID:24336748

  9. FLT3 mutations in canine acute lymphocytic leukemia

    International Nuclear Information System (INIS)

    FMS-like tyrosine kinase 3 (FLT3) is a commonly mutated protein in a variety of human acute leukemias. Mutations leading to constitutively active FLT3, including internal tandem duplications of the juxtamembrane domain (ITD), result in continuous cellular proliferation, resistance to apoptotic cell death, and a poorer prognosis. A better understanding of the molecular consequences of FLT3 activation would allow improved therapeutic strategies in these patients. Canine lymphoproliferative diseases, including lymphoma and acute leukemias, share evolutionarily conserved chromosomal aberrations and exhibit conserved mutations within key oncogenes when compared to their human counterparts. A small percentage of canine acute lymphocytic leukemias (ALL) also exhibit FLT3 ITD mutations. We molecularly characterized FLT3 mutations in two dogs and one cell line, by DNA sequencing, gene expression analysis via quantitative real-time PCR, and sensitivity to the FLT3 inhibitor lestaurtinib via in vitro proliferation assays. FLT 3 and downstream mediators of FLT3 activation were assessed by Western blotting. The canine B-cell leukemia cell line, GL-1, and neoplastic cells from 2/7 dogs diagnosed cytologically with ALL were found to have FLT3 ITD mutations and FLT3 mRNA up-regulation. Lestaurtinib, a small molecule FLT3 inhibitor, significantly inhibited the growth of GL-1 cells, while not affecting the growth of two other canine lymphoid cell lines without the FLT3 mutation. Finally, western blots were used to confirm the conserved downstream mediators of FLT3 activating mutations. These results show that ALL and FLT3 biology is conserved between canine and human patients, supporting the notion that canine ALL, in conjunction with the GL-1 cell line, will be useful in the development of a relevant large animal model to aid in the study of human FLT3 mutant leukemias

  10. Lack of mutation-histopathology correlation in a patient with Proteus syndrome.

    Science.gov (United States)

    Doucet, Meggie E; Bloomhardt, Hadley M; Moroz, Krzysztof; Lindhurst, Marjorie J; Biesecker, Leslie G

    2016-06-01

    Proteus syndrome (PS) is characterized by progressive, disproportionate, segmental overgrowth, and tumor susceptibility caused by a somatic mosaic AKT1 activating mutation. Each individual has unique manifestations making this disorder extremely heterogeneous. We correlated three variables in 38 tissue samples from a patient who died with PS: the gross affection status, the microscopic affection status, and the mutation level. The AKT1 mutation was measured using a PCR-based RFLP assay. Thirteen samples were grossly normal; six had detectable mutation (2-29%) although four of these six were histopathologically normal. Of the seven grossly normal samples that had no mutation, only four were histologically normal. The mutation level in the grossly abnormal samples was 3-35% and all but the right and left kidneys, skull, and left knee bone, with mutation levels of 19%, 15%, 26%, and 17%, respectively, had abnormal histopathology. The highest mutation level was in a toe bone sample whereas the lowest levels were in the soft tissue surrounding that toe, and an omental fat nodule. We also show here that PS overgrowth can be caused by cellular proliferation or by extracellular matrix expansion. Additionally, papillary thyroid carcinoma was identified, a tumor not previously associated with PS. We conclude that gross pathology and histopathology correlate poorly with mutation levels in PS, that overgrowth can be mediated by cellular proliferation or extracellular matrix expansion, and that papillary thyroid carcinoma is part of the tumor susceptibility of PS. New methods need to be developed to facilitate genotype-phenotype correlation in mosaic disorders. © 2016 Wiley Periodicals, Inc. PMID:27112325

  11. Cellular Automata

    OpenAIRE

    Bagnoli, Franco

    1998-01-01

    An introduction to cellular automata (both deterministic and probabilistic) with examples. Definition of deterministic automata, dynamical properties, damage spreading and Lyapunov exponents; probabilistic automata and Markov processes, nonequilibrium phase transitions, directed percolation, diffusion; simulation techniques, mean field. Investigation themes: life, epidemics, forest fires, percolation, modeling of ecosystems and speciation. They represent my notes for the school "Dynamical Mod...

  12. Competitive amplification of differentially melting amplicons (CADMA improves KRAS hotspot mutation testing in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Kristensen Lasse

    2012-11-01

    Full Text Available Abstract Background Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR. Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. Methods We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA. The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. Results The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. Conclusions The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

  13. Cellular senescence in aging primates.

    Science.gov (United States)

    Herbig, Utz; Ferreira, Mark; Condel, Laura; Carey, Dee; Sedivy, John M

    2006-03-01

    The aging of organisms is characterized by a gradual functional decline of all organ systems. Mammalian somatic cells in culture display a limited proliferative life span, at the end of which they undergo an irreversible cell cycle arrest known as replicative senescence. Whether cellular senescence contributes to organismal aging has been controversial. We investigated telomere dysfunction, a recently discovered biomarker of cellular senescence, and found that the number of senescent fibroblasts increases exponentially in the skin of aging baboons, reaching >15% of all cells in very old individuals. In addition, the same cells contain activated ataxia-telangiectasia mutated kinase and heterochromatinized nuclei, confirming their senescent status. PMID:16456035

  14. Cellular resilience.

    Science.gov (United States)

    Smirnova, Lena; Harris, Georgina; Leist, Marcel; Hartung, Thomas

    2015-01-01

    Cellular resilience describes the ability of a cell to cope with environmental changes such as toxicant exposure. If cellular metabolism does not collapse directly after the hit or end in programmed cell death, the ensuing stress responses promote a new homeostasis under stress. The processes of reverting "back to normal" and reversal of apoptosis ("anastasis") have been studied little at the cellular level. Cell types show astonishingly similar vulnerability to most toxicants, except for those that require a very specific target, metabolism or mechanism present only in specific cell types. The majority of chemicals triggers "general cytotoxicity" in any cell at similar concentrations. We hypothesize that cells differ less in their vulnerability to a given toxicant than in their resilience (coping with the "hit"). In many cases, cells do not return to the naive state after a toxic insult. The phenomena of "pre-conditioning", "tolerance" and "hormesis" describe this for low-dose exposures to toxicants that render the cell more resistant to subsequent hits. The defense and resilience programs include epigenetic changes that leave a "memory/scar" - an alteration as a consequence of the stress the cell has experienced. These memories might have long-term consequences, both positive (resistance) and negative, that contribute to chronic and delayed manifestations of hazard and, ultimately, disease. This article calls for more systematic analyses of how cells cope with toxic perturbations in the long-term after stressor withdrawal. A technical prerequisite for these are stable (organotypic) cultures and a characterization of stress response molecular networks. PMID:26536287

  15. Use of a Mismatch Amplification Mutation Assay PCR Method to Detect the Complex Vertebral Malformation in Some Chinese Holstein Sires%部分中国荷斯坦种公牛脊柱畸形综合征携带状况的检测和分析

    Institute of Scientific and Technical Information of China (English)

    王帅; 赵学明; 朱化彬; 杜卫华; 王栋; 郝海生; 王宗礼

    2011-01-01

    摘本研究旨在对部分中国荷斯坦种公牛脊柱畸形综合征(Complex vertebral malformation,CVM)致病基因的携带状况进行筛查.应用错配PCR突变分析技术(PCR mismatch amplification mutation assay,PCR-MAMA)建立了针对CVM致病基因的特异性检测方法.利用PCR-MAMA法检测了154头荷斯坦种公牛,发现了24头CVM阳性个体,阳性率为15.58%.结果显示,应对中国荷斯坦种公牛进行全面的针对CVM的检测.%This experiment was conducted to test some Chinese Holstein sires for complex vertebral malformation (CVM). In this study, a simple, rapid PCR mismatch amplification mutation assay (PCR- MAMA) was developed to detect the mutation allele. Out of 154 tested Holstein sires, 24 sires (15. 58%) were identified to be CVM carriers by PCR- MAMA. The results indicate that all the Chinese Holstein sires should be tested for CVM.

  16. The comet assay – how to recognise “good data”

    OpenAIRE

    William Barfield

    2015-01-01

    Testing of potentially genotoxic materials currently involves use of in vitro assays such as the Ames test (gene mutations), the chromosome aberration assay and in vitro micronucleus assay (predominantly using human peripheral lymphocytes to detect clastogenicity and/or aueuploidy) and the mouse lymphoma assay (gene mutations). In addition, one in vivo assay, predominately the in vivo micronucleus assay, is “normally” required to satisfy regulatory testing requirements and on occasion a secon...

  17. γδ T cell-mediated antibody-dependent cellular cytotoxicity with CD19 antibodies assessed by an impedance-based label-free real-time cytotoxicity assay

    OpenAIRE

    Ursula Jördis Eva Seidel; Fabian eVogt; Ludger eGrosse-Hovest; Gundram eJung; Rupert eHandgretinger; Peter eLang

    2014-01-01

    γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation (SCT) strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T ce...

  18. γδ T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay

    OpenAIRE

    Seidel, Ursula Jördis Eva; Vogt, Fabian; Grosse-Hovest, Ludger; Jung, Gundram; Handgretinger, Rupert; Lang, Peter

    2014-01-01

    γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells wi...

  19. Hormone assay

    International Nuclear Information System (INIS)

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  20. Functional characterization of a CRH missense mutation identified in an ADNFLE family.

    Directory of Open Access Journals (Sweden)

    Veronica Sansoni

    Full Text Available Nocturnal frontal lobe epilepsy has been historically considered a channelopathy caused by mutations in subunits of the neuronal nicotinic acetylcholine receptor or in a recently reported potassium channel. However, these mutations account for only a minority of patients, and the existence of at least a new locus for the disease has been demonstrated. In 2005, we detected two nucleotide variations in the promoter of the CRH gene coding for the corticotropin releasing hormone in 7 patients. These variations cosegregated with the disease and were demonstrated to alter the cellular levels of this hormone. Here, we report the identification in an Italian affected family of a novel missense mutation (hpreproCRH p.Pro30Arg located in the region of the CRH coding for the protein pro-sequence. The mutation was detected in heterozygosity in the two affected individuals. In vitro assays demonstrated that this mutation results in reduced levels of protein secretion in the short time thus suggesting that mutated people could present an altered capability to respond immediately to stress agents.

  1. Identification of Capsid Mutations That Alter the Rate of HIV-1 Uncoating in Infected Cells

    OpenAIRE

    Hulme, Amy E.; Kelley, Z; Okocha, Eneniziaogochukwu A.; Hope, Thomas J.

    2014-01-01

    After viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the cyclosporine (CsA) washout assay, in which TRIM-CypA-mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1-infected cells. Here we have extended this analysis to examine the effects of p24 capsid protein (p24CA) mutations and cellular environment on the kinetics of uncoating ...

  2. Epidemiology of rifampicin resistant tuberculosis and common mutations in rpoB gene of Mycobacterium tuberculosis: A retrospective study from six districts of Punjab (India) using Xpert MTB/RIF assay

    OpenAIRE

    Ramandeep Kaur; Neerja Jindal; Shilpa Arora; Shajla Kataria

    2016-01-01

    Background: Xpert MTB/RIF assay has revolutionized the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (RIF), a surrogate marker for multidrug-resistant TB (MDR-TB) in

  3. Radiation-induced mutation at minisatellite loci

    International Nuclear Information System (INIS)

    We are studying the radiation-induced increase of mutation rate in minisatellite loci in mice and humans. Minisatellite mutations were scored by multilocus DNA fingerprint analysis in the progeny of γ-irradiated and non-irradiated mice. The frequency of mutation in offspring of irradiated males was 1.7 higher that in the control group. Germline mutation at human minisatellite loci was studied among children born in heavily polluted areas of the Mogilev district of Belarus after the Chernobyl accident and in a control population. The frequency of mutation assayed both by DNA fingerprinting and by eight single locus probes was found to be two times higher in the exposed families than in the control group. Furthermore, mutation rate was correlated with the parental radiation dose for chronic exposure 137Cs, consistent with radiation-induction of germline mutation. The potential use of minisatellites in monitoring germline mutation in humans will be discussed

  4. Assay of the multiple energy-producing pathways of mammalian cells.

    Directory of Open Access Journals (Sweden)

    Barry R Bochner

    Full Text Available BACKGROUND: To elucidate metabolic changes that occur in diabetes, obesity, and cancer, it is important to understand cellular energy metabolism pathways and their alterations in various cells. METHODOLOGY AND PRINCIPAL FINDINGS: Here we describe a technology for simultaneous assessment of cellular energy metabolism pathways. The technology employs a redox dye chemistry specifically coupled to catabolic energy-producing pathways. Using this colorimetric assay, we show that human cancer cell lines from different organ tissues produce distinct profiles of metabolic activity. Further, we show that murine white and brown adipocyte cell lines produce profiles that are distinct from each other as well as from precursor cells undergoing differentiation. CONCLUSIONS: This technology can be employed as a fundamental tool in genotype-phenotype studies to determine changes in cells from shared lineages due to differentiation or mutation.

  5. Identification and Functional Testing of ERCC2 Mutations in a Multi-national Cohort of Patients with Familial Breast- and Ovarian Cancer.

    Science.gov (United States)

    Rump, Andreas; Benet-Pages, Anna; Schubert, Steffen; Kuhlmann, Jan Dominik; Janavičius, Ramūnas; Macháčková, Eva; Foretová, Lenka; Kleibl, Zdenek; Lhota, Filip; Zemankova, Petra; Betcheva-Krajcir, Elitza; Mackenroth, Luisa; Hackmann, Karl; Lehmann, Janin; Nissen, Anke; DiDonato, Nataliya; Opitz, Romy; Thiele, Holger; Kast, Karin; Wimberger, Pauline; Holinski-Feder, Elke; Emmert, Steffen; Schröck, Evelin; Klink, Barbara

    2016-08-01

    The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription). In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC) predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense), all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of "bonafide" breast cancer susceptibility genes. PMID:27504877

  6. Identification and Functional Testing of ERCC2 Mutations in a Multi-national Cohort of Patients with Familial Breast- and Ovarian Cancer

    Science.gov (United States)

    Kuhlmann, Jan Dominik; Janavičius, Ramūnas; Foretová, Lenka; Lhota, Filip; Zemankova, Petra; Betcheva-Krajcir, Elitza; Mackenroth, Luisa; Lehmann, Janin; Nissen, Anke; DiDonato, Nataliya; Opitz, Romy; Thiele, Holger; Kast, Karin; Wimberger, Pauline; Holinski-Feder, Elke; Emmert, Steffen

    2016-01-01

    The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription). In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC) predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense), all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of “bonafide” breast cancer susceptibility genes. PMID:27504877

  7. Radioreceptor assays

    International Nuclear Information System (INIS)

    Radioreceptor assay (RRA) is an analytical method using the specific interaction of some pharmaceuticals and endogenic substances (ligands) with specific receptors present in certin tissues of living organisms. RRA uses the principle of isotope dilution. The method is described in detail of the preparation of receptors, samples and radioligands, conditions of incubation, the separation of free and bound radioligand, and the mathematical evaluation of RRA. The sensitivity of RRA is measured in units to tens of pg. The specificity of RRA relates to a group of substances with similar pharmacological effect. RRA may be used for identifying neuroleptics, antidepressants, anxiolytics, ergot alkaloids, beta blockers, anticholinergic drugs, certain hormones and neuropeptides. (M.D.)

  8. Clinical implications of BRAF mutation test in colorectal cancer

    OpenAIRE

    Mojarad, Ehsan Nazemalhosseini; Farahani, Roya Kishani; HAGHIGHI, MAHDI MONTAZER; Aghdaei, Hamid Asadzadeh; Kuppen, Peter JK; Zali, Mohammad Reza

    2013-01-01

    Knowledge about the clinical significance of V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) mutations in colorectal cancer (CRC) is growing. BRAF encodes a protein kinase involved with intracellular signaling and cell division. The gene product is a downstream effector of Kirsten Ras 1(KRAS) within the RAS/RAF/MAPK cellular signaling pathway. Evidence suggests that BRAF mutations, like KRAS mutations, result in uncontrolled, non–growth factor-dependent cellular proliferation. Similar t...

  9. Angiogenesis Assays.

    Science.gov (United States)

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  10. Cellular Signaling Pathways and Their Clinical Reflections

    Directory of Open Access Journals (Sweden)

    N. Ceren Sumer-Turanligil

    2010-06-01

    Full Text Available Cellular signaling pathways have important roles in cellular growth, differentiation, inflammatory response and apoptosis and in regulation of cellular responses under various chemical stimulators. Different proteins which belong to these pathways may be exposed to loss-of-function or gain-of-function mutations; this may lead to many clinical phenotypes including primarily cancer. In this review information about basic working principles of these pathways and diseases related to them are included. [Archives Medical Review Journal 2010; 19(3.000: 180-191

  11. Radiopharmaceutical assays

    International Nuclear Information System (INIS)

    Under the laws in force, radiopharmaceuticals for human use must be among other features, non-pyrogenous and non-toxic. For this reason pyrogenity and toxicity assays are carried out. Pharmacokinetic studies may also be necessary in some cases. Products currently made at the Radiopharmaceutical Center, new products designed for certification and raw materials used to manufacture the above, were tested. A total 342 pyrogenity and toxicity tests, and four pharmacokinetic studies were conducted in 1996. To determine pyrogenity, the temperature animals were measured following intravenous administration of radiopharmaceuticals concerned: sodium pertechnetate, colloidal gold and sodium orthoradiohippurate from current production; pharmaceutic components of several new products, i.e. technetium generator, fibrinogen and microspheres. A total 327 products were tested, 96 percent of which met the requirements. To determine toxicity, the probit method was used, consisting of the administration of radiopharmaceutical doses for seven straight days, and checking for lethal effects. An overall 15 tests were carried out and 80 percent of products tested were found certifiable. Pharmacokinetic tests consisted of tropism on target organs and biodistribution in several organs using the tomographic method. (author)

  12. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  13. Effects of T592 phosphomimetic mutations on tetramer stability and dNTPase activity of SAMHD1 can not explain the retroviral restriction defect.

    Science.gov (United States)

    Bhattacharya, Akash; Wang, Zhonghua; White, Tommy; Buffone, Cindy; Nguyen, Laura A; Shepard, Caitlin N; Kim, Baek; Demeler, Borries; Diaz-Griffero, Felipe; Ivanov, Dmitri N

    2016-01-01

    SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro. PMID:27511536

  14. Evaluation of CFTR gene mutations in Adana

    Directory of Open Access Journals (Sweden)

    Ozlem Goruroglu Ozturk

    2013-04-01

    Full Text Available ABSTRACT Objective: Cystic fibrosis is the most common autosomal recessive inherited disorder seen in the white populations. It develops in result of mutations of cystic fibrosis transmembrane regulator (CFTR gene. Rate of these mutations vary in different geographical regions. In this study, we aimed to determine the frequency of CFTR gene mutations in Adana. Methods: DNA samples of 63 subjects (21 women, 42 men who were diagnosed as cystic fibrosis at Balcali Hospital of Cukurova University, were studied for 19 different CFTR mutations by the strip assay method which is based on reverse hybridization. Results: In cystic fibrosis diagnosed patients, 19 mutations were observed of which 9 were homozygous and 10 were heterozygous. ∆F508 frequency was found as 11.9%, and rate of homozygous was found as 66.7%. Mutation frequencies of W1282X and N1303K were found as 2.40% and 4.80% respectively and rate of homozygous mutations were 50% for both. I148T mutation frequency was found as 3.20% and all were heterozygous. For the whole 19 mutations, frequency of mutation in 63 subjects was 22.3%. Conclusion: Detection of CFTR gene mutations by the strip assay method by reverse hybridization is an easy, fast and informative method. However, due to improvability of the common mutations in probable cystic fibrosis patients because of heterogenity in this region, it is still a major problem and does not exclude cystic fibrosis diagnosis. But this problematic issue can be overcome by evaluating the whole exons of CFTR mutations by advanced molecular tecniques. Key words: CFTR, cystic fibrosis, molecular diagnosis, reverse hibridisation [Cukurova Med J 2013; 38(2.000: 202-208

  15. Pig-α基因突变检测方法在药物遗传毒性评价中的应用及研究进展%Development of Pig-α gene mutation assay in genotoxicity evaluation and research applications

    Institute of Scientific and Technical Information of China (English)

    邓媛元; 郭隽; 王雪; 李波

    2012-01-01

    International Conference of Harmonizition (ICH) guidelines allowed 3Rs (reduction, replacement, refinement) into genotoxicity tests and added new demands to genotoxicity tests. The method of Pig-a mutation assay in vivo has sprung up in recent years, which analyzes peripheral blood by flow cytometry. There are likely several advantages of this system compared to conventional approaches, including the need of a small amount of blood, a non-invasive, continuous and dynamic monitoring, and long period detection window. Adapting to current trends in toxicology research, this method can combine with the general toxicity tests with different endpoints by the principles of animal experiments 3Rs. Here, we reviewed the recent progress in Pig-a mutation assay.%ICH修订技术指导原则将动物实验的3Rs原则(减少、替代、优化)引入到遗传毒性试验中,对新试验方法体系提出了更高的要求.Pig-α基因突变检测是近年建立的遗传毒性体内试验方法,其特点是使用流式细胞仪对外周血红细胞进行分析,所需血液样品量较少,同时具有无创、连续、动态监测和检测窗口期长等优点.此外,该方法能够结合重复给药毒性试验进行多终点检测,符合动物实验3Rs原则,适应当前毒理学研究的发展趋势.本文对Pig-α基因突变检测方法及研究进展作一综述.

  16. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten;

    2010-01-01

    The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...

  17. Effect of dabrafenib on melanoma cell lines harbouring the BRAFV600D/R mutations

    International Nuclear Information System (INIS)

    Conventional therapeutic agents are largely unsatisfactory into the treatment of malignant melanoma. Recently, an innovative approach based on inhibitors of the mutated BRAF gene (which represents the most prevalent alteration in melanoma patients) appears very promising from the clinical point of view. On this regard, a new compound, dabrafenib (GSK2118436), has been demonstrated to be effective in patients carrying the BRAFV600E/K mutations. We here tested dabrafenib for its capability to inhibit cell growth on primary melanoma cell lines, established from patients' tumour tissues and carrying the BRAFV600D/R mutations. Three melanoma cell lines were tested: M257 wild-type BRAF, LCP BRAFV600R and WM266 BRAFV600D. The MTT assays were performed using standardized approaches. To evaluate the inhibition of MAPK pathway and the consequent inhibition of cellular proliferation, the phosphorylation of ERK was examined by Western Blot analysis performed on total protein extracts from cell lines after treatment with dabrafenib. Our experiments demonstrated an effective action of Dabrafenib (GSK2118436) and the inhibition of MAPK pathway in melanoma cell lines carrying BRAFV600D/R mutations. These results could be helpful to enlarge the number of melanoma patients who may benefit of a more effective targeted treatment

  18. Effect of dabrafenib on melanoma cell lines harbouring the BRAFV600D/R mutations

    Directory of Open Access Journals (Sweden)

    Gentilcore Giusy

    2013-01-01

    Full Text Available Abstract Background Conventional therapeutic agents are largely unsatisfactory into the treatment of malignant melanoma. Recently, an innovative approach based on inhibitors of the mutated BRAF gene (which represents the most prevalent alteration in melanoma patients appears very promising from the clinical point of view. On this regard, a new compound, dabrafenib (GSK2118436, has been demonstrated to be effective in patients carrying the BRAFV600E/K mutations. We here tested dabrafenib for its capability to inhibit cell growth on primary melanoma cell lines, established from patients' tumour tissues and carrying the BRAFV600D/R mutations. Methods Three melanoma cell lines were tested: M257 wild-type BRAF, LCP BRAFV600R and WM266 BRAFV600D. The MTT assays were performed using standardized approaches. To evaluate the inhibition of MAPK pathway and the consequent inhibition of cellular proliferation, the phosphorylation of ERK was examined by Western Blot analysis performed on total protein extracts from cell lines after treatment with dabrafenib. Results Our experiments demonstrated an effective action of Dabrafenib (GSK2118436 and the inhibition of MAPK pathway in melanoma cell lines carrying BRAFV600D/R mutations. Conclusion These results could be helpful to enlarge the number of melanoma patients who may benefit of a more effective targeted treatment.

  19. A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance.

    Science.gov (United States)

    Zeng, Xin; Montoute, Monica; Bee, Tiger W; Lin, Hong; Kallal, Lorena A; Liu, Yan; Agarwal, Pankaj; Wang, Dayuan; Lu, Quinn; Morrow, Dwight; Pope, Andrew J; Wu, Zining

    2016-03-01

    Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential. PMID:26656867

  20. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line.

    Science.gov (United States)

    Meng, Ran; Zhou, Jin; Sui, Meng; Li, ZhiYong; Feng, GuoSheng; Yang, BaoFeng

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment. PMID:20596959

  1. Oxidative stress action in cellular aging

    OpenAIRE

    Monique Cristine de Oliveira; João Paulo Ferreira Schoffen

    2010-01-01

    Various theories try to explain the biological aging by changing the functions and structure of organic systems and cells. During lifetime, free radicals in the oxidative stress lead to lipid peroxidation of cellular membranes, homeostasis imbalance, chemical residues formation, gene mutations in DNA, dysfunction of certain organelles, and the arise of diseases due to cell death and/or injury. This review describes the action of oxidative stress in the cells aging process, emphasizing the fac...

  2. Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

    Directory of Open Access Journals (Sweden)

    Nicolas Faller

    Full Text Available Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

  3. Clinical implications of BRAF mutation test in colorectal cancer

    Science.gov (United States)

    Mojarad, Ehsan Nazemalhosseini; Farahani, Roya Kishani; Haghighi, Mahdi Montazer; Aghdaei, Hamid Asadzadeh; Kuppen, Peter JK

    2013-01-01

    Knowledge about the clinical significance of V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) mutations in colorectal cancer (CRC) is growing. BRAF encodes a protein kinase involved with intracellular signaling and cell division. The gene product is a downstream effector of Kirsten Ras 1(KRAS) within the RAS/RAF/MAPK cellular signaling pathway. Evidence suggests that BRAF mutations, like KRAS mutations, result in uncontrolled, non–growth factor-dependent cellular proliferation. Similar to the rationale that KRAS mutation precludes effective treatment with anti-EGFR drugs. Recently, BRAF mutation testing has been introduced into routine clinical laboratories because its significance has become clearer in terms of effect on pathogenesis of CRC, utility in differentiating sporadic CRC from Lynch syndrome (LS), prognosis, and potential for predicting patient outcome in response to targeted drug therapy. In this review we describe the impact of BRAF mutations for these aspects. PMID:24834238

  4. Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline

    DEFF Research Database (Denmark)

    Giannoulatou, Eleni; McVean, Gilean; Taylor, Indira B;

    2013-01-01

    The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause...

  5. Toxicology and cellular effect of manufactured nanomaterials

    Science.gov (United States)

    Chen, Fanqing

    2014-07-22

    The increasing use of nanotechnology in consumer products and medical applications underlies the importance of understanding its potential toxic effects to people and the environment. Herein are described methods and assays to predict and evaluate the cellular effects of nanomaterial exposure. Exposing cells to nanomaterials at cytotoxic doses induces cell cycle arrest and increases apoptosis/necrosis, activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response. Certain nanomaterials induce genes indicative of a strong immune and inflammatory response within skin fibroblasts. Furthermore, the described multiwall carbon nanoonions (MWCNOs) can be used as a therapeutic in the treatment of cancer due to its cytotoxicity.

  6. Multiple oncogenic mutations related to targeted therapy in nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jian-Wei Zhang; Hong-Yuan Zhao; Yu-Xiang Ma; Zhi-Huang Hu; Pei-Yu Huang; Li Zhang; Tao Qin; Shao-Dong Hong; Jing Zhang; Wen-Feng Fang; Yuan-Yuan Zhao; Yun-Peng Yang; Cong Xue; Yan Huang

    2015-01-01

    Introduction:An increasing number of targeted drugs have been tested for the treatment of nasopharyngeal carcinoma (NPC). However, targeted therapy-related oncogenic mutations have not been fully evaluated. This study aimed to detect targeted therapy-related oncogenic mutations in NPC and to determine which targeted therapy might be potentially effective in treating NPC. Methods:By using the SNaPshot assay, a rapid detection method, 19 mutation hotspots in 6 targeted therapy-related oncogenes were examined in 70 NPC patients. The associations between oncogenic mutations and clinicopathologic factors were analyzed. Results:Among 70 patients, 12 (17.1%) had mutations in 5 oncogenes:7 (10.0%) had v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) mutation, 2 (2.8%) had epidermal growth factor receptor (EGFR) mutation, 1 (1.4%) had phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutation, 1 (1.4%) had Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, and 1 (1.4%) had simultaneous EGFR and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations. No significant differences were observed between oncogenic mutations and clinicopathologic characteristics. Additionally, these oncogenic mutations were not associated with tumor recurrence and metastasis. Conclusions:Oncogenic mutations are present in NPC patients. The efficacy of targeted drugs on patients with the related oncogenic mutations requires further validation.

  7. Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations

    International Nuclear Information System (INIS)

    Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds. Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot. Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAFV600E mutation. In BRAFV600E mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27Kip1. Specific inhibition of BRAF by RNAi in cells with BRAFV600E mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAFV600E mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2. Our results in thyroid cancer cells, namely those harbouring BRAFV600Emutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAFV600E mutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF

  8. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    Directory of Open Access Journals (Sweden)

    Jana eSlyskova

    2014-05-01

    Full Text Available Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER in peripheral blood cells based on modified comet assay protocols have been widely applied in human epidemiological studies. These provided some interesting observations of individual DNA repair activity being suppressed among cancer patients. However, extension of these results to cancer target tissues requires a different approach. Here we describe the evaluation of BER and NER activities in extracts from deep-frozen colon biopsies using an upgraded version of the in vitro comet-based DNA repair assay in which twelve reactions on one microscope slide can be performed. The aim of this report is to provide a detailed, easy-to-follow protocol together with results of optimization experiments. Additionally, results obtained by functional assays were analysed in the context of other cellular biomarkers, namely single nucleotide polymorphisms and gene expressions. We have shown that measuring DNA repair activity is not easily replaceable by genomic or transcriptomic approaches, but should be applied with the latter techniques in a complementary manner. The ability to measure DNA repair directly in cancer target tissues might finally answer questions about the tissue-specificity of DNA repair processes and their real involvement in the process of carcinogenesis.

  9. Survey of CF mutations in the clinical laboratory

    OpenAIRE

    Myers Angela; Nersesian Rhea; Mirkovic Borka; Huber Klaus; Saiki Randall; Bauer Kurt

    2002-01-01

    Abstract Background Since it is impossible to sequence the complete CFTR gene routinely, clinical laboratories must rely on test systems that screen for a panel of the most frequent mutations causing disease in a high percentage of patients. Thus, in a cohort of 257 persons that were referred to our laboratory for analysis of CF gene mutations, reverse line probe assays for the most common CF mutations were performed. These techniques were evaluated as routine first-line analyses of the CFTR ...

  10. Comprehensive Profiling of Radiosensitive Human Cell Lines with DNA Damage Response Assays Identifies the Neutral Comet Assay as a Potential Surrogate for Clonogenic Survival

    OpenAIRE

    Nahas, Shareef A.; Davies, Robert; Fike, Francesca; Nakamura, Kotoka; Du, Liutao; Kayali, Refik; Martin, Nathan T.; Concannon, Patrick; Gatti, Richard A.

    2011-01-01

    In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We c...

  11. Modelling cellular behaviour

    Science.gov (United States)

    Endy, Drew; Brent, Roger

    2001-01-01

    Representations of cellular processes that can be used to compute their future behaviour would be of general scientific and practical value. But past attempts to construct such representations have been disappointing. This is now changing. Increases in biological understanding combined with advances in computational methods and in computer power make it possible to foresee construction of useful and predictive simulations of cellular processes.

  12. p53 inactivation by point mutations and splice site mutations in human and mouse tumors

    OpenAIRE

    Magnússon, Kristinn P.

    1998-01-01

    The p53 tumor suppressor gene is frequently mutated in human tumors. p53 induces cell cycle arrest and/or apoptosis in response to cellular stress, such as DNA damage, hypoxia and certain activated oncogenes like c-myc. The status of p53 in Burkitt's Iymphoma (BL) cell lines was investigated. The majority of BL lines expressed mutated p53 protein. Functional reconstitution with exogenous wild type (wt) p53 induced apoptosis in a BL line that carried a mutated p53 gene. T...

  13. Comet assay on mice testicular cells

    OpenAIRE

    Anoop Kumar Sharma

    2015-01-01

    Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS)” has published classification criteria for germ cell mutagens (Speit et al., 2009). The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cel...

  14. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

    Science.gov (United States)

    Shinbrot, Eve; Henninger, Erin E; Weinhold, Nils; Covington, Kyle R; Göksenin, A Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M; Gibbs, Richard A; Sander, Chris; Pursell, Zachary F; Wheeler, David A

    2014-11-01

    Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

  15. Heterogeneous cellular networks

    CERN Document Server

    Hu, Rose Qingyang

    2013-01-01

    A timely publication providing coverage of radio resource management, mobility management and standardization in heterogeneous cellular networks The topic of heterogeneous cellular networks has gained momentum in industry and the research community, attracting the attention of standardization bodies such as 3GPP LTE and IEEE 802.16j, whose objectives are looking into increasing the capacity and coverage of the cellular networks. This book focuses on recent progresses,  covering the related topics including scenarios of heterogeneous network deployment, interference management i

  16. Cellular Reflectarray Antenna

    Science.gov (United States)

    Romanofsky, Robert R.

    2010-01-01

    The cellular reflectarray antenna is intended to replace conventional parabolic reflectors that must be physically aligned with a particular satellite in geostationary orbit. These arrays are designed for specified geographical locations, defined by latitude and longitude, each called a "cell." A particular cell occupies nominally 1,500 square miles (3,885 sq. km), but this varies according to latitude and longitude. The cellular reflectarray antenna designed for a particular cell is simply positioned to align with magnetic North, and the antenna surface is level (parallel to the ground). A given cellular reflectarray antenna will not operate in any other cell.

  17. Novel mutations and the emergence of a common mutation in the SDHD gene causing familial paraganglioma.

    Science.gov (United States)

    Milunsky, J M; Maher, T A; Michels, V V; Milunsky, A

    2001-05-15

    Familial paragangliomas (PGL) are slow-growing, highly vascular, generally benign neoplasms, usually of the head and neck, that arise from neural crest cells. This rare autosomal dominant disorder is highly penetrant and influenced by genomic imprinting through paternal transmission. Timely detection of these tumors may afford the affected individual the opportunity to avoid the potential serious morbidity associated with surgical removal and the mortality that may accompany local and distant metastases. Linkage to two distinct chromosomal loci, 11q13.1 and 11q23, has been previously reported. Recently, germline mutations in SDHD, a mitochondrial complex II gene on chromosome 11q23, have been demonstrated. We evaluated members of seven families with PGL, five previously studied and shown to have linkage to chromosome 11q23. The entire coding region of the SDHD gene was sequenced and yielded four novel mutations and one mutation shared in three of our unrelated families. Novel mutations found included a truncating mutation in exon 2, as well as a missense mutation, a deletion, and an insertion in exon 4. Three of our families had a common mutation in exon 3 (P81L) that has been reported and thought to be a founder mutation. A restriction enzyme assay was developed for initial screening of this mutation. Molecular analysis is now available and recommended for presymptomatic diagnosis in those at-risk individuals and for confirmatory diagnosis in those having PGL. PMID:11343322

  18. A DSPP Mutation Causing Dentinogenesis Imperfecta and Characterization of the Mutational Effect

    Directory of Open Access Journals (Sweden)

    Sook-Kyung Lee

    2013-01-01

    Full Text Available Mutations in the DSPP gene have been identified in nonsyndromic hereditary dentin defects, but the genotype-phenotype correlations are not fully understood. Recently, it has been demonstrated that the mutations of DSPP affecting the IPV leader sequence result in mutant DSPP retention in rough endoplasmic reticulum (ER. In this study, we identified a Korean family with dentinogenesis imperfecta type III. To identify the disease causing mutation in this family, we performed mutational analysis based on candidate gene sequencing. Exons and exon-intron boundaries of DSPP gene were sequenced, and the effects of the identified mutation on the pre-mRNA splicing and protein secretion were investigated. Candidate gene sequencing revealed a mutation (c.50C > T, p.P17L in exon 2 of the DSPP gene. The splicing assay showed that the mutation did not influence pre-mRNA splicing. However, the mutation interfered with protein secretion and resulted in the mutant protein remaining largely in the ER. These results suggest that the mutation affects ER-to-Golgi apparatus export and results in the reduction of secreted DSPP and ER overload. This may induce cell stress and damage processing and/or transport of dentin matrix proteins or other critical proteins.

  19. Inherited cardiomyopathies caused by troponin mutations

    Institute of Scientific and Technical Information of China (English)

    Qun-Wei Lu; Xiao-Yan Wu; Sachio Morimoto

    2013-01-01

    Genetic investigations of cardiomyopathy in the recent two decades have revealed a large number of mutations in the genes encoding sarcomeric proteins as a cause of inherited hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), or restrictive cardiomyopathy (RCM). Most functional analyses of the effects of mutations on cardiac muscle contraction have revealed significant changes in the Ca2+-regulatory mechanism, in which cardiac troponin (cTn) plays important structural and functional roles as a key regulatory protein. Over a hundred mutations have been identified in all three subunits of cTn, i.e., cardiac troponins T, I, and C. Recent studies on cTn mutations have provided plenty of evidence that HCM- and RCM-linked mutations increase cardiac myofilament Ca2+ sensitivity, while DCM-linked mutations decrease it. This review focuses on the functional consequences of mutations found in cTn in terms of cardiac myofilament Ca2+ sensitivity, ATPase activity, force generation, and cardiac troponin I phosphorylation, to understand potential molecular and cellular pathogenic mechanisms of the three types of inherited cardiomyopathy.

  20. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Energy Technology Data Exchange (ETDEWEB)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim; (OHSU- Cancer Instit.); (ARIAD)

    2010-09-07

    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  1. The Cobas® EGFR Mutation Test v2 assay.

    Science.gov (United States)

    Brown, Paul

    2016-02-01

    Paul Brown speaks to Gemma Westcott, Commissioning Editor: Paul Brown has served as the Head of Roche Molecular Diagnostics at Roche Diagnostics Corporation since February 2010 having previously held a variety of positions within Roche Pharma. After completing his doctorate in organic chemistry he was awarded a post-doctoral fellowship at the California Institute of Technology in Pasadena, CA, USA, but soon returned to his native UK to join Roche Pharma Research. Paul's first post within Roche was as group leader, doing drug discovery and making new small molecule drugs, but later moved into the business part of the company. Since then, he has enjoyed roles such as lifecycle leader for brands such as Tamiflu(®) and Xenical, vice president of sales and marketing of the pharmaceutical division and most recently general manager of Roche Pharma, Sweden. PMID:26838018

  2. Cellular oncogenes in neoplasia.

    OpenAIRE

    Chan, V T; McGee, J O

    1987-01-01

    In recent years cellular homologues of many viral oncogenes have been identified. As these genes are partially homologous to viral oncogenes and are activated in some tumour cell lines they are termed "proto-oncogenes". In tumour cell lines proto-oncogenes are activated by either quantitative or qualitative changes in gene structure: activation of these genes was originally thought to be a necessary primary event in carcinogenesis, but activated cellular oncogenes, unlike viral oncogenes, do ...

  3. Cellular Cardiomyoplasty: Clinical Application

    OpenAIRE

    Chachques, J. (J.); Acar, C; J. Herreros; Trainini, J. (Jorge); Prosper, F.; D’Attellis, N. (N.); Fabiani, J. N.; Carpentier, A

    2004-01-01

    Myocardial regeneration can be induced with the implantation of a variety of myogenic and angiogenic cell types. More than 150 patients have been treated with cellular cardiomyoplasty worldwide, 18 patients have been treated by our group. Cellular cardiomyoplasty seems to reduce the size and fibrosis of infarct scars, limit postischemic remodelling, and restore regional myocardial contractility. Techniques for skeletal myoblasts culture and ex vivo expansion using auto...

  4. Wide spectrum of developmental brain disorders from megalencephaly to focal cortical dysplasia and pigmentary mosaicism caused by mutations of MTOR

    Science.gov (United States)

    Solovieff, Nadia; Goold, Carleton; Jansen, Laura A.; Menon, Suchithra; Timms, Andrew E.; Conti, Valerio; Biag, Jonathan D.; Adams, Carissa; Boyle, Evan August; Collins, Sarah; Ishak, Gisele; Poliachik, Sandra; Girisha, Katta M.; Yeung, Kit San; Chung, Brian Hon Yin; Rahikkala, Elisa; Gunter, Sonya A.; McDaniel, Sharon S.; Macmurdo, Colleen Forsyth; Bernstein, Jonathan A.; Martin, Beth; Leary, Rebecca; Mahan, Scott; Liu, Shanming; Weaver, Molly; Doerschner, Michael; Jhangiani, Shalini; Muzny, Donna M.; Boerwinkle, Eric; Gibbs, Richard A.; Lupski, James R.; Shendure, Jay; Saneto, Russell P.; Novotny, Edward J.; Wilson, Christopher J.; Sellers, William R.; Morrissey, Michael; Hevner, Robert F.; Ojemann, Jeffrey G.; Guerrini, Renzo; Murphy, Leon O.; Winckler, Wendy; Dobyns, William B.

    2016-01-01

    Importance Focal cortical dysplasia (FCD), hemimegalencephaly (HMEG) and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. Collectively, these disorders are associated with significant childhood morbidity and mortality. FCD, in particular, represents the most frequent cause of intractable focal epilepsy in children. Objective To identify the underlying molecular etiology of FCD, HMEG, and diffuse megalencephaly. Design, Setting and Participants We performed whole exome sequencing (WES) on eight children with FCD or HMEG using standard depth (~50-60X) sequencing in peripheral samples (blood, saliva or skin) from the affected child and their parents, and deep (~150-180X) sequencing in affected brain tissue. We used both targeted sequencing and WES to screen a cohort of 93 children with molecularly unexplained diffuse or focal brain overgrowth (42 with FCD-HMEG, and 51 with diffuse megalencephaly). Histopathological and functional assays of PI3K-AKT-MTOR pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. Main Outcomes and Measures Whole exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. Results We identified low-level mosaic mutations of MTOR in brain tissue in four children with FCD type 2a with alternative allele fractions ranging from 0.012–0.086. We also identified intermediate level mosaic mutation of MTOR (p.Thr1977Ile) in three unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin that resembles hypomelanosis of Ito. Finally, we identified a constitutional de novo mutation of MTOR (p.Glu1799Lys) in three unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in two children with FCD type 2a from whom multiple affected brain tissue samples were available revealed a gradient of alternate allele

  5. Molecular and cellular fundamentals of stochastic radiation action

    International Nuclear Information System (INIS)

    Description of basic radiobiological processes in order to assess the risk of carcinogenesis due to radiotherapeutic procedures; demonstration of direct radiation-induced damage to DNA and indirect effects due to radiolysis of cellular water; description of repair mechanisms; radiation- induced chromosomal aberrations, genome mutations and disturbances of the cell cycle and their contributions to the carcinogenic effects of radiotherapy.(MG)

  6. Application of natural and amplification created restriction sites for the diagnosis of PKU mutations.

    OpenAIRE

    Eiken, H. G.; Odland, E; Boman, H.; Skjelkvåle, L; Engebretsen, L F; Apold, J.

    1991-01-01

    PCR amplification, either conventional, or as site directed mutagenesis using primers with mismatched 3'-ends, followed by restriction endonuclease digestion, provides rapid, non-isotope assays of known mutations in the human phenylalanine hydroxylase gene. Such assays were shown to have the potential to detect all of the 18 presently reported phenylketonuria mutations. The practical applicability of this approach was demonstrated for eight mutations in Norwegian phenylketonuria patients, amo...

  7. Application of natural and amplification created restriction sites for the diagnosis of PKU mutations.

    Science.gov (United States)

    Eiken, H G; Odland, E; Boman, H; Skjelkvåle, L; Engebretsen, L F; Apold, J

    1991-01-01

    PCR amplification, either conventional, or as site directed mutagenesis using primers with mismatched 3'-ends, followed by restriction endonuclease digestion, provides rapid, non-isotope assays of known mutations in the human phenylalanine hydroxylase gene. Such assays were shown to have the potential to detect all of the 18 presently reported phenylketonuria mutations. The practical applicability of this approach was demonstrated for eight mutations in Norwegian phenylketonuria patients, among them the most common ones. Images PMID:1851292

  8. Predicting Resistance Mutations Using Protein Design Algorithms

    Energy Technology Data Exchange (ETDEWEB)

    Frey, K.; Georgiev, I; Donald, B; Anderson, A

    2010-01-01

    Drug resistance resulting from mutations to the target is an unfortunate common phenomenon that limits the lifetime of many of the most successful drugs. In contrast to the investigation of mutations after clinical exposure, it would be powerful to be able to incorporate strategies early in the development process to predict and overcome the effects of possible resistance mutations. Here we present a unique prospective application of an ensemble-based protein design algorithm, K*, to predict potential resistance mutations in dihydrofolate reductase from Staphylococcus aureus using positive design to maintain catalytic function and negative design to interfere with binding of a lead inhibitor. Enzyme inhibition assays show that three of the four highly-ranked predicted mutants are active yet display lower affinity (18-, 9-, and 13-fold) for the inhibitor. A crystal structure of the top-ranked mutant enzyme validates the predicted conformations of the mutated residues and the structural basis of the loss of potency. The use of protein design algorithms to predict resistance mutations could be incorporated in a lead design strategy against any target that is susceptible to mutational resistance.

  9. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  10. Effect of cellular mobility on immune response

    Science.gov (United States)

    Pandey, R. B.; Mannion, R.; Ruskin, H. J.

    2000-08-01

    Mobility of cell types in our HIV immune response model is subject to an intrinsic mobility and an explicit directed mobility, which is governed by Pmob. We investigate how restricting the explicit mobility, while maintaining the innate mobility of a viral-infected cell, affects the model's results. We find that increasing the explicit mobility of the immune system cells leads to viral dominance for certain levels of viral mutation. We conclude that increasing immune system cellular mobility indirectly increases the virus’ inherent mobility.

  11. Cellular and molecular basis of cerebellar development

    Science.gov (United States)

    Martinez, Salvador; Andreu, Abraham; Mecklenburg, Nora; Echevarria, Diego

    2013-01-01

    Historically, the molecular and cellular mechanisms of cerebellar development were investigated through structural descriptions and studying spontaneous mutations in animal models and humans. Advances in experimental embryology, genetic engineering, and neuroimaging techniques render today the possibility to approach the analysis of molecular mechanisms underlying histogenesis and morphogenesis of the cerebellum by experimental designs. Several genes and molecules were identified to be involved in the cerebellar plate regionalization, specification, and differentiation of cerebellar neurons, as well as the establishment of cellular migratory routes and the subsequent neuronal connectivity. Indeed, pattern formation of the cerebellum requires the adequate orchestration of both key morphogenetic signals, arising from distinct brain regions, and local expression of specific transcription factors. Thus, the present review wants to revisit and discuss these morphogenetic and molecular mechanisms taking place during cerebellar development in order to understand causal processes regulating cerebellar cytoarchitecture, its highly topographically ordered circuitry and its role in brain function. PMID:23805080

  12. Cellular and Molecular Basis of Cerebellar Development

    Directory of Open Access Journals (Sweden)

    Salvador eMartinez

    2013-06-01

    Full Text Available Historically, the molecular and cellular mechanisms of cerebellar development were investigated through structural descriptions and studying spontaneous mutations in animal models and humans. Advances in experimental embryology, genetic engineering and neuroimaging techniques render today the possibility to approach the analysis of molecular mechanisms underlying histogenesis and morphogenesis of the cerebellum by experimental designs. Several genes and molecules were identified to be involved in the cerebellar plate regionalization, specification and differentiation of cerebellar neurons, as well as the establishment of cellular migratory routes and the subsequent neuronal connectivity. Indeed, pattern formation of the cerebellum requires the adequate orchestration of both key morphogenetic signals, arising from distinct brain regions, and local expression of specific transcription factors. Thus, the present review wants to revisit and discuss these morphogenetic and molecular mechanisms taking place during cerebellar development in order to understand causal processes regulating cerebellar cytoarchitecture, its highly topographically ordered circuitry and its role in brain function.

  13. A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

    DEFF Research Database (Denmark)

    Kjær, L.; Riley, C.H.; Westman, M.; Høgdall, E.; Bjerrum, O.W.; Hasselbalch, H.

    2012-01-01

    present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel...

  14. Effect of vitamin E on cytotoxicity, DNA single strand breaks, chromosomal aberrations, and mutation in Chinese hamster V-79 cells exposed to ultraviolet-B light

    Energy Technology Data Exchange (ETDEWEB)

    Sugiyama, M.; Tsuzuki, K.; Matsumoto, K.; Ogura, R. (Kurume Univ., Fukuoka (Japan). School of Medicine)

    1992-07-01

    The effect of pretreatment with vitamin E on cytotoxicity, DNA single strand breaks, and chromosomal aberrations as well as on mutation induced by ultraviolet-B light (UV-B) was investigated in Chinese hamster V-79 cells. Cellular pretreatment with non-toxic levels of 25 {mu}M {alpha}-tocopherol succinate (vitamin E) for 24h prior to exposure resulted in a 10-fold increase in cellular levels of {alpha}-tocopherol. Using a colony-forming assay, this pretreatment decreased the cytotoxicity of UV-B light. However, alkaline elution assays demonstrated that pretreatment with vitamin E did not affect the number of DNA single strand breaks caused by UV-B light. UV-B exposure produced a dose-dependent induction of chromosomal aberrations and mutations at the HGPRT locus, and neither of these actions of UV-B was influenced by pretreatment with the vitamin. These results suggest that vitamin E protects cells from UV-B-induced cytotoxicity, possibly through its ability to scavenge free radicals. The genotoxicity induced by UV-B light may not correlate directly with the cytotoxic action of this wavelength region in sunlight. (author).

  15. Effect of vitamin E on cytotoxicity, DNA single strand breaks, chromosomal aberrations, and mutation in Chinese hamster V-79 cells exposed to ultraviolet-B light

    International Nuclear Information System (INIS)

    The effect of pretreatment with vitamin E on cytotoxicity, DNA single strand breaks, and chromosomal aberrations as well as on mutation induced by ultraviolet-B light (UV-B) was investigated in Chinese hamster V-79 cells. Cellular pretreatment with non-toxic levels of 25 μM α-tocopherol succinate (vitamin E) for 24h prior to exposure resulted in a 10-fold increase in cellular levels of α-tocopherol. Using a colony-forming assay, this pretreatment decreased the cytotoxicity of UV-B light. However, alkaline elution assays demonstrated that pretreatment with vitamin E did not affect the number of DNA single strand breaks caused by UV-B light. UV-B exposure produced a dose-dependent induction of chromosomal aberrations and mutations at the HGPRT locus, and neither of these actions of UV-B was influenced by pretreatment with the vitamin. These results suggest that vitamin E protects cells from UV-B-induced cytotoxicity, possibly through its ability to scavenge free radicals. The genotoxicity induced by UV-B light may not correlate directly with the cytotoxic action of this wavelength region in sunlight. (author)

  16. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  17. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  18. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  19. Analysis of Hexanitrostilbene (HNS) and Dipicryethane (DPE) for Mutagenicity by the Ames/Salmonella Assay

    Energy Technology Data Exchange (ETDEWEB)

    Wu, R; Felton, J

    2007-10-12

    The Ames/Salmonella assay, developed by Professor Bruce Ames at the University of California, Berkeley, is a rapid and sensitive assay for detecting mutagenicity of various chemical compounds (Maron and Ames, 1983). It is a widely accepted short-term assay for detecting chemicals that induce mutations in the histidine (his) gene of Salmonella typhimurium. This is a reverse mutation assay that detects the mutational reversion of his-dependent Salmonella to the his-independent counterpart. Thereby, mutagenic compounds will increase the frequency of occurrence of his-independent bacterial colonies. The assay utilizes the specific genetically constructed strains of bacteria either with or without mammalian metabolic activation enzymes (S9), Aroclor induced rat liver homogenate to assess the mutagenicity of different compounds. In this study, we will use the Ames/Salmonella assay to investigate the mutagenicity of Hexanitrostilbene (HNS) from both Bofors and Pantex, and Dipicryethane (DPE).

  20. Tricyclic antidepressant radioreceptor assay

    International Nuclear Information System (INIS)

    A receptor assay for tricyclic antidepressants described here is based on the ability of these drugs to compete with [3H]-3-guinuclidnyl benzilate (3H-QNB) for binding to muscarinic cholinergic receptors in rat brain membranes. The assay is sensitive, in that it can detect, for example, 2ng/ml nortriptyline in plasma. Seven plasma samples from depressed patients treated with nortriptyline were assayed with the radioreceptor and gas liquid chromatographic methods, and the results from these two methods were almost identical. This assay should be used cautiously, if at all, in patients treated with other drugs that have potent anticholinergic effects. (Auth.)

  1. How do oncoprotein mutations rewire protein-protein interaction networks?

    Science.gov (United States)

    Bowler, Emily H; Wang, Zhenghe; Ewing, Rob M

    2015-01-01

    The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein-protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein-protein interaction networks and drive the cancer phenotype. PMID:26325016

  2. Irregular Cellular Learning Automata.

    Science.gov (United States)

    Esnaashari, Mehdi; Meybodi, Mohammad Reza

    2015-08-01

    Cellular learning automaton (CLA) is a recently introduced model that combines cellular automaton (CA) and learning automaton (LA). The basic idea of CLA is to use LA to adjust the state transition probability of stochastic CA. This model has been used to solve problems in areas such as channel assignment in cellular networks, call admission control, image processing, and very large scale integration placement. In this paper, an extension of CLA called irregular CLA (ICLA) is introduced. This extension is obtained by removing the structure regularity assumption in CLA. Irregularity in the structure of ICLA is needed in some applications, such as computer networks, web mining, and grid computing. The concept of expediency has been introduced for ICLA and then, conditions under which an ICLA becomes expedient are analytically found. PMID:25291810

  3. Architected Cellular Materials

    Science.gov (United States)

    Schaedler, Tobias A.; Carter, William B.

    2016-07-01

    Additive manufacturing enables fabrication of materials with intricate cellular architecture, whereby progress in 3D printing techniques is increasing the possible configurations of voids and solids ad infinitum. Examples are microlattices with graded porosity and truss structures optimized for specific loading conditions. The cellular architecture determines the mechanical properties and density of these materials and can influence a wide range of other properties, e.g., acoustic, thermal, and biological properties. By combining optimized cellular architectures with high-performance metals and ceramics, several lightweight materials that exhibit strength and stiffness previously unachievable at low densities were recently demonstrated. This review introduces the field of architected materials; summarizes the most common fabrication methods, with an emphasis on additive manufacturing; and discusses recent progress in the development of architected materials. The review also discusses important applications, including lightweight structures, energy absorption, metamaterials, thermal management, and bioscaffolds.

  4. Cellular Homeostasis and Aging.

    Science.gov (United States)

    Hartl, F Ulrich

    2016-06-01

    Aging and longevity are controlled by a multiplicity of molecular and cellular signaling events that interface with environmental factors to maintain cellular homeostasis. Modulation of these pathways to extend life span, including insulin-like signaling and the response to dietary restriction, identified the cellular machineries and networks of protein homeostasis (proteostasis) and stress resistance pathways as critical players in the aging process. A decline of proteostasis capacity during aging leads to dysfunction of specific cell types and tissues, rendering the organism susceptible to a range of chronic diseases. This volume of the Annual Review of Biochemistry contains a set of two reviews addressing our current understanding of the molecular mechanisms underlying aging in model organisms and humans. PMID:27050288

  5. Radioreceptor assay for epidermal growth factor

    International Nuclear Information System (INIS)

    An established cell line of human lung fibroblasts with a high number of surface receptors for mouse epidermal growth factor (mEGF) was used to deveop a simple and highly sensitive radioreceptor assay for EGF. 125I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF

  6. A new in vivo capillary assay of coagulation in invertebrates

    Directory of Open Access Journals (Sweden)

    C Bajzek

    2012-10-01

    Full Text Available Greater understanding of insect clotting requires better tests that can be performed in whole, living insects. We found we could collect hemolymph bleeding from wounded Drosophila larvae in microcapillary tubes. The capillary assay showed a difference in the amount of bleeding between feeding and wandering stage third instar larvae, and performed well with clotting mutations. This new in vivo coagulation assay will be helpful for studies of coagulation in Drosophila and other invertebrates.

  7. Nanoparticles for Use in Enzyme Assays.

    Science.gov (United States)

    Kim, Young-Pil; Kim, Hak-Sung

    2016-02-01

    Nanoparticles (NPs) have created new ways to enhance the performance of classical biosensors in analytical sciences. NPs with unprecedented physiochemical properties can serve both as excellent carriers of bioreceptors and as signal enhancers, leading to improved assay platforms with high sensitivity and selectivity. Because enzymes play central roles in many cellular functions, specific and precise assays of their functions are of great significance in medical science and biotechnology. Here we review recent advances in NP-based biosensors and their use in enzyme assays. With fast and specific responses to enzyme-mediated reactions, NPs transduce and amplify the initial responses into various types of signals, such as electrochemical, optical and magnetic ones. Translation of their potential should lead to functionalized NPs finding wide applications in diagnostics, drug development and biotechnology. PMID:26662229

  8. Wireless Cellular Mobile Communications

    Directory of Open Access Journals (Sweden)

    V. Zalud

    2002-12-01

    Full Text Available In this article is briefly reviewed the history of wireless cellularmobile communications, examined the progress in current secondgeneration (2G cellular standards and discussed their migration to thethird generation (3G. The European 2G cellular standard GSM and itsevolution phases GPRS and EDGE are described somewhat in detail. Thethird generation standard UMTS taking up on GSM/GPRS core network andequipped with a new advanced access network on the basis of codedivision multiple access (CDMA is investigated too. A sketch of theperspective of mobile communication beyond 3G concludes this article.

  9. Cellular tolerance to pulsed heating

    Science.gov (United States)

    Simanovski, Dimitrii; Sarkar, M.; Irani, A.; O'Connell-Rodwell, C.; Contag, C.; Schwettman, H. Alan; Palanker, D.

    2005-04-01

    Many laser therapies involve significant heating of tissue with pulses varying from picoseconds to minutes in duration. In some of the applications heating is a primary goal, while in others it is an undesirable side effect. In both cases, if a hyperthermia is involved, the knowledge about the threshold temperature leading to irreversible cellular damage is critically important. We study the dependence of the threshold temperature on duration of the heat exposure in the range of 0.3 ms to 5 seconds. Thin layer of cells cultured in a Petri dish was exposed to a pulsed CO2 laser radiation. Laser beam was focused onto sample providing Gaussian intensity distribution in the focal plane with a beam diameter (2w) 2-10 mm. Surface temperature in the central part of the focal spot (1mm in diameter) was measured by thermal infrared (IR) emission from the sample, recorded with a fast IR detector. For pulses shorter than 1 s the temperature profile across the focal spot was found to closely correspond to the radial distribution of the laser beam intensity, thus allowing for accurate determination of temperature at any given distance from the center of the spot. Immediate cellular damage was assessed using vital staining with the live/dead fluorescent assay. Threshold temperatures were found to vary from 65 °C at 5 s of heating to 160 °C at pulses of 0.3 ms in duration. The shorter end of this range was limited by vaporization, which occurs during the laser pulse and results in mechanical damage to cells. Dependence of the maximal temperature on pulse duration could be approximated by Arrhenius law with activation energy being about 1 eV.

  10. Translating partitioned cellular automata into classical type cellular automata

    OpenAIRE

    Poupet, Victor

    2008-01-01

    Partitioned cellular automata are a variant of cellular automata that was defined in order to make it very simple to create complex automata having strong properties such as number conservation and reversibility (which are often difficult to obtain on cellular automata). In this article we show how a partitioned cellular automaton can be translated into a regular cellular automaton in such a way that these properties are conserved.

  11. Breast tumor specific mutation in GATA3 affects physiological mechanisms regulating transcription factor turnover

    International Nuclear Information System (INIS)

    The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus. Immunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ERα agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). GATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERα and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation

  12. Cellular functions of p53 and p53 gene family members p63 and p73

    OpenAIRE

    Nadir Koçak; İbrahim Halil Yıldırım; Seval Cing Yıldırım

    2011-01-01

    p53 is a transcription factor that regulates multiple cellular processes that are also important in cellular fates such as cell cycle arrest or programmed cell death. Induction of growth arrest or cell death by p53 prevents the replication of damaged DNA and proliferation of genetically abnormal cells. Therefore, inactivation of p53 by mutation or deletion is also important in ensuring the cellular homeostasis. However, studies showed that p53 deficient mice and cells such as Saos-2 cells are...

  13. Genetic Dominance & Cellular Processes

    Science.gov (United States)

    Seager, Robert D.

    2014-01-01

    In learning genetics, many students misunderstand and misinterpret what "dominance" means. Understanding is easier if students realize that dominance is not a mechanism, but rather a consequence of underlying cellular processes. For example, metabolic pathways are often little affected by changes in enzyme concentration. This means that…

  14. Radioactivity of cellular concrete

    International Nuclear Information System (INIS)

    The natural radioactivity of cellular concrete is discussed. Some data on the concentrations of 40K, 226Ra and 232Th in building materials in Poland are given. The results of dose rates measurements in living quarters as well as outside are presented. (A.S.)

  15. The New Cellular Immunology

    Science.gov (United States)

    Claman, Henry N.

    1973-01-01

    Discusses the nature of the immune response and traces many of the discoveries that have led to the present state of knowledge in immunology. The new cellular immunology is directing its efforts toward improving health by proper manipulation of the immune mechanisms of the body. (JR)

  16. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  17. Mutation distributions and clinical correlations of PIK3CA gene mutations in breast cancer.

    Science.gov (United States)

    Dirican, Ebubekir; Akkiprik, Mustafa; Özer, Ayşe

    2016-06-01

    Breast cancer (BCa) is the most common cancer and the second cause of death among women. Phosphoinositide 3-kinase (PI3K) signaling pathway has a crucial role in the cellular processes such as cell survival, growth, division, and motility. Moreover, oncogenic mutations in the PI3K pathway generally involve the activation phosphatidylinositol-4,5-bisphosphate 3-kinase-catalytic subunit alpha (PIK3CA) mutation which has been identified in numerous BCa subtypes. In this review, correlations between PIK3CA mutations and their clinicopathological parameters on BCa will be described. It is reported that PIK3CA mutations which have been localized mostly on exon 9 and 20 hot spots are detected 25-40 % in BCa. This relatively high frequency can offer an advantage for choosing the best treatment options for BCa. PIK3CA mutations may be used as biomarkers and have been major focus of drug development in cancer with the first clinical trials of PI3K pathway inhibitors currently in progress. Screening of PIK3CA gene mutations might be useful genetic tests for targeted therapeutics or diagnosis. Increasing data about PIK3CA mutations and its clinical correlations with BCa will help to introduce new clinical applications in the near future. PMID:26921096

  18. Mutation directional selection sheds light on prion pathogenesis

    International Nuclear Information System (INIS)

    Highlights: → Most pathogenic mutations possess strong directional selection, i.e., enhancing hydrophobicity or decreasing negative and increasing positive charge. → Mutation-induced changes may strengthen the interactions between PrP and facilitating factors. → The findings also have significant implications for exploring potential regions involved in the conformational transition from PrPC to PrPSc. -- Abstract: As mutations in the PRNP gene account for human hereditary prion diseases (PrDs), it is crucial to elucidating how these mutations affect the central pathogenic conformational transition of normal cellular prion protein (PrPC) to abnormal scrapie isoform (PrPSc). Many studies proposed that these pathogenic mutations may make PrP more susceptible to conformational change through altering its structure stability. By evaluating the most recent observations regarding pathogenic mutations, it was found that the pathogenic mutations do not exert a uniform effect on the thermodynamic stability of the human PrP's structure. Through analyzing the reported PrDs-related mutations, we found that 25 out of 27 mutations possess strong directional selection, i.e., enhancing hydrophobicity or decreasing negative and increasing positive charge. Based on the triggering role reported by previous studies of facilitating factors in PrPC conversion, e.g., lipid and polyanion, we proposed that the mutation-induced changes may strengthen the interaction between PrP and facilitating factors, which will accelerate PrP conversion and cause PrDs.

  19. Characterization of pathogenic germline mutations in human Protein Kinases

    Directory of Open Access Journals (Sweden)

    Orengo Christine A

    2011-07-01

    Full Text Available Abstract Background Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. Results Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. Conclusions Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

  20. Illuminating cellular physiology: recent developments.

    Science.gov (United States)

    Brovko, Lubov Y; Griffiths, Mansel W

    2007-01-01

    Bioluminescent methods are gaining more and more attention among scientists due to their sensitivity, selectivity and simplicity; coupled with the fact that the bioluminescence can be monitored both in vitro and in vivo. Since the discovery of bioluminescence in the 19th century, enzymes involved in the bioluminescent process have been isolated and cloned. The bioluminescent reactions in several different organisms have also been fully characterized and used as reporters in a wide variety of biochemical assays. From the 1990s it became clear that bioluminescence can be detected and quantified directly from inside a living cell. This gave rise to numerous possibilities for the in vivo monitoring of intracellular processes non-invasively using bioluminescent molecules as reporters. This review describes recent developments in the area of bioluminescent imaging for cell biology. Newly developed imaging methods allow transcriptional/translational regulation, signal transduction, protein-protein interaction, oncogenic transformation, cell and protein trafficking, and target drug action to be monitored in vivo in real-time with high temporal and spatial resolution; thus providing researchers with priceless information on cellular functions. Advantages and limitations of these novel bioluminescent methods are discussed and possible future developments identified. PMID:17725230

  1. Deletion mutations of bacteriophage

    International Nuclear Information System (INIS)

    Resolution of mutation mechanism with structural changes of DNA was discussed through the studies using bacteriophage lambda. One of deletion mutations inductions of phage lambda is the irradiation of ultraviolet ray. It is not clear if the inductions are caused by errors in reparation of ultraviolet-induced damage or by the activation of int gene. Because the effective site of int gene lies within the regions unnecessary for existing, it is considered that int gene is connected to deletion mutations induction. A certain system using prophage complementarity enables to detect deletion mutations at essential hereditary sites and to solve the relations of deletion mutations with other recombination system, DNA reproduction and repairment system. Duplication and multiplication of hereditary elements were discussed. If lambda deletion mutations of the system, which can control recombination, reproduction and repairment of added DNA, are constructed, mutations mechanism with great changes of DNA structure can be solved by phage lambda. (Ichikawa, K.)

  2. An insight in to effect of dose, dose rate and confounding factors on radiation induced DNA damage and repair using comet assay

    International Nuclear Information System (INIS)

    Ionizing radiation is known to induce a variety of DNA lesions such as single strand breaks (SSBs), double strand breaks (DSBs), and oxidative damage to bases, interstrand cross-links and locally multiplies damaged sites (LMDs). However, the most dangerous DNA lesions which are responsible for the origin of lethal effects, mutagenesis, genomic instability and carcinogenesis are the DSBs and LMDs. Humans are at high risk of exposure to low doses of ionizing radiation either through environmental or occupational exposures. It is known that following exposure to doses below 10 cGy mammalian cells adapt to subsequent higher doses of ionizing radiation exposures a phenomenon known as adaptive response. Neither the changes induced by low dose ionizing radiation nor the biochemical pathways that signal this low dose radiation effect are well studied. The genetic effect of ionizing radiation depends on the radiation dose as well as on the dose rate at which it is delivered. The radiation induced cellular effects such as chromosome aberrations, sister chromatid exchange, micronucleus formation, transformation, mutations and changes in gene expression can cause cancer, cell death or damage can transmitted to subsequent generations. Contradictory reports exist in literature about variation in genetic response as function of dose and dose rates. There are different methods available to detect the DNA damage such as Neutral and Alkaline elution assay, DNA unwinding assay, Comet assay, Halo assay, FISH-comet assay, gamma-H2AX. Comet assay is a valuable technique which allows detection of DNA damage and repair at single cell level and provides a unique opportunity to investigate intercellular differences in any eukaryotic cell population. Thus, there is need to evaluate the utility and accuracy of different techniques used for estimation of radiation induced DNA damage. Here, we report our observations on the effect of low-dose, low dose rates, low dose limit, type of radiation

  3. CPTAC Assay Portal: a repository of targeted proteomic assays

    Energy Technology Data Exchange (ETDEWEB)

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  4. p53 mutations increase resistance to ionizing radiation

    International Nuclear Information System (INIS)

    Mouse and human tumors of diverse origin frequently have somatically acquired mutations or rearrangements of the p53 gene, or they have lost one or both copies of the gene. Although wild-type p53 protein is believed to function as a tumor-suppressor gene, it is as yet unclear how p53 mutations lead to neoplastic development. Wild-type p53 has been postulated to play a role in DNA repair, suggesting that expression of mutant forms of p53 might alter cellular resistance to the DNA damage caused by γ radiation. Moreover, p53 is thought to function as a cell cycle checkpoint after irradiation, also suggesting that mutant p53 might change the cellular proliferative response to radiation. The authors have used transgenic mice expressing one of two mutant alleles of p53 to test this prediction. Their results show that expression of both mutant variants of the mouse p53 gene significantly increases the cellular resistance of a variety of hematopoietic cell lineages to γ radiation. These observations provide direct evidence that p53 mutations affect the cellular response to DNA damage, either by increasing DNA repair processes or, possibly, by increasing cellular tolerance to DNA damage. The association of p53 mutations with increased radioresistance suggests possible mechanisms through which alterations in the p53 gene might lead to oncogenic transformation. 53 refs., 5 figs

  5. Cellular regulation of the dopamine transporter

    DEFF Research Database (Denmark)

    Eriksen, Jacob

    2010-01-01

    The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. DAT and its trafficking...... single-membrane spanning protein Tac, thereby creating an extracellular antibody epitope. Upon expression in HEK293 cells this TacDAT fusion protein displayed functional properties similar to the wild type transporter. In an ELISA based internalization assay, TacDAT intracellular accumulation was...

  6. New concept: cellular senescence in pathophysiology of cholangiocarcinoma.

    Science.gov (United States)

    Sasaki, Motoko; Nakanuma, Yasuni

    2016-05-01

    Cholangiocarcinoma, a malignant tumor arising in the hepatobiliary system, presents with poor prognosis because of difficulty in its early detection/diagnosis. Recent progress revealed that cellular senescence may be involved in the pathophysiology of cholangiocarcinoma. Cellular senescence is defined as permanent growth arrest caused by several cellular injuries, such as oncogenic mutations and oxidative stress. "Oncogene-induced" and/or stress-induced senescence may occur in the process of multi-step cholangiocarcinogenesis, and overexpression of a polycomb group protein EZH2 may play a role in the escape from, and/or bypassing of, senescence. Furthermore, senescent cells may play important roles in tumor development and progression via the production of senescence-associated secretory phenotypes. Cellular senescence may be a new target for the prevention, early diagnosis, and therapy of cholangiocarcinoma in the near future. PMID:26680649

  7. Comet assay to measure DNA repair: approach and applications

    OpenAIRE

    Azqueta, Amaya; SLYSKOVA, JANA; Langie, Sabine A. S.; O’Neill Gaivão, Isabel; Collins, Andrew

    2014-01-01

    Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of ...

  8. Development of an in vivo assay for non-targeted radiation effects based on response rather than dose

    International Nuclear Information System (INIS)

    An adaptive response may be defined as the effect of a small priming dose of radiation modifying the anticipated cellular response of the same tissues so as to alter the predicted response to a larger dose of radiation. We and others have demonstrated that at low radiation doses (less than 0.5 Gy) the lethal and mutational effect of the radiation is mainly, possibly entirely, due to the non-targeted effects. This is the dose range for priming doses in adaptive response protocols. In an associated presentation from our group, we demonstrate that the adaptive response may be explicable as a non targeted (bystander) response. In this paper we present data from exposed people, showing that a simple assay using blood can demonstrate variation in the extent and type of non-targeted effects and that exposure to radiation can modulate the subsequent non-targeted response to a later dose. The serum from blood samples of exposed individuals was harvested, diluted in tissue culture medium and added to reporter cells. The toxicity or growth promoting activity of the serum was measured using a clonogenic assay coupled with immunocytochemical measurement of various proteins involved in apoptosis or growth. While the assay has only been applied to human blood so far, it is expected that it would be useful for monitoring response to low doses in fish or mammals. There is already evidence that bystander effects are controlled by both genetic and epigenetic (lifestyle) factors. These data could support the development of a simple blood based assay to predict overall response of critical species to low doses of radiation taking all the low dose factors into account. A key element of the assay is that it allows response to be measured and correlates response rather than dose with harm. (author)

  9. Rapid screen for truncating ATM mutations by PTT-ELISA

    Energy Technology Data Exchange (ETDEWEB)

    Du Liutao; Lai, C.-H. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, CA 90095 (United States); Concannon, Patrick [Department of Biochemistry and Molecular Genetics, University of Virginia, VA 22908 (United States); Gatti, Richard A. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, CA 90095 (United States); Department of Human Genetics, David Geffen School of Medicine at UCLA, CA 90095 (United States)], E-mail: rgatti@mednet.ucla.edu

    2008-04-02

    Mutations in the ataxia-telangiectasia mutated (ATM) gene are responsible for the autosomal recessive genetic disorder, ataxia-telangiectasia (A-T). Approximately 80% of ATM mutations found in A-T patients results in truncations, which can be detected by Protein Truncation Test (PTT). Conventional PTT uses SDS-PAGE electrophoresis to detect mobility of radiolabeled truncated protein fragments. In this study, we developed a non-radioactive Protein Truncation Test which utilizes an enzyme-linked immunosorbent assay (PTT-ELISA) to detect ATM mutations in eight overlapping fragments. N- and C-terminal epitopes (c-myc and V5, respectively) were introduced into transcription/translation products, which could then be detected by Sandwich ELISA. Using this assay, we screened 9 newly diagnosed A-T patients consecutively. Of the 18 expected mutations, 14 truncating mutations were independently identified by cDNA direct sequencing and/or DNA dHPLC analysis. PTT-ELISA detected all of these 14. Four mutations were novel. The PTT-ELISA provides a rapid method for detecting truncating mutations in large genes and should be considered prior to using more laborious or costly methods, such as direct sequencing.

  10. Molecular and Cellular Signaling

    CERN Document Server

    Beckerman, Martin

    2005-01-01

    A small number of signaling pathways, no more than a dozen or so, form a control layer that is responsible for all signaling in and between cells of the human body. The signaling proteins belonging to the control layer determine what kinds of cells are made during development and how they function during adult life. Malfunctions in the proteins belonging to the control layer are responsible for a host of human diseases ranging from neurological disorders to cancers. Most drugs target components in the control layer, and difficulties in drug design are intimately related to the architecture of the control layer. Molecular and Cellular Signaling provides an introduction to molecular and cellular signaling in biological systems with an emphasis on the underlying physical principles. The text is aimed at upper-level undergraduates, graduate students and individuals in medicine and pharmacology interested in broadening their understanding of how cells regulate and coordinate their core activities and how diseases ...

  11. The Quiescent Cellular State is Arf/p53-Dependent and Associated with H2AX Downregulation and Genome Stability

    Directory of Open Access Journals (Sweden)

    Mitsuko Masutani

    2012-05-01

    Full Text Available Cancer is a disease associated with genomic instability and mutations. Excluding some tumors with specific chromosomal translocations, most cancers that develop at an advanced age are characterized by either chromosomal or microsatellite instability. However, it is still unclear how genomic instability and mutations are generated during the process of cellular transformation and how the development of genomic instability contributes to cellular transformation. Recent studies of cellular regulation and tetraploidy development have provided insights into the factors triggering cellular transformation and the regulatory mechanisms that protect chromosomes from genomic instability.

  12. Electromagnetic cellular interactions

    Czech Academy of Sciences Publication Activity Database

    Cifra, Michal; Fields, J. S.; Farhadi, A.

    2011-01-01

    Roč. 105, č. 3 (2011), 223-246. ISSN 0079-6107. [36th International Congress of Physiological Sciences (IUPS2009). Kyoto, 27.07.2009-01.08.2009] R&D Projects: GA ČR(CZ) GPP102/10/P454 Institutional research plan: CEZ:AV0Z20670512 Keywords : bioelectric phenomena * cellular biophysics Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 3.203, year: 2011

  13. Magnetic Cellular Switches

    OpenAIRE

    Overby, Darryl R.; Alenghat, Francis J.; Montoya-Zavala, Martín; Bei, HuCheng; Oh, Philmo; Karavitis, John; Ingber, Donald E.

    2004-01-01

    This paper focuses on the development of magnetic cellular switches to enable magnetic control of intracellular functions in living mammalian cells, including receptor signal transduction and gene transcription. Our approach takes advantage of the mechanosensitivity of adenosine 3′,5′-monophosphate (cAMP) induction and downstream transcription controlled by the cAMP regulatory element (CRE) to engineer gene constructs that optically report gene expression in living cells. We activate transcri...

  14. Cellular therapy in Tuberculosis

    Directory of Open Access Journals (Sweden)

    Shreemanta K. Parida

    2015-03-01

    Full Text Available Cellular therapy now offer promise of potential adjunct therapeutic options for treatment of drug-resistant tuberculosis (TB. We review here the role of Mesenchymal stromal cells, (MSCs, as well as other immune effector cells in the therapy of infectious diseases with a focus on TB. MSCs represent a population of tissue-resident non-hematopoietic adult progenitor cells which home into injured tissues increase the proliferative potential of broncho-alveolar stem cells and restore lung epithelium. MSCs have been shown to be immune-modulatory and anti-inflammatory mediated via cell-cell contacts as well as soluble factors. We discuss the functional profile of MSCs and their potential use for adjunct cellular therapy of multi-drug resistant TB, with the aim of limiting tissue damage, and to convert unproductive inflammatory responses into effective anti-pathogen directed immune responses. Adjunct cellular therapy could potentially offer salvage therapy options for patients with drug-resistant TB, increase clinically relevant anti-M.tuberculosis directed immune responses and possibly shorten the duration of anti-TB therapy.

  15. Cellular therapy in tuberculosis.

    Science.gov (United States)

    Parida, Shreemanta K; Madansein, Rajhmun; Singh, Nalini; Padayatchi, Nesri; Master, Iqbal; Naidu, Kantharuben; Zumla, Alimuddin; Maeurer, Markus

    2015-03-01

    Cellular therapy now offer promise of potential adjunct therapeutic options for treatment of drug-resistant tuberculosis (TB). We review here the role of Mesenchymal stromal cells, (MSCs), as well as other immune effector cells in the therapy of infectious diseases with a focus on TB. MSCs represent a population of tissue-resident non-hematopoietic adult progenitor cells which home into injured tissues increase the proliferative potential of broncho-alveolar stem cells and restore lung epithelium. MSCs have been shown to be immune-modulatory and anti-inflammatory mediated via cell-cell contacts as well as soluble factors. We discuss the functional profile of MSCs and their potential use for adjunct cellular therapy of multi-drug resistant TB, with the aim of limiting tissue damage, and to convert unproductive inflammatory responses into effective anti-pathogen directed immune responses. Adjunct cellular therapy could potentially offer salvage therapy options for patients with drug-resistant TB, increase clinically relevant anti-M.tuberculosis directed immune responses and possibly shorten the duration of anti-TB therapy. PMID:25809753

  16. Quantum cellular automata

    Science.gov (United States)

    Porod, Wolfgang; Lent, Craig S.; Bernstein, Gary H.

    1994-06-01

    The Notre Dame group has developed a new paradigm for ultra-dense and ultra-fast information processing in nanoelectronic systems. These Quantum Cellular Automata (QCA's) are the first concrete proposal for a technology based on arrays of coupled quantum dots. The basic building block of these cellular arrays is the Notre Dame Logic Cell, as it has been called in the literature. The phenomenon of Coulomb exclusion, which is a synergistic interplay of quantum confinement and Coulomb interaction, leads to a bistable behavior of each cell which makes possible their use in large-scale cellular arrays. The physical interaction between neighboring cells has been exploited to implement logic functions. New functionality may be achieved in this fashion, and the Notre Dame group invented a versatile majority logic gate. In a series of papers, the feasibility of QCA wires, wire crossing, inverters, and Boolean logic gates was demonstrated. A major finding is that all logic functions may be integrated in a hierarchial fashion which allows the design of complicated QCA structures. The most complicated system which was simulated to date is a one-bit full adder consisting of some 200 cells. In addition to exploring these new concepts, efforts are under way to physically realize such structures both in semiconductor and metal systems. Extensive modeling work of semiconductor quantum dot structures has helped identify optimum design parameters for QCA experimental implementations.

  17. The neutral comet assay

    International Nuclear Information System (INIS)

    The single-cell gel electrophoresis (or comet) assay is a rapid and sensitive fluorescence microscopic method that is used for the detection of DNA damage at the individual cell level. Comet assay under neutral conditions allows the detection of DNA double-strand breaks, considered to the biologically relevant radiation-induced lesion. In this report we describe modifications of the neutral comet method and its use for studying DNA damage and its repair in irradiated human lymphocytes. (author)

  18. Environment Aware Cellular Networks

    KAUST Repository

    Ghazzai, Hakim

    2015-02-01

    The unprecedented rise of mobile user demand over the years have led to an enormous growth of the energy consumption of wireless networks as well as the greenhouse gas emissions which are estimated currently to be around 70 million tons per year. This significant growth of energy consumption impels network companies to pay huge bills which represent around half of their operating expenditures. Therefore, many service providers, including mobile operators, are looking for new and modern green solutions to help reduce their expenses as well as the level of their CO2 emissions. Base stations are the most power greedy element in cellular networks: they drain around 80% of the total network energy consumption even during low traffic periods. Thus, there is a growing need to develop more energy-efficient techniques to enhance the green performance of future 4G/5G cellular networks. Due to the problem of traffic load fluctuations in cellular networks during different periods of the day and between different areas (shopping or business districts and residential areas), the base station sleeping strategy has been one of the main popular research topics in green communications. In this presentation, we present several practical green techniques that provide significant gains for mobile operators. Indeed, combined with the base station sleeping strategy, these techniques achieve not only a minimization of the fossil fuel consumption but also an enhancement of mobile operator profits. We start with an optimized cell planning method that considers varying spatial and temporal user densities. We then use the optimal transport theory in order to define the cell boundaries such that the network total transmit power is reduced. Afterwards, we exploit the features of the modern electrical grid, the smart grid, as a new tool of power management for cellular networks and we optimize the energy procurement from multiple energy retailers characterized by different prices and pollutant

  19. Validation of the cell cycle G2 delay assay in assessing ionizing radiation sensitivity and breast cancer risk

    International Nuclear Information System (INIS)

    Genetic variations in cell cycle checkpoints and DNA repair genes are associated with prolonged cell cycle G2 delay following ionizing radiation (IR) treatment and breast cancer risk. However, different studies reported conflicting results examining the association between post-IR cell cycle delay and breast cancer risk utilizing four different parameters: cell cycle G2 delay index, %G2–M, G2/G0–G1, and (G2/G0–G1)/S. Therefore, we evaluated whether different parameters may influence study results using a data set from 118 breast cancer cases and 225 controls as well as lymphoblastoid and breast cancer cell lines with different genetic defects. Our results suggest that cell cycle G2 delay index may serve as the best parameter in assessing breast cancer risk, genetic regulation of IR-sensitivity, and mutations of ataxia telangiectasia mutated (ATM) and TP53. Cell cycle delay in 21 lymphoblastoid cell lines derived from BRCA1 mutation carriers was not different from that in controls. We also showed that IR-induced DNA-damage signaling, as measured by phosphorylation of H2AX on serine 139 (γ-H2AX) was inversely associated with cell cycle G2 delay index. In summary, the cellular responses to IR are extremely complex; mutations or genetic variations in DNA damage signaling, cell cycle checkpoints, and DNA repair contribute to cell cycle G2 delay and breast cancer risk. The cell cycle G2 delay assay characterized in this study may help identify subpopulations with elevated risk of breast cancer or susceptibility to adverse effects in normal tissue following radiotherapy

  20. Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding.

    Science.gov (United States)

    McReynolds, Andrea C; Karra, Aroon S; Li, Yan; Lopez, Elias Daniel; Turjanski, Adrian G; Dioum, Elhadji; Lorenz, Kristina; Zaganjor, Elma; Stippec, Steve; McGlynn, Kathleen; Earnest, Svetlana; Cobb, Melanie H

    2016-03-29

    The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183. ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligonucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site. PMID:26950759

  1. Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

    Science.gov (United States)

    Itsara, Leslie S; Kennedy, Scott R; Fox, Edward J; Yu, Selina; Hewitt, Joshua J; Sanchez-Contreras, Monica; Cardozo-Pelaez, Fernando; Pallanck, Leo J

    2014-02-01

    The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations. PMID:24516391

  2. Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

    Directory of Open Access Journals (Sweden)

    Leslie S Itsara

    2014-02-01

    Full Text Available The accumulation of somatic mitochondrial DNA (mtDNA mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5, an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine, did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.

  3. Mucopolysaccharidosis type VII: Characterization of mutations and molecular heterogeneity

    OpenAIRE

    Tomatsu, Shunji; Fukuda, Seiji; Sukegawa, Kazuko; Ikedo, Yuko; Yamada, Shinji; Yamada, Yukiji; Sasaki, Toshiya; Okamoto, Hiroyuki; Kuwahara, Takashi; Yamaguchi, Seiji; Kiman, Tadashi; Shintaku, Haruo; Isshiki, Gen; Orii, Tadao

    1991-01-01

    We identified two different exonic point mutations causing β-glucuronidase (βGl) deficiency in two Japanese patients with mucopolysaccharidosis type VII (MPSVII). Enzyme assay of lysates of the lymphocytes and cultured fibroblasts showed little residual activity. The βGl-specific mRNA levels were normal, as determined by northern blot analysis. Mutated cDNA clones, including the entire coding sequence, were isolated using the polymerase chain reaction (PCR) products derived from βGl-deficient...

  4. Mutations in the FHA-domain of ectopically expressed NBS1 lead to radiosensitization and to no increase in somatic mutation rates via a partial suppression of homologous recombination

    International Nuclear Information System (INIS)

    Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy. (author)

  5. Application of statistical process control to qualitative molecular diagnostic assays

    LENUS (Irish Health Repository)

    O'Brien, Cathal P.

    2014-11-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  6. Understanding TERT Promoter Mutations: A Common Path to Immortality.

    Science.gov (United States)

    Bell, Robert J A; Rube, H Tomas; Xavier-Magalhães, Ana; Costa, Bruno M; Mancini, Andrew; Song, Jun S; Costello, Joseph F

    2016-04-01

    Telomerase (TERT) activation is a fundamental step in tumorigenesis. By maintaining telomere length, telomerase relieves a main barrier on cellular lifespan, enabling limitless proliferation driven by oncogenes. The recently discovered, highly recurrent mutations in the promoter ofTERTare found in over 50 cancer types, and are the most common mutation in many cancers. Transcriptional activation ofTERT, via promoter mutation or other mechanisms, is the rate-limiting step in production of active telomerase. AlthoughTERTis expressed in stem cells, it is naturally silenced upon differentiation. Thus, the presence ofTERTpromoter mutations may shed light on whether a particular tumor arose from a stem cell or more differentiated cell type. It is becoming clear thatTERTmutations occur early during cellular transformation, and activate theTERTpromoter by recruiting transcription factors that do not normally regulateTERTgene expression. This review highlights the fundamental and widespread role ofTERTpromoter mutations in tumorigenesis, including recent progress on their mechanism of transcriptional activation. These somatic promoter mutations, along with germline variation in theTERTlocus also appear to have significant value as biomarkers of patient outcome. Understanding the precise molecular mechanism ofTERTactivation by promoter mutation and germline variation may inspire novel cancer cell-specific targeted therapies for a large number of cancer patients.Mol Cancer Res; 14(4); 315-23. ©2016 AACR. PMID:26941407

  7. A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

    Directory of Open Access Journals (Sweden)

    Chen Zhang

    2013-01-01

    Full Text Available Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis. In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient.

  8. Effective Temperature of Mutations

    Science.gov (United States)

    Derényi, Imre; Szöllősi, Gergely J.

    2015-02-01

    Biological macromolecules experience two seemingly very different types of noise acting on different time scales: (i) point mutations corresponding to changes in molecular sequence and (ii) thermal fluctuations. Examining the secondary structures of a large number of microRNA precursor sequences and model lattice proteins, we show that the effects of single point mutations are statistically indistinguishable from those of an increase in temperature by a few tens of kelvins. The existence of such an effective mutational temperature establishes a quantitative connection between robustness to genetic (mutational) and environmental (thermal) perturbations.

  9. Assay method and compositions

    International Nuclear Information System (INIS)

    Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine(3H)-methyl. The O-methylated (3H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin-3H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin-3H and raising the pH of the aqueous periodate phase from which O-methylated (3H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

  10. Failover in cellular automata

    CERN Document Server

    Kumar, Shailesh

    2010-01-01

    A cellular automata (CA) configuration is constructed that exhibits emergent failover. The configuration is based on standard Game of Life rules. Gliders and glider-guns form the core messaging structure in the configuration. The blinker is represented as the basic computational unit, and it is shown how it can be recreated in case of a failure. Stateless failover using primary-backup mechanism is demonstrated. The details of the CA components used in the configuration and its working are described, and a simulation of the complete configuration is also presented.

  11. Cellular-scale hydrodynamics

    DEFF Research Database (Denmark)

    Abkarian, Manouk; Faivre, Magalie; Horton, Renita; Smistrup, Kristian; Best-Popescu, Catherine A; Stone, Howard A.

    2008-01-01

    Microfluidic tools are providing many new insights into the chemical, physical and physicochemical responses of cells. Both suspension-level and single-cell measurements have been studied. We review our studies of these kinds of problems for red blood cells with particular focus on the shapes of ...... mechanical effects on suspended cells can be studied systematically in small devices, and how these features can be exploited to develop methods for characterizing physicochemical responses and possibly for the diagnosis of cellular-scale changes to environmental factors....

  12. Cellular mechanics and motility

    Science.gov (United States)

    Hénon, Sylvie; Sykes, Cécile

    2015-10-01

    The term motility defines the movement of a living organism. One widely known example is the motility of sperm cells, or the one of flagellar bacteria. The propulsive element of such organisms is a cilium(or flagellum) that beats. Although cells in our tissues do not have a flagellum in general, they are still able to move, as we will discover in this chapter. In fact, in both cases of movement, with or without a flagellum, cell motility is due to a dynamic re-arrangement of polymers inside the cell. Let us first have a closer look at the propulsion mechanism in the case of a flagellum or a cilium, which is the best known, but also the simplest, and which will help us to define the hydrodynamic general conditions of cell movement. A flagellum is sustained by cellular polymers arranged in semi-flexible bundles and flagellar beating generates cell displacement. These polymers or filaments are part of the cellular skeleton, or "cytoskeleton", which is, in this case, external to the cellular main body of the organism. In fact, bacteria move in a hydrodynamic regime in which viscosity dominates over inertia. The system is thus in a hydrodynamic regime of low Reynolds number (Box 5.1), which is nearly exclusively the case in all cell movements. Bacteria and their propulsion mode by flagella beating are our unicellular ancestors 3.5 billion years ago. Since then, we have evolved to form pluricellular organisms. However, to keep the ability of displacement, to heal our wounds for example, our cells lost their flagellum, since it was not optimal in a dense cell environment: cells are too close to each other to leave enough space for the flagella to accomplish propulsion. The cytoskeleton thus developed inside the cell body to ensure cell shape changes and movement, and also mechanical strength within a tissue. The cytoskeleton of our cells, like the polymers or filaments that sustain the flagellum, is also composed of semi-flexible filaments arranged in bundles, and also in

  13. Radiolabelled Cellular Blood Elements

    International Nuclear Information System (INIS)

    This volume contains the abstracts of the 5th International Symposion on Radiolabelling of Cellular Blood Elements to be held in Vienna, Austria, September 10-14, 1989. The Meeting is the fifth in a series of meetings designed to discuss the basics and clinical application of radiolabelling techniques. In these days, beside the search for new labelling agents and extending the knowledge in clinical use, the use of monoclonal antibodies is a big new challenge. All reviewed contributions that have been accepted for presentation are contained in this volume. (authors) 58 of them are of INIS scope

  14. Determination of somatic mutations in human erythrocytes by cytometry

    International Nuclear Information System (INIS)

    Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab

  15. Determination of somatic mutations in human erythrocytes by cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, R.H.; Langlois, R.G.; Bigbee, W.L.

    1985-06-21

    Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab.

  16. A multi-detection assay for malaria transmitting mosquitoes.

    Science.gov (United States)

    Lee, Yoosook; Weakley, Allison M; Nieman, Catelyn C; Malvick, Julia; Lanzaro, Gregory C

    2015-01-01

    The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays. PMID:25867057

  17. Repair and mutagenesis in procaryotes as cellular responses to ambiental agents

    International Nuclear Information System (INIS)

    The correct and incorrect mechanisms of DNA repair are discussed, as well as the cellular responses induced by the DNA lesions; the reductone mollecular effects; the cellular interactions among irradiated populations of microorganisms and the utilization of microbial assays for the detection of oncogenic activities of chemicals. (M.A.)

  18. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  19. Rover waste assay system

    International Nuclear Information System (INIS)

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  20. Xeroderma Pigmentosum-Trichothiodystrophy overlap patient with novel XPD/ERCC2 mutation

    DEFF Research Database (Denmark)

    Kralund, Henrik H; Ousager, Lilian; Jaspers, Nicolaas G;

    2013-01-01

    Xeroderma Pigmentosum (XP), Trichothiodystrophy (TTD) and Cockayne Syndrome (CS) are rare, recessive disorders caused by mutational defects in the Nucleotide Excision Repair (NER) pathway and/or disruption of basic cellular DNA transcription. To date, a multitude of mutations in the XPD/ERCC2 gen...

  1. Gestational mutations in radiation carcinogenesis

    Science.gov (United States)

    Meza, R.; Luebeck, G.; Moolgavkar, S.

    Mutations in critical genes during gestation could increase substantially the risk of cancer. We examine the consequences of such mutations using the Luebeck-Moolgavkar model for colorectal cancer and the Lea-Coulson modification of the Luria-Delbruck model for the accumulation of mutations during gestation. When gestational mutation rates are high, such mutations make a significant contribution to cancer risk even for adult tumors. Furthermore, gestational mutations ocurring at distinct times during emryonic developmemt lead to substantially different numbers of mutated cells at birth, with early mutations leading to a large number (jackpots) of mutated cells at birth and mutation occurring late leading to only a few mutated cells. Thus gestational mutations could confer considerable heterogeneity of the risk of cancer. If the fetus is exposed to an environmental mutagen, such as ionizing radiation, the gestational mutation rate would be expected to increase. We examine the consequences of such exposures during gestation on the subsequent development of cancer.

  2. Ubiquitin Metabolism Affects Cellular Response to Volatile Anesthetics in Yeast

    OpenAIRE

    Wolfe, Darren; Reiner, Thomas; Keeley, Jessica L.; Pizzini, Mark; Keil, Ralph L.

    1999-01-01

    To investigate the mechanism of action of volatile anesthetics, we are studying mutants of the yeast Saccharomyces cerevisiae that have altered sensitivity to isoflurane, a widely used clinical anesthetic. Several lines of evidence from these studies implicate a role for ubiquitin metabolism in cellular response to volatile anesthetics: (i) mutations in the ZZZ1 gene render cells resistant to isoflurane, and the ZZZ1 gene is identical to BUL1 (binds ubiquitin ligase), which appears to be invo...

  3. The GARP complex is required for cellular sphingolipid homeostasis

    DEFF Research Database (Denmark)

    Fröhlich, Florian; Petit, Constance; Kory, Nora;

    2015-01-01

    (GARP) complex, which functions in endosome-to-Golgi retrograde vesicular transport, as a critical player in sphingolipid homeostasis. GARP deficiency leads to accumulation of sphingolipid synthesis intermediates, changes in sterol distribution, and lysosomal dysfunction. A GARP complex mutation...... the phenotypes of GARP-deficient yeast or mammalian cells. Together, these data show that GARP is essential for cellular sphingolipid homeostasis and suggest a therapeutic strategy for the treatment of PCCA2....

  4. Neutral Comet Assay

    OpenAIRE

    2013-01-01

    The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The ...

  5. Lateral flow strip assay

    Science.gov (United States)

    Miles, Robin R.; Benett, William J.; Coleman, Matthew A.; Pearson, Francesca S.; Nasarabadi, Shanavaz L.

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  6. Automated phantom assay system

    International Nuclear Information System (INIS)

    This paper describes an automated phantom assay system developed for assaying phantoms spiked with minute quantities of radionuclides. The system includes a computer-controlled linear-translation table that positions the phantom at exact distances from a spectrometer. A multichannel analyzer (MCA) interfaces with a computer to collect gamma spectral data. Signals transmitted between the controller and MCA synchronize data collection and phantom positioning. Measured data are then stored on disk for subsequent analysis. The automated system allows continuous unattended operation and ensures reproducible results

  7. Frequent DPH3 promoter mutations in skin cancers.

    Science.gov (United States)

    Denisova, Evgeniya; Heidenreich, Barbara; Nagore, Eduardo; Rachakonda, P Sivaramakrishna; Hosen, Ismail; Akrap, Ivana; Traves, Víctor; García-Casado, Zaida; López-Guerrero, José Antonio; Requena, Celia; Sanmartin, Onofre; Serra-Guillén, Carlos; Llombart, Beatriz; Guillén, Carlos; Ferrando, Jose; Gimeno, Enrique; Nordheim, Alfred; Hemminki, Kari; Kumar, Rajiv

    2015-11-01

    Recent reports suggested frequent occurrence of cancer associated somatic mutations within regulatory elements of the genome. Based on initial exome sequencing of 21 melanomas, we report frequent somatic mutations in skin cancers in a bidirectional promoter of diphthamide biosynthesis 3 (DPH3) and oxidoreductase NAD-binding domain containing 1 (OXNAD1) genes. The UV-signature mutations occurred at sites adjacent and within a binding motif for E-twenty six/ternary complex factors (Ets/TCF), at -8 and -9 bp from DPH3 transcription start site. Follow up screening of 586 different skin lesions showed that the DPH3 promoter mutations were present in melanocytic nevi (2/114; 2%), melanoma (30/304; 10%), basal cell carcinoma of skin (BCC; 57/137; 42%) and squamous cell carcinoma of skin (SCC; 12/31; 39%). Reporter assays carried out in one melanoma cell line for DPH3 and OXNAD1 orientations showed statistically significant increased promoter activity due to -8/-9CC > TT tandem mutations; although, no effect of the mutations on DPH3 and OXNAD1 transcription in tumors was observed. The results from this study show occurrence of frequent somatic non-coding mutations adjacent to a pre-existing binding site for Ets transcription factors within the directional promoter of DPH3 and OXNAD1 genes in three major skin cancers. The detected mutations displayed typical UV signature; however, the functionality of the mutations remains to be determined. PMID:26416425

  8. Identification and functional analysis of novel THAP1 mutations.

    Science.gov (United States)

    Lohmann, Katja; Uflacker, Nils; Erogullari, Alev; Lohnau, Thora; Winkler, Susen; Dendorfer, Andreas; Schneider, Susanne A; Osmanovic, Alma; Svetel, Marina; Ferbert, Andreas; Zittel, Simone; Kühn, Andrea A; Schmidt, Alexander; Altenmüller, Eckart; Münchau, Alexander; Kamm, Christoph; Wittstock, Matthias; Kupsch, Andreas; Moro, Elena; Volkmann, Jens; Kostic, Vladimir; Kaiser, Frank J; Klein, Christine; Brüggemann, Norbert

    2012-02-01

    Mutations in THAP1 have been associated with dystonia 6 (DYT6). THAP1 encodes a transcription factor that represses the expression of DYT1. To further evaluate the mutational spectrum of THAP1 and its associated phenotype, we sequenced THAP1 in 567 patients with focal (n = 461), segmental (n = 68), or generalized dystonia (n = 38). We identified 10 novel variants, including six missense substitutions within the DNA-binding Thanatos-associated protein domain (Arg13His, Lys16Glu, His23Pro, Lys24Glu, Pro26Leu, Ile80Val), a 1bp-deletion downstream of the nuclear localization signal (Asp191Thrfs*9), and three alterations in the untranslated regions. The effect of the missense variants was assessed using prediction tools and luciferase reporter gene assays. This indicated the Ile80Val substitution as a benign variant. The subcellular localization of Asp191Thrfs*9 suggests a disturbed nuclear import for this mutation. Thus, we consider six of the 10 novel variants as pathogenic mutations accounting for a mutation frequency of 1.1%. Mutation carriers presented mainly with early onset dystonia (<12 years in five of six patients). Symptoms started in an arm or neck and spread to become generalized in three patients or segmental in two patients. Speech was affected in four mutation carriers. In conclusion, THAP1 mutations are rare in unselected dystonia patients and functional analysis is necessary to distinguish between benign variants and pathogenic mutations. PMID:21847143

  9. High Resolution Melting Analysis for Detecting p53 Gene Mutations in Patients with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Zhihong CHEN

    2011-10-01

    Full Text Available Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

  10. PAP-1, the mutated gene underlying the RP9 form of dominant retinitis pigmentosa, is a splicing factor

    International Nuclear Information System (INIS)

    PAP-1 is an in vitro phosphorylation target of the Pim-1 oncogene. Although PAP-1 binds to Pim-1, it is not a substrate for phosphorylation by Pim-1 in vivo. PAP-1 has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). However, RP9 is a rare disease and only two missense mutations have been described, so the report of a link between PAP-1 and RP9 was tentative. The precise cellular role of PAP-1 was also unknown at that time. We now report that PAP-1 localizes in nuclear speckles containing the splicing factor SC35 and interacts directly with another splicing factor, U2AF35. Furthermore, we used in vitro and in vivo splicing assays to show that PAP-1 has an activity, which alters the pattern of pre-mRNA splicing and that this activity is dependent on the phosphorylation state of PAP-1. We used the same splicing assay to examine the activities of two mutant forms of PAP-1 found in RP9 patients. The results showed that while one of the mutations, H137L, had no effect on splicing activity compared with that of wild-type PAP-1, the other, D170G, resulted in both a defect in splicing activity and a decreased proportion of phosphorylated PAP-1. The D170G mutation may therefore cause RP by altering splicing of retinal genes through a decrease in PAP-1 phosphorylation. These results demonstrate that PAP-1 has a role in pre-mRNA splicing and, given that three other splicing factors have been implicated in adRP, this finding provides compelling further evidence that PAP-1 is indeed the RP9 gene

  11. Algorithms for Detecting Significantly Mutated Pathways in Cancer

    Science.gov (United States)

    Vandin, Fabio; Upfal, Eli; Raphael, Benjamin J.

    Recent genome sequencing studies have shown that the somatic mutations that drive cancer development are distributed across a large number of genes. This mutational heterogeneity complicates efforts to distinguish functional mutations from sporadic, passenger mutations. Since cancer mutations are hypothesized to target a relatively small number of cellular signaling and regulatory pathways, a common approach is to assess whether known pathways are enriched for mutated genes. However, restricting attention to known pathways will not reveal novel cancer genes or pathways. An alterative strategy is to examine mutated genes in the context of genome-scale interaction networks that include both well characterized pathways and additional gene interactions measured through various approaches. We introduce a computational framework for de novo identification of subnetworks in a large gene interaction network that are mutated in a significant number of patients. This framework includes two major features. First, we introduce a diffusion process on the interaction network to define a local neighborhood of "influence" for each mutated gene in the network. Second, we derive a two-stage multiple hypothesis test to bound the false discovery rate (FDR) associated with the identified subnetworks. We test these algorithms on a large human protein-protein interaction network using mutation data from two recent studies: glioblastoma samples from The Cancer Genome Atlas and lung adenocarcinoma samples from the Tumor Sequencing Project. We successfully recover pathways that are known to be important in these cancers, such as the p53 pathway. We also identify additional pathways, such as the Notch signaling pathway, that have been implicated in other cancers but not previously reported as mutated in these samples. Our approach is the first, to our knowledge, to demonstrate a computationally efficient strategy for de novo identification of statistically significant mutated subnetworks. We

  12. Dominant β-catenin mutations cause intellectual disability with recognizable syndromic features

    OpenAIRE

    Tucci, Valter; Kleefstra, Tjitske; Hardy, Andrea; Heise, Ines; Maggi, Silvia; Willemsen, Marjolein H.; Hilton, Helen; Esapa, Chris; Simon, Michelle; Buenavista, Maria-Teresa; McGuffin, Liam; Vizor, Lucie; Dodero, Luca; Tsaftaris, Sotirios; Romero, Rosario

    2014-01-01

    The recent identification of multiple dominant mutations in the gene encoding β-catenin in both humans and mice has enabled exploration of the molecular and cellular basis of β-catenin function in cognitive impairment. In humans, β-catenin mutations that cause a spectrum of neurodevelopmental disorders have been identified. We identified de novo β-catenin mutations in patients with intellectual disability, carefully characterized their phenotypes, and were able to define a r...

  13. Mapping Mutations on Phylogenies

    DEFF Research Database (Denmark)

    Nielsen, Rasmus

    2005-01-01

    This chapter provides a short review of recent methodologies developed for mapping mutations on phylogenies. Mapping of mutations, or character changes in general, using the maximum parsimony principle has been one of the most powerful tools in phylogenetics, and it has been used in a variety of...

  14. EFFECT OF THE ANTIMUTAGENS VANILLIN AND CINNAMALDEHYDE ON THE SPONTANEOUS MUTATION SPECTRA OF SALMONELLA TA104

    Science.gov (United States)

    Effect of the Antimutagens Vanillin and Cinnamaldehyde on the / Spontaneous Mutation Spectra of Salmonella TAlO4 Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that, when added to assay plates, reduced the spontaneous mutant frequency in Salmonella typhi...

  15. Integrated cellular systems

    Science.gov (United States)

    Harper, Jason C.

    The generation of new three-dimensional (3D) matrices that enable integration of biomolecular components and whole cells into device architectures, without adversely altering their morphology or activity, continues to be an expanding and challenging field of research. This research is driven by the promise that encapsulated biomolecules and cells can significantly impact areas as diverse as biocatalysis, controlled delivery of therapeutics, environmental and industrial process monitoring, early warning of warfare agents, bioelectronics, photonics, smart prosthetics, advanced physiological sensors, portable medical diagnostic devices, and tissue/organ replacement. This work focuses on the development of a fundamental understanding of the biochemical and nanomaterial mechanisms that govern the cell directed assembly and integration process. It was shown that this integration process relies on the ability of cells to actively develop a pH gradient in response to evaporation induced osmotic stress, which catalyzes silica condensation within a thin 3D volume surrounding the cells, creating a functional bio/nano interface. The mechanism responsible for introducing functional foreign membrane-bound proteins via proteoliposome addition to the silica-lipid-cell matrix was also determined. Utilizing this new understanding, 3D cellular immobilization capabilities were extended using sol-gel matrices endowed with glycerol, trehalose, and media components. The effects of these additives, and the metabolic phase of encapsulated S. cerivisiase cells, on long-term viability and the rate of inducible gene expression was studied. This enabled the entrapment of cells within a novel microfluidic platform capable of simultaneous colorimetric, fluorescent, and electrochemical detection of a single analyte, significantly improving confidence in the biosensor output. As a complementary approach, multiphoton protein lithography was utilized to engineer 3D protein matrices in which to

  16. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H;

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  17. Instrument for assaying radiation

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  18. Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology

    OpenAIRE

    Hovhannisyan Galina G

    2010-01-01

    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...

  19. Multiplex, Quantitative, Real-Time PCR Assay for Cytomegalovirus and Human DNA

    OpenAIRE

    Sanchez, Jason L.; Storch, Gregory A.

    2002-01-01

    We created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copie...

  20. Bacterial Stationary-State Mutagenesis and Mammalian Tumorigenesis as Stress-Induced Cellular Adaptations and the Role of Epigenetics

    OpenAIRE

    Karpinets, TV; Greenwood, DJ; Pogribny, IP; Samatova, NF

    2006-01-01

    Mechanisms of cellular adaptation may have some commonalities across different organisms. Revealing these common mechanisms may provide insight in the organismal level of adaptation and suggest solutions to important problems related to the adaptation. An increased rate of mutations, referred as the mutator phenotype, and beneficial nature of these mutations are common features of the bacterial stationary-state mutagenesis and of the tumorigenic transformations in mammalian cells. We argue th...

  1. Mutational signatures of de-differentiation in functional non-coding regions of melanoma genomes.

    Directory of Open Access Journals (Sweden)

    Stephen C J Parker

    Full Text Available Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular

  2. Genotype MTBDR plus assay for molecular detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Soniya Sharma

    2014-01-01

    Full Text Available Aim: This study was performed for the rapid identification of Mycobacterium tuberculosis complex and its resistance to rifampicin and isoniazid, directly from the sputum samples of pulmonary tuberculosis patients. Materials and Methods: A commercially available genotype MTBDR plus assay was used for the identification and detection of mutations in Mycobacterial isolates. A total of 100 sputum samples of pulmonary tuberculosis patients were analyzed by using the genotype MTBDR plus assay. The MTBDR plus assay is designed to detect the mutations in the hotspot region of rpoB gene, katG and regulatory region of inhA gene. Results: The genotype MTBDR plus assay detected 22% multidrug resistant (MDR, 2% rifampicin (RMP monoresistant and 1% isoniazid (INH monoresistant isolates. In 22 MDR isolates, the codons most frequently involved in RMP-associated mutations were codon 531 (54.55%, 516 (31.82% and 526 (13.63%, and 90.90% of MDR isolates showed KatG S315T mutations and 9.1% showed inhA C-15T mutations associated with INH resistance. Conclusion: The new genotype MTBDR plus assay represents a rapid, reliable tool for the detection of MDR-TB, wherein results are obtained in 5 h allowing early and appropriate treatment, which is essential to cut the transmission path and reduce the spread of MDR-TB. The genotype MTBDR plus assay can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.

  3. Multiuser Cellular Network

    CERN Document Server

    Bao, Yi; Chen, Ming

    2011-01-01

    Modern radio communication is faced with a problem about how to distribute restricted frequency to users in a certain space. Since our task is to minimize the number of repeaters, a natural idea is enlarging coverage area. However, coverage has restrictions. First, service area has to be divided economically as repeater's coverage is limited. In this paper, our fundamental method is to adopt seamless cellular network division. Second, underlying physics content in frequency distribution problem is interference between two close frequencies. Consequently, we choose a proper frequency width of 0.1MHz and a relevantly reliable setting to apply one frequency several times. We make a few general assumptions to simplify real situation. For instance, immobile users yield to homogenous distribution; repeaters can receive and transmit information in any given frequency in duplex operation; coverage is mainly decided by antenna height. Two models are built up to solve 1000 users and 10000 users situations respectively....

  4. Modeling and cellular studies

    International Nuclear Information System (INIS)

    Testing the applicability of mathematical models with carefully designed experiments is a powerful tool in the investigations of the effects of ionizing radiation on cells. The modeling and cellular studies complement each other, for modeling provides guidance for designing critical experiments which must provide definitive results, while the experiments themselves provide new input to the model. Based on previous experimental results the model for the accumulation of damage in Chlamydomonas reinhardi has been extended to include various multiple two-event combinations. Split dose survival experiments have shown that models tested to date predict most but not all the observed behavior. Stationary-phase mammalian cells, required for tests of other aspects of the model, have been shown to be at different points in the cell cycle depending on how they were forced to stop proliferating. These cultures also demonstrate different capacities for repair of sublethal radiation damage

  5. Engineering Cellular Metabolism

    DEFF Research Database (Denmark)

    Nielsen, Jens; Keasling, Jay

    2016-01-01

    Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds......, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges...... of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation....

  6. SPOP Mutations in Prostate Cancer across Demographically Diverse Patient Cohorts

    Directory of Open Access Journals (Sweden)

    Mirjam Blattner

    2014-01-01

    Full Text Available BACKGROUND: Recurrent mutations in the Speckle-Type POZ Protein (SPOP gene occur in up to 15% of prostate cancers. However, the frequency and features of cancers with these mutations across different populations is unknown. OBJECTIVE: To investigate SPOP mutations across diverse cohorts and validate a series of assays employing high-resolution melting (HRM analysis and Sanger sequencing for mutational analysis of formalin-fixed paraffin-embedded material. DESIGN, SETTING, AND PARTICIPANTS: 720 prostate cancer samples from six international cohorts spanning Caucasian, African American, and Asian patients, including both prostate-specific antigen-screened and unscreened populations, were screened for their SPOP mutation status. Status of SPOP was correlated to molecular features (ERG rearrangement, PTEN deletion, and CHD1 deletion as well as clinical and pathologic features. RESULTS AND LIMITATIONS: Overall frequency of SPOP mutations was 8.1% (4.6% to 14.4%, SPOP mutation was inversely associated with ERG rearrangement (P < .01, and SPOP mutant (SPOPmut cancers had higher rates of CHD1 deletions (P < .01. There were no significant differences in biochemical recurrence in SPOPmut cancers. Limitations of this study include missing mutational data due to sample quality and lack of power to identify a difference in clinical outcomes. CONCLUSION: SPOP is mutated in 4.6% to 14.4% of patients with prostate cancer across different ethnic and demographic backgrounds. There was no significant association between SPOP mutations with ethnicity, clinical, or pathologic parameters. Mutual exclusivity of SPOP mutation with ERG rearrangement as well as a high association with CHD1 deletion reinforces SPOP mutation as defining a distinct molecular subclass of prostate cancer.

  7. Analysis of cellular phosphatidylinositol (3,4,5)-trisphosphate levels and distribution using confocal fluorescent microscopy.

    Science.gov (United States)

    Palmieri, Michelle; Nowell, Cameron J; Condron, Melanie; Gardiner, James; Holmes, Andrew B; Desai, Jayesh; Burgess, Antony W; Catimel, Bruno

    2010-11-01

    We have developed an immunocytochemistry method for the semiquantitative detection of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) at the cell plasma membrane. This protocol combines the use of a glutathione S-transferase-tagged pleckstrin homology (PH) domain of the general phosphoinositides-1 receptor (GST-GRP1PH) with fluorescence confocal microscopy and image segmentation using cell mask software analysis. This methodology allows the analysis of PI(3,4,5)P3 subcellular distribution in resting and epidermal growth factor (EGF)-stimulated HEK293T cells and in LIM1215 (wild-type phosphoinositide 3-kinase (PI3K)) and LIM2550 (H1047R mutation in PI3K catalytic domain) colonic carcinoma cells. Formation of PI(3,4,5)P3 was observed 5min following EGF stimulation and resulted in an increase of the membrane/cytoplasm fluorescence ratio from 1.03 to 1.53 for HEK293T cells and from 2.2 to 3.3 for LIM1215 cells. Resting LIM2550 cells stained with GST-GRP1PH had an elevated membrane/cytoplasm fluorescence ratio of 9.8, suggesting constitutive PI3K activation. The increase in the membrane/cytoplasm fluorescent ratio was inhibited in a concentration-dependent manner by the PI3K inhibitor LY294002. This cellular confocal imaging assay can be used to directly assess the effects of PI3K mutations in cancer cell lines and to determine the potential specificity and effectiveness of PI3K inhibitors in cancer cells. PMID:20599646

  8. Clinical relevance of sensitive and quantitative STAT3 mutation analysis using next-generation sequencing in T-cell large granular lymphocytic leukemia

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Larsen, Martin; Rewes, Annika; Frederiksen, Henrik; Thomassen, Mads; Møller, Michael Boe

    2014-01-01

    , therefore, represents a promising marker in T-LGL diagnostics. We developed a new quantitative next-generation sequencing assay that allows sensitive analysis of the STAT3 gene. The assay was used to study the utility of STAT3 mutation analysis as a diagnostic tool in T-LGL. The study included 16 T......-LGL patients. A total of 15 mutations, including 2 new mutations (G618R and K658R), were detected in 12 patients (75%), with mutation levels ranging from 2.5% to 45.6% mutation-positive alleles. Next-generation sequencing detected 50% more mutations than Sanger sequencing. Blood samples from 20 healthy blood...... donors all tested negative, thus demonstrating the specificity of the assay. The results also indicated that mutation levels in blood and bone marrow are not systematically different, and next-generation sequencing-based STAT3 mutation analysis represents a sensitive method for monitoring residual...

  9. Molecular nature of spontaneous and radiation-induced mutations with allelic loss in human cells

    International Nuclear Information System (INIS)

    An assay system was developed for dissecting the second step in loss of function mutations and for determining the potency of physical and chemical agents to produce such mutations. Results obtained suggest that somatic recombination and/or deletion occurs close to the border between the heterochromatic and euchromatic regions of chromosome 16q. (author)

  10. Hereditary and acquired p53 gene mutations in childhood acute lymphoblastic leukemia.

    OpenAIRE

    Felix, C A; Nau, M M; Takahashi, T.; Mitsudomi, T.; Chiba, I.; Poplack, D G; Reaman, G H; Cole, D E; Letterio, J J; Whang-Peng, J

    1992-01-01

    The p53 gene was examined in primary lymphoblasts of 25 pediatric patients with acute lymphoblastic leukemia by the RNase protection assay and by single strand conformation polymorphism analysis in 23 of 25 cases. p53 mutations were found to occur, but at a low frequency (4 of 25). While all four mutations were identified by single strand conformation polymorphism, the comparative sensitivity of RNase protection was 50% (2 of 4). Heterozygosity was retained at mutated codons in 3 of 4 cases. ...

  11. Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Kazuko Sakai

    Full Text Available Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10(-5. The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100. KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100. DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

  12. Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning

    OpenAIRE

    Tabone, Tania; Sallmann, Georgina; Webb, Elizabeth; Cotton, Richard G.H.

    2006-01-01

    We report the development of a simple and inexpensive assay for the detection of DNA polymorphisms and mutations that is based on the modification of mismatched bases by potassium permanganate. Unlike the chemical cleavage of mismatch assay, which also exploits the reactivity of potassium permanganate to detect genomic variants, the assay we describe here does not require a cleavage manipulation and therefore does not require expensive or toxic chemicals or a separation step, as mismatches ar...

  13. The State of Cellular Probes

    OpenAIRE

    Yim, Youngbin

    2003-01-01

    Cellular probe technology is one of several potentially promising technologies for obtaining accurate travel time information. In 1996, the Federal Communications Commission (FCC) mandated E911 requirements that cellular location be provided when 911 emergency calls come in to emergency management authorities. The E911 requirements allow 50 -300 meters from the emergency call location, depending on the type of cellular phone technology used and whether handset-based or network-based solutions...

  14. Never-ageing cellular senescence

    OpenAIRE

    Ogrunc, Müge; d’Adda di Fagagna, Fabrizio

    2011-01-01

    Cellular senescence was historically discovered as a form of cellular ageing of in vitro cultured cells. It has been under the spotlight following the evidence of oncogene-induced senescence in vivo and its role as a potent tumour suppressor mechanism. Presently, a PubMed search using keywords ‘cellular senescence and cancer’ reveals 8398 number of references (by April 2011) showing that while our knowledge of senescence keeps expanding, the complexity of the phenomenon keeps us – researchers...

  15. Dual isotope assays

    International Nuclear Information System (INIS)

    Dual isotope assays for thyroid function are performed by carrying out a radio-immunoassay for two of thyroxine (T4), tri-iodothyronine (T3), thyroid stimulating hormone (TSH), and thyroxine binding globulin (TBG), by a method wherein a version of one of the thyroid components, preferably T4 or T3 is labelled with Selenium-75 and the version of the other thyroid component is labelled with a different radionuclide, preferably Iodine-125. (author)

  16. Automated uranium assays

    International Nuclear Information System (INIS)

    Precise, timely inventories of enriched uranium stocks are vital to help prevent the loss, theft, or diversion of this material for illicit use. A wet-chemistry analyzer has been developed at LLL to assist in these inventories by performing automated analyses of uranium samples from different stages in the nuclear fuel cycle. These assays offer improved accuracy, reduced costs, significant savings in manpower, and lower radiation exposure for personnel compared with present techniques

  17. Disagreement between Human Papillomavirus Assays

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte Møller;

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four...... assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41......-65 years (n = 2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays. While 4% of primary screening samples showed abnormal cytology, 6 to 10% were discordant on any pair of assays. A literature review corroborated our findings...

  18. Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays.

    Directory of Open Access Journals (Sweden)

    Lena Poulsen

    Full Text Available BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%. Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.

  19. Active Cellular Nematics

    Science.gov (United States)

    Duclos, Guillaume; Erlenkaemper, Christoph; Garcia, Simon; Yevick, Hannah; Joanny, Jean-François; Silberzan, Pascal; Biology inspired physics at mesoscales Team; Physical approach of biological problems Team

    We study the emergence of a nematic order in a two-dimensional tissue of apolar elongated fibroblast cells. Initially, these cells are very motile and the monolayer is characterized by giant density fluctuations, a signature of far-from-equilibrium systems. As the cell density increases because of proliferation, the cells align with each other forming large perfectly oriented domains while the cellular movements slow down and eventually freeze. Therefore topological defects characteristic of nematic phases remain trapped at long times, preventing the development of infinite domains. By analogy with classical non-active nematics, we have investigated the role of boundaries and we have shown that cells confined in stripes of width smaller than typically 500 µm are perfectly aligned in the stripe direction. Experiments performed in cross-shaped patterns show that both the number of cells and the degree of alignment impact the final orientation. Reference: Duclos G., Garcia S., Yevick H.G. and Silberzan P., ''Perfect nematic order in confined monolayers of spindle-shaped cells'', Soft Matter, 10, 14, 2014

  20. Real-time electrocatalytic sensing of cellular respiration.

    Science.gov (United States)

    Yip, Nga-Chi; Rawson, Frankie J; Tsang, Chi Wai; Mendes, Paula M

    2014-07-15

    In the present work we develop a real-time electrochemical mediator assay to enable the assessment of cell numbers and chemical toxicity. This allowed us to monitor metabolism down to a single cell in a low cost easy to use rapid assay which is not possible with current technology. The developed assay was based on the determination of oxygen. This was made possible via the use of electrochemical mediator ferrocene carboxylic acid (FcA). The FcA showed distinctive catalytic properties in interacting with reactive oxygen species generated from oxygen when compared to ferrocene methanol (FcMeOH). A deeper insight into the chemistry controlling this behaviour is provided. The behaviour is then taken advantage of to develop a cellular aerobic respiration assay. We describe the properties of the FcA system to detect, in real-time, the oxygen consumption of Escherichia coli DH5-α (E. coli). We demonstrated that the FcA-based oxygen assay is highly sensitive, and using a population of cells, oxygen consumption rates could be calculated down to a single cell level. More importantly, the results can be accomplished in minutes, considerably outperforming current commercially available biooxygen demand assays. The developed assay is expected to have a significant impact in diverse fields and industries, ranging from environmental toxicology through to pharmaceutical and agrochemical industries. PMID:24607581

  1. Flower colour mutations

    International Nuclear Information System (INIS)

    In the floriculture trade there is always a demand for new ornamental varieties. Flower colour is one of the most important components. Induced somatic mutation techniques using ionizing radiation and other mutagens have successfully produced many promising varieties in different ornamental plants by bringing about genetic changes. Induced mutation is a chance process. It is not known what flower colour change is likely to occur after mutagen treatment. Attempts are being made to induce a direct mutation for the flower colour of ornamental plants. For a better understanding of the exact mechanisms involved in the origin and evolution of somatic flower colour mutations at the molecular level, much attention has been paid to comparative analyses of the original cultivars and their induced mutants. Efforts are being made to identify the flower pigments and to prepare a colour chart which will be helpful in inducing the desired novelties in ornamental plants using induced genetic manipulation. 8 refs, 3 figs

  2. Driver Gene Mutations in Stools of Colorectal Carcinoma Patients Detected by Targeted Next-Generation Sequencing.

    Science.gov (United States)

    Armengol, Gemma; Sarhadi, Virinder K; Ghanbari, Reza; Doghaei-Moghaddam, Masoud; Ansari, Reza; Sotoudeh, Masoud; Puolakkainen, Pauli; Kokkola, Arto; Malekzadeh, Reza; Knuutila, Sakari

    2016-07-01

    Detection of driver gene mutations in stool DNA represents a promising noninvasive approach for screening colorectal cancer (CRC). Amplicon-based next-generation sequencing (NGS) is a good option to study mutations in many cancer genes simultaneously and from a low amount of DNA. Our aim was to assess the feasibility of identifying mutations in 22 cancer driver genes with Ion Torrent technology in stool DNA from a series of 65 CRC patients. The assay was successful in 80% of stool DNA samples. NGS results showed 83 mutations in cancer driver genes, 29 hotspot and 54 novel mutations. One to five genes were mutated in 75% of cases. TP53, KRAS, FBXW7, and SMAD4 were the top mutated genes, consistent with previous studies. Of samples with mutations, 54% presented concomitant mutations in different genes. Phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway genes were mutated in 70% of samples, with 58% having alterations in KRAS, NRAS, or BRAF. Because mutations in these genes can compromise the efficacy of epidermal growth factor receptor blockade in CRC patients, identifying mutations that confer resistance to some targeted treatments may be useful to guide therapeutic decisions. In conclusion, the data presented herein show that NGS procedures on stool DNA represent a promising tool to detect genetic mutations that could be used in the future for diagnosis, monitoring, or treating CRC. PMID:27155048

  3. Low prevalence of germline hMSH6 mutations in colorectal cancer families from Spain

    Institute of Scientific and Technical Information of China (English)

    Ana Sánchez de Abajo; Trinidad Caldes; Miguel de la Hoya; Alicia Tosar; Javier Godino; Juan Manuel Fernández; Jose Lopez Asenjo; Beatriz Perez Villamil; Pedro Perez Segura; Eduardo Diaz-Rubio

    2005-01-01

    AIM: To investigate the prevalence and penetrance of hMSH6 mutations in Spanish HNPCC families that was negative for mutation in hMLH1 or hMSH2.METHODS: We used PCR-based DGGE assay and direct Sequencing to screen for hMSH6 gene in 91 HNPCC families.RESULTS: we have identified 10 families with germ-line mutations in the DNA sequence. These mutations included two intronic variation, three missense mutation, one nonsense mutation, and four silent mutations. Among the 10 germ-line mutations identified in the Spanish cohort,8 were novel, perhaps, suggesting different mutational spectra in the Spanish population. Detailed pedigrees were constructed for the three families with a possible pathogenic hMSH6 mutation. The two silent mutations H388H and L758L, detected in a person affected of colorectal cancer at age 29, produce loss of the wild-type allele in the tumor sample. Immunohistochemical analysis showed that expression of MSH6 protein was lost only in the tumors from the carriers of V878A and Q263X mutations.CONCLUSION: Altogether, our results indicate that disease-causing germ-line mutations of hMSH6 are very less frequent in Spanish HNPCC families.

  4. Genetic mutations associated with metastatic clear cell renal cell carcinoma

    Science.gov (United States)

    Wu, Qingjian; Li, Fengjie; Zhao, Jiang; Wu, Kaijin; Qu, Cunye; Chen, Yibu; Li, Meng; Chen, Xuelian; Stucky, Andres; Zhong, Jiangjian; Li, Longkun; Zhong, Jiang F.

    2016-01-01

    Metastasis is the major cause of death among cancer patients, yet early detection and intervention of metastasis could significantly improve their clinical outcomes. We have sequenced and analyzed RNA (Expression) and DNA (Mutations) from the primary tumor (PT), tumor extension (TE) and lymphatic metastatic (LM) sites of patients with clear cell renal cell carcinoma (CCRCC) before treatment. Here, we report a three-nucleotide deletion near the C-region of Plk5 that is specifically associated with the lymphatic metastasis. This mutation is un-detectable in the PT, becomes detectable in the TE and dominates the LM tissue. So while only a few primary cancer cells carry this mutation, the majority of metastatic cells have this mutation. The increasing frequency of this mutation in metastatic tissue suggests that this Plk5 deletion could be used as an early indicator of CCRCC metastasis, and be identified by low cost PCR assay. A large scale clinical trial could reveal whether a simple PCR assay for this mutation at the time of nephrectomy could identify and stratify high-risk CCRCC patients for treatments. PMID:26908440

  5. 47 CFR 22.909 - Cellular markets.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Cellular markets. 22.909 Section 22.909... Cellular Radiotelephone Service § 22.909 Cellular markets. Cellular markets are standard geographic areas used by the FCC for administrative convenience in the licensing of cellular systems. Cellular...

  6. The role of mutation in the new cancer paradigm

    Directory of Open Access Journals (Sweden)

    Prehn Richmond T

    2005-04-01

    Full Text Available Abstract The almost universal belief that cancer is caused by mutation may gradually be giving way to the belief that cancer begins as a cellular adaptation that involves the local epigenetic silencing of various genes. In my own interpretation of the new epigenetic paradigm, the genes epigenetically suppressed are genes that normally serve in post-embryonic life to suppress and keep suppressed those other genes upon which embryonic development depends. Those other genes, if not silenced or suppressed in the post-embryonic animal, become, I suggest, the oncogenes that are the basis of neoplasia. Mutations that occur in silenced genes supposedly go unrepaired and are, therefore, postulated to accumulate, but such mutations probably play little or no causative role in neoplasia because they occur in already epigenetically silenced genes. These mutations probably often serve to make the silencing, and therefore the cancer, epigenetically irreversible.

  7. Mitochondrial DNA mutations in single human blood cells.

    Science.gov (United States)

    Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

    2015-09-01

    Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. PMID:26149767

  8. Mutations in Lettuce Improvement

    OpenAIRE

    2012-01-01

    Lettuce is a major vegetable in western countries. Mutations generated genetic variations and played an important role in the domestication of the crop. Many traits derived from natural and induced mutations, such as dwarfing, early flowering, male sterility, and chlorophyll deficiency, are useful in physiological and genetic studies. Mutants were also used to develop new lettuce products including miniature and herbicide-tolerant cultivars. Mutant analysis was critical in lettuce genomic stu...

  9. Mutation breeding in peas

    International Nuclear Information System (INIS)

    The pea as an ancient crop plant still today has wide uses and is an import source of food protein. It is also an important object for genetic studies and as such has been widely used in mutation induction experiments. However, in comparison with cereals this ancient crop plant (like several other grain legumes) has gained relatively little from advances in breeding. The review focuses on the prospects of genetic improvement of pea by induced mutations, discusses principles and gives methodological information. (author)

  10. Genotoxicity Assessment of Erythritol by Using Short-term Assay

    OpenAIRE

    Chung, Young-Shin; Lee, Michael

    2013-01-01

    Erythritol is a sugar alcohol that is widely used as a natural sugar substitute. Thus, the safety of its usage is very important. In the present study, short-term genotoxicity assays were conducted to evaluate the potential genotoxic effects of erythritol. According to the OECD test guidelines, the maximum test dose was 5,000 μg/plate in bacterial reverse mutation tests, 5,000 μg/ml in cell-based assays, and 5,000 mg/kg for in vivo testing. An Ames test did not reveal any positive results. No...

  11. Mutation Breeding in Sugarcane

    International Nuclear Information System (INIS)

    The present position of sugar industry particularly cane sugar production in the world has been discussed. The role of African Countries which can contribute more than the present 11% to world cane sugar production is presented. The breeding methods employed in cane growing court-tries indicate the biparental crossing and selection in F1 has been the major method used to develop varieties. Due to cytogenetical peculiarities, thousands of seedlings are grown to select the desirable genotype. Mutations or sports has been a source of variation for selection in nature. Induced mutations have only enhanced the mutation rate and has enabled the plant breeders to get better variation for selection. Though many mutagens have been used gamma rays have been most effective. Induced mutations for nonflowering, spineless leaf-sheath, higher sugar content, yield md resistance to diseases like smut and downy mildew have been reported. The methods of making mutated tissues express itself have been indicated. Mutation breeding holds out promise in sugarcane in that the basic variety or genotype can be kept intact and a few characters changed as desired by the plant breeder provided proper selection methods are employed. (author)

  12. KRAS mutations: analytical considerations.

    Science.gov (United States)

    Herreros-Villanueva, Marta; Chen, Chih-Chieh; Yuan, Shyng-Shiou F; Liu, Ta-Chih; Er, Tze-Kiong

    2014-04-20

    Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer death globally. Significant improvements in survival have been made in patients with metastasis by new therapies. For example, Cetuximab and Panitumumab are monoclonal antibodies that inhibit the epidermal growth receptor (EGFR). KRAS mutations in codon 12 and 13 are the recognized biomarkers that are analyzed in clinics before the administration of anti-EGFR therapy. Genetic analyses have revealed that mutations in KRAS predict a lack of response to Panitumumab and Cetuximab in patients with metastatic CRC (mCRC). Notably, it is estimated that 35-45% of CRC patients harbor KRAS mutations. Therefore, KRAS mutation testing should be performed in all individuals with the advanced CRC in order to identify the patients who will not respond to the monoclonal EGFR antibody inhibitors. New techniques for KRAS testing have arisen rapidly, and each technique has advantages and disadvantages. Herein, we review the latest published literature specific to KRAS mutation testing techniques. Since reliability and feasibility are important issues in clinical analyses. Therefore, this review also summarizes the effectiveness and limitations of numerous KRAS mutation testing techniques. PMID:24534449

  13. Subquivers of mutation-acyclic quivers are mutation-acyclic

    CERN Document Server

    Warkentin, Matthias

    2011-01-01

    Quiver mutation plays a crucial role in the definition of cluster algebras by Fomin and Zelevinsky. It induces an equivalence relation on the set of all quivers without loops and two-cycles. A quiver is called mutation-acyclic if it is mutation-equivalent to an acyclic quiver. The aim of this note is to show that full subquivers of mutation-acyclic quivers are mutation-acyclic.

  14. Hyperpolarized NMR Probes for Biological Assays

    Directory of Open Access Journals (Sweden)

    Sebastian Meier

    2014-01-01

    Full Text Available During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

  15. In vitro and in vivo assays of protein kinase CK2 activity.

    Science.gov (United States)

    Prudent, Renaud; Sautel, Céline F; Moucadel, Virginie; Laudet, Béatrice; Filhol, Odile; Cochet, Claude

    2010-01-01

    Protein kinase CK2 (formerly casein kinase 2) is recognized as a central component in the control of the cellular homeostasis; however, much remains unknown regarding its regulation and its implication in cellular transformation and carcinogenesis. Moreover, study of CK2 function and regulation in a cellular context is complicated by the dynamic multisubunit architecture of this protein kinase. Although a number of robust techniques are available to assay CK2 activity in vitro, there is a demand for sensitive and specific assays to evaluate its activity in living cells. We hereby provide a detailed description of several assays for monitoring the CK2 activity and its subunit interaction in living cells. The guidelines presented herein should enable researchers in the field to establish strategies for cellular screenings of CK2 inhibitors. PMID:21050938

  16. Radiorespirometic assay device

    International Nuclear Information System (INIS)

    A radiorespirometic assay device is described in which the presence of microorganisms in a sample is determined by placing the sample in contact with a metabolisable radioactive labelled substrate, collecting any gas evolved, exposing a photosensitive material to the gas and determining if a spot is produced on the material. A spot indicates the presence of radioactivity showing that the substrate has been metabolized by a microorganism. Bacteria may be detected in body fluids, hospital operating rooms, water, food, cosmetics and drugs. (U.K.)

  17. Radon assay for SNO+

    International Nuclear Information System (INIS)

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+

  18. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  19. MIMO Communication for Cellular Networks

    CERN Document Server

    Huang, Howard; Venkatesan, Sivarama

    2012-01-01

    As the theoretical foundations of multiple-antenna techniques evolve and as these multiple-input multiple-output (MIMO) techniques become essential for providing high data rates in wireless systems, there is a growing need to understand the performance limits of MIMO in practical networks. To address this need, MIMO Communication for Cellular Networks presents a systematic description of MIMO technology classes and a framework for MIMO system design that takes into account the essential physical-layer features of practical cellular networks. In contrast to works that focus on the theoretical performance of abstract MIMO channels, MIMO Communication for Cellular Networks emphasizes the practical performance of realistic MIMO systems. A unified set of system simulation results highlights relative performance gains of different MIMO techniques and provides insights into how best to use multiple antennas in cellular networks under various conditions. MIMO Communication for Cellular Networks describes single-user,...

  20. Fetal ACL Fibroblasts Exhibit Enhanced Cellular Properties Compared with Adults

    OpenAIRE

    Stalling, Simone S.; Nicoll, Steven B.

    2008-01-01

    Fetal tendons and skin heal regeneratively without scar formation. Cells isolated from these fetal tissues exhibit enhanced cellular migration and collagen production in comparison to cells from adult tissue. We determined whether fetal and adult fibroblasts isolated from the anterior cruciate ligament (ACL), a tissue that does not heal regeneratively, exhibit differences in cell migration rates and collagen elaboration. An in vitro migration assay showed fetal ACL fibroblasts migrated twice ...

  1. A Cellular Model for Screening Neuronal Nitric Oxide Synthase Inhibitors

    OpenAIRE

    Fang, Jianguo; Silverman, Richard B.

    2009-01-01

    Nitric oxide synthase (NOS) inhibitors are potential drug candidates because it has been well demonstrated that excessive production of NO critically contributes to a range of diseases. Most inhibitors have been screened in vitro using recombinant enzymes, leading to the discovery of a variety of potent compounds. To make inhibition studies more physiologically relevant and bridge the gap between the in vitro assay and in vivo studies, we report here a cellular model for screening NOS inhibit...

  2. Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

    OpenAIRE

    Lee, Katherine J.; Mwongela, Simon M.; Kottegoda, Sumith; Borland, Laura; Nelson, Allison R.; Sims, Christopher E.; Allbritton, Nancy L.

    2008-01-01

    Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sph...

  3. Cellular response after irradiation: Cell cycle control and apoptosis

    International Nuclear Information System (INIS)

    The importance of apoptotic death was assessed in a set of experiments, involving eight human tumour cell lines (breast cancer, bladder carcinoma, medulloblastoma). Various aspects of the quantitative study of apoptosis and methods based on the detection of DNA fragmentation (in situ tailing and comet assay) are described and discussed. Data obtained support the hypothesis that apoptosis is not crucial for cellular radiosensitivity and that the relationship between p53 functionality or clonogenic survival and apoptosis may bee cell type specific. (author)

  4. Telomere-independent cellular senescence in human fetal cardiomyocytes

    OpenAIRE

    Ball, Andrew J.; Levine, F

    2005-01-01

    Fetal cardiomyocytes have been proposed as a potential source of cell-based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT-PCR. Cells were found to senesce after 20-25 population doublings, as determined by growth arrest, morphological changes and senescence-associated beta-galactosidase activity. Using the telomeric repeat amplification protocol assay...

  5. Mutation directional selection sheds light on prion pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Liang [Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Shandong University of Technology, Zibo 255049 (China); Ji, Hong-Fang, E-mail: jhf@sdut.edu.cn [Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Shandong University of Technology, Zibo 255049 (China)

    2011-07-01

    Highlights: {yields} Most pathogenic mutations possess strong directional selection, i.e., enhancing hydrophobicity or decreasing negative and increasing positive charge. {yields} Mutation-induced changes may strengthen the interactions between PrP and facilitating factors. {yields} The findings also have significant implications for exploring potential regions involved in the conformational transition from PrP{sup C} to PrP{sup Sc}. -- Abstract: As mutations in the PRNP gene account for human hereditary prion diseases (PrDs), it is crucial to elucidating how these mutations affect the central pathogenic conformational transition of normal cellular prion protein (PrP{sup C}) to abnormal scrapie isoform (PrP{sup Sc}). Many studies proposed that these pathogenic mutations may make PrP more susceptible to conformational change through altering its structure stability. By evaluating the most recent observations regarding pathogenic mutations, it was found that the pathogenic mutations do not exert a uniform effect on the thermodynamic stability of the human PrP's structure. Through analyzing the reported PrDs-related mutations, we found that 25 out of 27 mutations possess strong directional selection, i.e., enhancing hydrophobicity or decreasing negative and increasing positive charge. Based on the triggering role reported by previous studies of facilitating factors in PrP{sup C} conversion, e.g., lipid and polyanion, we proposed that the mutation-induced changes may strengthen the interaction between PrP and facilitating factors, which will accelerate PrP conversion and cause PrDs.

  6. Mutation rates and the evolution of germline structure.

    Science.gov (United States)

    Scally, Aylwyn

    2016-07-19

    Genome sequencing studies of de novo mutations in humans have revealed surprising incongruities in our understanding of human germline mutation. In particular, the mutation rate observed in modern humans is substantially lower than that estimated from calibration against the fossil record, and the paternal age effect in mutations transmitted to offspring is much weaker than expected from our long-standing model of spermatogenesis. I consider possible explanations for these discrepancies, including evolutionary changes in life-history parameters such as generation time and the age of puberty, a possible contribution from undetected post-zygotic mutations early in embryo development, and changes in cellular mutation processes at different stages of the germline. I suggest a revised model of stem-cell state transitions during spermatogenesis, in which 'dark' gonial stem cells play a more active role than hitherto envisaged, with a long cycle time undetected in experimental observations. More generally, I argue that the mutation rate and its evolution depend intimately on the structure of the germline in humans and other primates.This article is part of the themed issue 'Dating species divergences using rocks and clocks'. PMID:27325834

  7. Variation in RNA virus mutation rates across host cells.

    Directory of Open Access Journals (Sweden)

    Marine Combe

    2014-01-01

    Full Text Available It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10(-6 to 10(-4 substitutions per nucleotide per round of copying (s/n/r and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV, which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10(-5 s/n/r. Cell immortalization through p53 inactivation and oxygen levels (1-21% did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.

  8. Depression of p53-independent Akt survival signals after high-LET radiation in mutated p53 cells

    Science.gov (United States)

    Ohnishi, Takeo; Takahashi, Akihisa; Nakagawa, Yosuke

    Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses the activities of serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signals were analyzed with Western blotting analysis 1 h, 2 h, 3 h and 6 h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis.Akt-related protein levels were decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G _{2}/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and depresses cell growth by suppressing Akt-related signals, even in the mp53 cells.

  9. BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells.

    Science.gov (United States)

    Woolery, Kamisha T; Mohamed, Mai; Linger, Rebecca J; Dobrinski, Kimberly P; Roman, Jesse; Kruk, Patricia A

    2015-01-01

    Familial history remains the strongest risk factor for developing ovarian cancer (OC) and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE) cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (-1800) to (-900) region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC. PMID:26357657

  10. BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Kamisha T. Woolery

    2015-01-01

    Full Text Available Familial history remains the strongest risk factor for developing ovarian cancer (OC and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β. Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (−1800 to (−900 region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

  11. Canberra waste assay systems

    International Nuclear Information System (INIS)

    Canberra Industries, Inc. (USA) offers complete solutions and turnkey systems for qualifying and quantifying drums of radioactive waste. Different Waste Assay Systems are available for low-, medium-, and high activity barrels. Such systems scan the segments of the drums. The gamma-emitting nuclides present in the radioactive waste are identified from the spectra of the gamma-rays emitted and their activities are quantified. The spectrum of each segment of the waste container is analysed separately and the results are summed for total drum inventory reporting. The Waste Assay specific Canberra software is an easy-to-use menu driven program. It offers automatic procedures for routine work but interactive mode is also available to investigate particular cases. The way of analysis and the format and contents of the report can be customized. Additional Quality Control program module monitors system parameters. The measured and calculated values of each waste container are saved into a relational database. Canberra offers mathematical efficiency calibration services for different drum types, with different waste densities and different activities. This new theoretical approach of efficiency calibration eliminates the need of calibration drums, thus saving costs. For field applications Canberra developed a portable In-Situ Object Counting System. This system determines efficiency calibration for any kind of objects after the user enters its size and density, furthermore the type of shielding and the distance between the detector and the object. (author)

  12. Single-Molecule Imaging of Cellular Signaling

    Science.gov (United States)

    De Keijzer, Sandra; Snaar-Jagalska, B. Ewa; Spaink, Herman P.; Schmidt, Thomas

    Single-molecule microscopy is an emerging technique to understand the function of a protein in the context of its natural environment. In our laboratory this technique has been used to study the dynamics of signal transduction in vivo. A multitude of signal transduction cascades are initiated by interactions between proteins in the plasma membrane. These cascades start by binding a ligand to its receptor, thereby activating downstream signaling pathways which finally result in complex cellular responses. To fully understand these processes it is important to study the initial steps of the signaling cascades. Standard biological assays mostly call for overexpression of the proteins and high concentrations of ligand. This sets severe limits to the interpretation of, for instance, the time-course of the observations, given the large temporal spread caused by the diffusion-limited binding processes. Methods and limitations of single-molecule microscopy for the study of cell signaling are discussed on the example of the chemotactic signaling of the slime-mold Dictyostelium discoideum. Single-molecule studies, as reviewed in this chapter, appear to be one of the essential methodologies for the full spatiotemporal clarification of cellular signaling, one of the ultimate goals in cell biology.

  13. Human T lymphotropic virus type-1 p30II alters cellular gene expression to selectively enhance signaling pathways that activate T lymphocytes

    Directory of Open Access Journals (Sweden)

    Feuer Gerold

    2004-11-01

    Full Text Available Abstract Background Human T-lymphotropic virus type-1 (HTLV-1 is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in the virus life cycle or HTLV-1 pathogenesis. Proviral clones of the virus with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. Exogenous expression of p30II differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and represses tax/rex RNA nuclear export. Results Herein, we further characterized the role of p30II in regulation of cellular gene expression, using stable p30II expression system employing lentiviral vectors to test cellular gene expression with Affymetrix U133A arrays, representing ~33,000 human genes. Reporter assays in Jurkat T cells and RT-PCR in Jurkat and primary CD4+ T-lymphocytes were used to confirm selected gene expression patterns. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling, apoptosis and cell cycle in p30II expressing Jurkat T cells. In all categories, p30II appeared to be an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes. Conclusions We are the first to demonstrate that p30II, while repressing the expression of many genes, selectively activates key gene pathways involved in T-cell signaling/activation. Collectively, our data suggests that this complex retrovirus, associated with lymphoproliferative diseases, relies upon accessory gene products to modify cellular environment to promote clonal expansion of the virus genome and thus maintain proviral loads in vivo.

  14. Cellularized Bilayer Pullulan-Gelatin Hydrogel for Skin Regeneration.

    Science.gov (United States)

    Nicholas, Mathew N; Jeschke, Marc G; Amini-Nik, Saeid

    2016-05-01

    Skin substitutes significantly reduce the morbidity and mortality of patients with burn injuries and chronic wounds. However, current skin substitutes have disadvantages related to high costs and inadequate skin regeneration due to highly inflammatory wounds. Thus, new skin substitutes are needed. By combining two polymers, pullulan, an inexpensive polysaccharide with antioxidant properties, and gelatin, a derivative of collagen with high water absorbency, we created a novel inexpensive hydrogel-named PG-1 for "pullulan-gelatin first generation hydrogel"-suitable for skin substitutes. After incorporating human fibroblasts and keratinocytes onto PG-1 using centrifugation over 5 days, we created a cellularized bilayer skin substitute. Cellularized PG-1 was compared to acellular PG-1 and no hydrogel (control) in vivo in a mouse excisional skin biopsy model using newly developed dome inserts to house the skin substitutes and prevent mouse skin contraction during wound healing. PG-1 had an average pore size of 61.69 μm with an ideal elastic modulus, swelling behavior, and biodegradability for use as a hydrogel for skin substitutes. Excellent skin cell viability, proliferation, differentiation, and morphology were visualized through live/dead assays, 5-bromo-2'-deoxyuridine proliferation assays, and confocal microscopy. Trichrome and immunohistochemical staining of excisional wounds treated with the cellularized skin substitute revealed thicker newly formed skin with a higher proportion of actively proliferating cells and incorporation of human cells compared to acellular PG-1 or control. Excisional wounds treated with acellular or cellularized hydrogels showed significantly less macrophage infiltration and increased angiogenesis 14 days post skin biopsy compared to control. These results show that PG-1 has ideal mechanical characteristics and allows ideal cellular characteristics. In vivo evidence suggests that cellularized PG-1 promotes skin regeneration and may

  15. p53: Biology and role for cellular radiosensitivity

    International Nuclear Information System (INIS)

    Purpose: p53 is the most commonly mutated gene in human tumors with large impact on cellular biology and response to radiation. Many excellent reviews are available on various aspects but for several years none about the role of p53 for radiosensitivity. The latter is the aim of the present paper. Methods: Review of the literature. Results: p53 is a regulator of apoptosis mainly in hematopoetic tissue. In normal tissue and solid tumors presumably other functions have more impact on the cellular response. p53 controls cell-cycle progression after irradiation and also DNA-repair, namely homologous and non-homologous recombination. Mutations of p53 alter these functions which may be responsible for an enhanced cellular and tumor radioresistance. At present only few reports were able to show that under tightly controlled conditions loss of p53 wild-type function leads to enhanced radioresistance. A general proof is still lacking. Conclusion: The emerging picture in the year 2000 shows p53 as a central protein in a multi-enzyme multi-function network which is far from being fully understood. Although p53 appears to be a major regulator it is certainly not the unreplacable component the loss of which uniformly determines radioresistance. Only further understanding of modifiers and cooperators in the cell and in the specific tissue context will elucidate p53's role for radiosensitivity and radiotherapy. (orig.)

  16. Deciphering the spectrum of somatic mutations in the entire mitochondrial DNA genome.

    Science.gov (United States)

    Chen, X Z; Fang, Y; Shi, Y H; Cui, J H; Li, L Y; Xu, Y C; Ling, B

    2015-01-01

    The mitochondrion is a crucial intracellular organelle responsible for regulating cellular energy metabolism, producing free radicals, initiating and executing the apoptotic pathways. Previous studies have shown that somatic mutations in mitochondrial DNA are associated with various tumors, which may be involved during carcinogenesis and tumor progression. To examine the mutation pattern in cancer, 625 reported somatic mutations in the mitochondrial DNA genome were analyzed. We found that, except for deletions and insertions, most somatic mutations were point mutations, accounting for 89.44% of somatic mutations. Transition was the predominant form of somatic mutation in the entire mitochondrial DNA genome, accounting for 87.12% of point mutations, most of which were homoplastic. Frequency statistics analysis of point mutations indicated that, except for 3 tRNA genes, the mutations were distributed on all resting genes and in the D-loop region, with the latter showing the highest frequency of somatic mutation (19.34%), followed by the tRNA leucine 2 gene and non-coding regions between base pairs 5892 and 5903, while 13 coding-region genes and 2 rRNA genes showed a relatively lower frequency of somatic point mutations. Nonsynonymous mutations and terminal amino acid changes were the primary point somatic mutations detected from 13 coding-region genes, which may cause mitochondrial dysfunction in cancer cells. We found that the somatic mutations may affect the mitochondrial DNA genome; the non-coding region should be examined to identify somatic mutations as potential diagnostic biomarkers for early detection of cancer. PMID:25966205

  17. Actual problems of cellular cardiomyoplasty

    Directory of Open Access Journals (Sweden)

    Bulat Kaupov

    2010-04-01

    Full Text Available The paper provides review of cellular technologies used incardiology, describes types of cellular preparations depending onsources of cells and types of compounding cells. The generalmechanisms of therapies with stem cells applications are described.Use of cellular preparations for treatment of cardiovascular diseasesand is improvement of the forecast at patients with heartinsufficiency of various genesis is considered as alternative topractice with organ transplantations. Efforts of biotechnologicallaboratories are directed on search of optimum population of cellsfor application in cardiology and studying of mechanisms andfactors regulating function of cardiac stem cells.

  18. Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations.

    Science.gov (United States)

    Castella, Maria; Pujol, Roser; Callén, Elsa; Trujillo, Juan P; Casado, José A; Gille, Hans; Lach, Francis P; Auerbach, Arleen D; Schindler, Detlev; Benítez, Javier; Porto, Beatriz; Ferro, Teresa; Muñoz, Arturo; Sevilla, Julián; Madero, Luis; Cela, Elena; Beléndez, Cristina; de Heredia, Cristina Díaz; Olivé, Teresa; de Toledo, José Sánchez; Badell, Isabel; Torrent, Montserrat; Estella, Jesús; Dasí, Angeles; Rodríguez-Villa, Antonia; Gómez, Pedro; Barbot, José; Tapia, María; Molinés, Antonio; Figuera, Angela; Bueren, Juan A; Surrallés, Jordi

    2011-04-01

    Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCA mutations, we determined a FANCA mutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCA mutation is not the result of a mutational "hot-spot" but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCA and 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCA mutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations. PMID:21273304

  19. Mutations in lettuce improvement.

    Science.gov (United States)

    Mou, Beiquan

    2011-01-01

    Lettuce is a major vegetable in western countries. Mutations generated genetic variations and played an important role in the domestication of the crop. Many traits derived from natural and induced mutations, such as dwarfing, early flowering, male sterility, and chlorophyll deficiency, are useful in physiological and genetic studies. Mutants were also used to develop new lettuce products including miniature and herbicide-tolerant cultivars. Mutant analysis was critical in lettuce genomic studies including identification and cloning of disease-resistance genes. Mutagenesis combined with genomic technology may provide powerful tools for the discovery of novel gene alleles. In addition to radiation and chemical mutagens, unconventional approaches such as tissue or protoplast culture, transposable elements, and space flights have been utilized to generate mutants in lettuce. Since mutation breeding is considered nontransgenic, it is more acceptable to consumers and will be explored more in the future for lettuce improvement. PMID:22287955

  20. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is...... adapted to receive one or more replaceable solid support(s) (40) onto which chemical entities (41) are attached, said device comprising a base (1, 60, 80, 300, 400, 10, 70, 140, 20, 90, 120, 150, 30, 100), one or more inlet(s) (5), one or more outlet(s) (6). The base and the solid support (40) defines......, when operatively connected, one or more chambers (21) comprising the chemical entities (41), the inlet(s) (5) and outlet(s) (6) and chambers (21) being in fluid connection. The device further comprise means for providing differing chemical conditions in each chamber (21)....

  1. Assay of oestrogen

    International Nuclear Information System (INIS)

    A particular problem with the direct radioimmunoassay of unconjugated oestriol in pregnancy is caused by the increased amount of steroid-binding proteins present in pregnancy serum and plasma. The steroid-binding proteins react with oestriol and 125I-labelled oestriol during the assay procedure and the steroid-protein bound 125I-labelled oestriol is precipitated along with the antibody-bound 125I-labelled oestriol by the ammonium sulphate solution separation system. A novel method is described whereby progesterone (1-20 μg/ml) is used to block the action of steroid-binding proteins in pregnancy serum and plasma samples, thus minimizing interference in a direct radioimmunoassay for unconjugated oestriol using a specific anti-oestriol serum. (U.K.)

  2. From cellular to tissue scales by asymptotic limits of thermostatted kinetic models

    Science.gov (United States)

    Bianca, Carlo; Dogbe, Christian; Lemarchand, Annie

    2016-02-01

    Tumor growth strictly depends on the interactions occurring at the cellular scale. In order to obtain the linking between the dynamics described at tissue and cellular scales, asymptotic methods have been employed, consisting in deriving tissue equations by suitable limits of mesoscopic models. In this paper, the evolution at the cellular scale is described by thermostatted kinetic theory that include conservative, nonconservative (proliferation, destruction and mutations), stochastic terms, and the role of external agents. The dynamics at the tissue scale (cell-density evolution) is obtained by performing a low-field scaling and considering the related convergence of the rescaled framework when the scaling parameter goes to zero.

  3. Dynamic modeling of cellular response to DNA damage based on p53 stress response networks

    Institute of Scientific and Technical Information of China (English)

    Jinpeng Qi; Yongsheng Ding; Shihuang Shao

    2009-01-01

    Under acute perturbations from the outside, cells can trigger self-defensive mechanisms to fight against genome stress. To investigate the cellular response to continuous ion radiation (IR), a dynamic model for p53 stress response networks at the cellular level is proposed. The model can successfully be used to simulate the dynamic processes of double-strand breaks (DSBs) generation and their repair, switch-like ataxia telangiectasia mutated (ATM) activation, oscillations occurring in the p53-MDM2 feedback loop, as well as toxins elimination triggered by p53 stress response networks. Especially, the model can predict the plausible outcomes of cellular response under different IR dose regimes.

  4. Granuloma pouch assay for mutagenicity testing.

    Science.gov (United States)

    Maier, P

    1980-11-01

    The Granuloma Pouch Assay (GPA) is an animal model in which mutagenic and carcinogenic effects of a testcompound can be detected in rapidly dividing fibroblasts of a granulation tissue in adult male rats. Growth of this tissue was initiated with a small amount of croton oil at the inside wall of a subcutaneous air pouch on the back of the animals. The test compound can be injected either into the pouch (local) or administered by systemic routes. Alkali labile DNA-lesions, chromosome aberrations, sister chromatid exchanges, point mutations and tumor development in situ were determined. The comparison of mutation frequencies after local and systemic administration of testcompounds, provide an estimation of the pharmacokinetic characteristics and the mutagenic potency of the chemical. The local application route allows the detection of locally active mutagens and of compounds which require activation by P-448 dependent mono-oxygenases. Liver mediated proximate metabolites are detectable when they are transformed into ultimate carcinogens in extrahepatic cells whereas chemicals with a strong organ specific activity are not. PMID:7235991

  5. Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations

    Directory of Open Access Journals (Sweden)

    Leary Jeffry J

    2002-05-01

    Full Text Available Abstract Background The thymidine kinase (tk mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. Methods A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. Results Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture. Conclusions This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a mutations may be modulated by other viral polypeptides cooperating with Pol, and (b the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

  6. PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA

    OpenAIRE

    Kim, Seung Tae; Lira, Maruja; Deng, Shibing; Lee, Sujin; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Mao, Mao; Heo, Jin Seok; Kwon, Wooil; Jang, Kee-Taek; Lee, Jeeyun; Park, Joon Oh

    2015-01-01

    PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, an...

  7. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder. The ...... binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  8. Anti-GD(2) with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia.

    Science.gov (United States)

    Sorkin, Linda S; Otto, Mario; Baldwin, William M; Vail, Emily; Gillies, Stephen D; Handgretinger, Rupert; Barfield, Raymond C; Ming Yu, Hui; Yu, Alice L

    2010-04-01

    Monoclonal antibodies against GD(2) ganglioside, such as ch14.18, the human-mouse chimeric antibody, have been shown to be effective for the treatment of neuroblastoma. However, treatment is associated with generalized, relatively opiate-resistant pain. We investigated if a point mutation in ch14.18 antibody (hu14.18K332A) to limit complement-dependent cytotoxicity (CDC) would ameliorate the pain behavior, while preserving antibody-dependent cellular cytotoxicity (ADCC). In vitro, CDC and ADCC were measured using europium-TDA assay. In vivo, allodynia was evaluated by measuring thresholds to von Frey filaments applied to the hindpaws after injection of either ch14.18 or hu14.18K332 into wild type rats or rats with deficient complement factor 6. Other rats were pretreated with complement factor C5a receptor antagonist and tested following ch14.18 injection. The mutation reduces the antibody's ability to activate complement, while maintaining its ADCC capabilities. Injection of hu14.18K322 (1 or 3mg/kg) produced faster resolving allodynia than that engendered by ch14.18 (1mg/kg). Injection of ch14.18 (1mg/kg) into rats with C6 complement deficiency further reduced antibody-induced allodynia, while pre-treatment with complement factor C5a receptor antagonist completely abolished ch14.18-induced allodynia. These findings showed that mutant hu14.18 K322 elicited less allodynia than ch14.18 and that ch14.18-elicited allodynia is due to activation of the complement cascade: in part, to formation of membrane attack complex, but more importantly to release of complement factor C5a. Development of immunotherapeutic agents with decreased complement-dependent lysis while maintaining cellular cytotoxicity may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of therapeutic antibodies. PMID:20171010

  9. Mutational myriad of tumor suppressor p53 in Filipino breast cancer: results and perspectives in molecular pathology and epidemiology

    International Nuclear Information System (INIS)

    The p53 tumor suppressor is by far the most widely mutated gene in human cancers. p53 encodes a 53-kDa phosphoprotein, transcription-activator whose targets include genes and gene products that orchestrate genomic stability, cellular response to DNA damage, cell cycle progression apoptosis and aging (senescence). Analysis of the p53 gene profile has previously resulted in identifying several cancer-causative factors in the human setting, as well as, in creating a unique molecular profile of a tumor useful in the design of tailored-therapies for individual cancer patients. Our results in screening for p53 abnormalities in 140 Filipino patients with primary breast lesions confined from 1997-1998 in 5 major hospitals in Manila reveal that p53 plays an important role in the development and progression of breast cancer in at least 48% of all cases. Two methods of p53 analysis are employed, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction-temporal temperature gradient electrophoresis (PCR-TTGE). Inter-comparisons of method exhibit 63.3% concordance in 21 fresh breast carcinoma samples, with ELISA demonstrating 14% false-positives and 10% false-negatives. Only mutations in exon 7 (p=0.063) in the tumor samples how significant correlation with abnormal cellular elevation of p53. PCR-TTGE screening in a large series of 140 patients show that most genetic lesions are localized in exons 5 (41% of the total cases) and 6 (27% of the total cases). No mutations are, however, detected in the transactivation (exons 2-4) and oligomerization (exons 10-11) domains. Invasive carcinomas (stages II and III) are characterized with more frequent and diverse genetic alterations compared with benign tumors, most significantly at exon 5B (p=0.066) and at independently multiple sites (p=0.066). Earlier-onset cases (age of diagnosis < 50 yrs), known to be more clinico-pathologically aggressive, are diagnosed harboring more frequent p53 mutations centered at exon 7 (p=0

  10. Origami interleaved tube cellular materials

    International Nuclear Information System (INIS)

    A novel origami cellular material based on a deployable cellular origami structure is described. The structure is bi-directionally flat-foldable in two orthogonal (x and y) directions and is relatively stiff in the third orthogonal (z) direction. While such mechanical orthotropicity is well known in cellular materials with extruded two dimensional geometry, the interleaved tube geometry presented here consists of two orthogonal axes of interleaved tubes with high interfacial surface area and relative volume that changes with fold-state. In addition, the foldability still allows for fabrication by a flat lamination process, similar to methods used for conventional expanded two dimensional cellular materials. This article presents the geometric characteristics of the structure together with corresponding kinematic and mechanical modeling, explaining the orthotropic elastic behavior of the structure with classical dimensional scaling analysis. (paper)

  11. Cellular mechanisms during vascular development

    OpenAIRE

    Blum, Yannick

    2012-01-01

    The vascular system is an essential organ in vertebrate animals and provides the organism with enough oxygen and nutrients. It is composed of an interconnected network of blood vessels, which form using a number of different morphogenetic mechanisms. Angiogenesis describes the formation of new blood vessels from preexisting vessels. A number of molecular pathways have been shown to be essential during angiogenesis. However, cellular architecture of blood vessels as well as cellular mechanisms...

  12. Predictive Modelling of Cellular Load

    OpenAIRE

    Carolan, Emmett; McLoone, Seamus; Farrell, Ronan

    2015-01-01

    This work examines the temporal dynamics of cellular load in four Irish regions. Large scale underutilisation of network resources is identified both at the regional level and at the level of individual cells. Cellular load is modeled and prediction intervals are generated. These prediction intervals are used to put an upper bound on usage in a particular cell at a particular time. Opportunities for improvements in network utilization by incorporating these upper bounds on usage are identifie...

  13. Cellular automaton for chimera states

    OpenAIRE

    García-Morales, Vladimir

    2016-01-01

    A minimalistic model for chimera states is presented. The model is a cellular automaton (CA) which depends on only one adjustable parameter, the range of the nonlocal coupling, and is built from elementary cellular automata and the majority (voting) rule. This suggests the universality of chimera-like behavior from a new point of view: Already simple CA rules based on the majority rule exhibit this behavior. After a short transient, we find chimera states for arbitrary initial conditions, the...

  14. qpure: A tool to estimate tumor cellularity from genome-wide single-nucleotide polymorphism profiles.

    Directory of Open Access Journals (Sweden)

    Sarah Song

    Full Text Available Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 ([Formula: see text]-value=0.0001 between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 ([Formula: see text]-value [Formula: see text] 2.2e-16 between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 ([Formula: see text]-value=0.004 between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.

  15. A multiparametric computational algorithm for comprehensive assessment of genetic mutations in mucopolysaccharidosis type IIIA (Sanfilippo syndrome.

    Directory of Open Access Journals (Sweden)

    Krastyu G Ugrinov

    Full Text Available Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo syndrome is a Lysosomal Storage Disease caused by cellular deficiency of N-sulfoglucosamine sulfohydrolase (SGSH. Given the large heterogeneity of genetic mutations responsible for the disease, a comprehensive understanding of the mechanisms by which these mutations affect enzyme function is needed to guide effective therapies. We developed a multiparametric computational algorithm to assess how patient genetic mutations in SGSH affect overall enzyme biogenesis, stability, and function. 107 patient mutations for the SGSH gene were obtained from the Human Gene Mutation Database representing all of the clinical mutations documented for Sanfilippo syndrome. We assessed each mutation individually using ten distinct parameters to give a comprehensive predictive score of the stability and misfolding capacity of the SGSH enzyme resulting from each of these mutations. The predictive score generated by our multiparametric algorithm yielded a standardized quantitative assessment of the severity of a given SGSH genetic mutation toward overall enzyme activity. Application of our algorithm has identified SGSH mutations in which enzymatic malfunction of the gene product is specifically due to impairments in protein folding. These scores provide an assessment of the degree to which a particular mutation could be treated using approaches such as chaperone therapies. Our multiparametric protein biogenesis algorithm advances a key understanding in the overall biochemical mechanism underlying Sanfilippo syndrome. Importantly, the design of our multiparametric algorithm can be tailored to many other diseases of genetic heterogeneity for which protein misfolding phenotypes may constitute a major component of disease manifestation.

  16. Embryonic stem cells as an ectodermal cellular model of human p63-related dysplasia syndromes.

    NARCIS (Netherlands)

    Rostagno, P.; Wolchinsky, Z.; Vigano, A.M.; Shivtiel, S.; Zhou, H.; Bokhoven, J.H.L.M. van; Ferone, G.; Missero, C.; Mantovani, R.; Aberdam, D.; Virolle, T.

    2010-01-01

    Heterozygous mutations in the TP63 transcription factor underlie the molecular basis of several similar autosomal dominant ectodermal dysplasia (ED) syndromes. Here we provide a novel cellular model derived from embryonic stem (ES) cells that recapitulates in vitro the main steps of embryonic skin d

  17. Pyrazinoic Acid Decreases the Proton Motive Force, Respiratory ATP Synthesis Activity, and Cellular ATP Levels▿†

    OpenAIRE

    Lu, P.; Haagsma, A.C.; Pham, H.; Maaskant, J. J.; Mol, S; Lill, H.; Bald, D

    2011-01-01

    Pyrazinoic acid, the active form of the first-line antituberculosis drug pyrazinamide, decreased the proton motive force and respiratory ATP synthesis rates in subcellular mycobacterial membrane assays. Pyrazinoic acid also significantly lowered cellular ATP levels in Mycobacterium bovis BCG. These results indicate that the predominant mechanism of killing by this drug may operate by depletion of cellular ATP reserves.

  18. PathogenMip Assay: A Multiplex Pathogen Detection Assay

    OpenAIRE

    Akhras, Michael S.; Sreedevi Thiyagarajan; Villablanca, Andrea C.; Davis, Ronald W; Pål Nyrén; Nader Pourmand

    2007-01-01

    The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The ...

  19. Mutations in BIN1 Associated with Centronuclear Myopathy Disrupt Membrane Remodeling by Affecting Protein Density and Oligomerization

    Science.gov (United States)

    Wu, Tingting; Shi, Zheng; Baumgart, Tobias

    2014-01-01

    The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease. PMID:24755653

  20. p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.

    Directory of Open Access Journals (Sweden)

    Virginia Andreotti

    Full Text Available BACKGROUND: The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. METHODOLOGY/PRINCIPAL FINDINGS: Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii single copy, chromosomally located p53-responsive and control luminescence reporters, iii enhanced chemical uptake using modified ABC-transporters, iv small-volume formats for treatment and dual-luciferase assays, and v opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1. CONCLUSIONS/SIGNIFICANCE: We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.

  1. Bovine Papillomavirus Clastogenic Effect Analyzed in Comet Assay

    OpenAIRE

    Araldi, R. P.; Melo, T. C.; N. Diniz; J. Mazzuchelli-de-Souza; R.F. Carvalho; Beçak, W.; Stocco, R. C.

    2013-01-01

    Bovine papillomavirus (BPV) is an oncogenic virus related to serious livestock diseases. Oncoproteins encoded by BPV are involved in several steps of cellular transformation and have been reported as presenting clastogenic effects in peripheral lymphocytes and primary culture cells. The aim of this study was to evaluate the clastogenic potential of BPV types 1, 2, and 4 by comet assay. Peripheral blood was collected from 37 bovines, 32 infected with different levels of papillomatosis (12 anim...

  2. Novel Phosphorylation Assay Based on Multi-Functionalized Soluble Nanopolymer

    OpenAIRE

    Iliuk, Anton; Martinez, Juan S.; Hall, Mark C.; Tao, W. Andy

    2011-01-01

    Quantitative phosphorylation analysis is essential to understanding cellular signal transductions. Here we present a novel technology for the highly efficient assay of protein phosphorylation in high throughput format without the use of phospho-specific antibodies. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO that is multi-functionalized with titanium (IV)ions for specific binding to phosphoproteins, and with biotin groups that allow for enzyme-linked spectrometr...

  3. Improved Dual-Luciferase Reporter Assays for Nuclear Receptors

    OpenAIRE

    Paguio, Aileen; Stecha, Pete; Wood, Keith V.; Fan, Frank

    2010-01-01

    Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptor...

  4. Multiscale Model of Colorectal Cancer Using the Cellular Potts Framework

    Science.gov (United States)

    Osborne, James M

    2015-01-01

    Colorectal cancer (CRC) is one of the major causes of death in the developed world and forms a canonical example of tumorigenesis. CRC arises from a string of mutations of individual cells in the colorectal crypt, making it particularly suited for multiscale multicellular modeling, where mutations of individual cells can be clearly represented and their effects readily tracked. In this paper, we present a multicellular model of the onset of colorectal cancer, utilizing the cellular Potts model (CPM). We use the model to investigate how, through the modification of their mechanical properties, mutant cells colonize the crypt. Moreover, we study the influence of mutations on the shape of cells in the crypt, suggesting possible cell- and tissue-level indicators for identifying early-stage cancerous crypts. Crucially, we discuss the effect that the motility parameters of the model (key factors in the behavior of the CPM) have on the distribution of cells within a homeostatic crypt, resulting in an optimal parameter regime that accurately reflects biological assumptions. In summary, the key results of this paper are 1) how to couple the CPM with processes occurring on other spatial scales, using the example of the crypt to motivate suitable motility parameters; 2) modeling mutant cells with the CPM; 3) and investigating how mutations influence the shape of cells in the crypt. PMID:26461973

  5. Molecular and cellular constraints on proteins

    Science.gov (United States)

    Kortemme, Tanja

    Engineering proteins with new sequences, structures and functions has many exciting practical applications, and provides new ways to dissect design principles for function. Recent successes in computational protein design provide a cause for optimism. Yet many functions are currently too complex to engineer predictively, and successful design of new biological activities also requires an understanding of the functional pressures acting on proteins in the context of cells and organisms. I will present two vignettes describing our progress with dissecting both molecular and cellular constraints on protein function. In the first, we characterized the cost and benefit of protein production upon sequence perturbations in a classic system for gene regulation, the lac operon. Our results were unexpected in light of the common assumption that the dominant fitness costs are due to protein expression. Instead, we discovered a direct linear relationship between cost and lacpermease activity, not protein or mRNA production. The magnitude of the cost of permease activity, relative to protein production, has consequences for regulation. Our model predicts an advantage of direct regulation of protein activity (not just expression), providing a new explanation for the long-known mechanism of ``inducer exclusion'' that inhibits transport through the permease. Similar pressures and cost/benefit tradeoffs may be key to engineering synthetic systems with improved fitness. In the second vignette, I will describe our recent efforts to develop computational approaches that predict protein sequences consistent with multiple functional conformations. We expect such ``multi-constraint'' models to improve predictions of functional sequences determined by deep mutational scanning in bacteria, to provide insights into how the balance between functional conformations shapes sequence space, and to highlight molecular and cellular constraints that cannot be captured by the model.

  6. Assaying Cell Cycle Status Using Flow Cytometry.

    Science.gov (United States)

    Kim, Kang Ho; Sederstrom, Joel M

    2015-01-01

    In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation-specific marker (Ki-67) and cellular DNA content, which discriminate resting/quiescent cell populations (G0 cell) and quantify cell cycle distribution (G1, S, or G2/M), respectively. The second is based on differential staining of DNA and RNA through co-staining of Hoechst 33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, two additional methods for cell proliferation assays with recent updates of newly developed fluorophores, which allow multiplex analysis of cell cycle status, cell proliferation, and a gene of interest using flow cytometry, are outlined. PMID:26131851

  7. Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay.

    Science.gov (United States)

    Varma-Basil, Mandira; El-Hajj, Hiyam; Colangeli, Roberto; Hazbón, Manzour Hernando; Kumar, Sujeet; Bose, Mridula; Bobadilla-del-Valle, Miriam; García, Lourdes García; Hernández, Araceli; Kramer, Fred Russell; Osornio, Jose Sifuentes; Ponce-de-León, Alfredo; Alland, David

    2004-12-01

    We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis. PMID:15583274

  8. Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients

    DEFF Research Database (Denmark)

    Petersen, S. M.; Dandanell, M.; Rasmussen, L. J.;

    2013-01-01

    /MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and......Background: Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as...... asses the effect on splicing. Results: We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c. 588 + 5G > A...

  9. Mutations in galactosemia

    Energy Technology Data Exchange (ETDEWEB)

    Reichardt, J.K.V. [Univ. of Southern California School of Medicine, Los Angeles, CA (United States)

    1995-10-01

    This Letter raises four issues concerning two papers on galactosemia published in the March 1995 of the Journal. First, table 2 in the paper by Elsas et al. incorrectly attributes seven galactose-l-phosphate uridyl transferase (GALT) mutations (S135L, L195P, K285N, N314D, R333W, R333G, and K334R). The table also fails to mention that others have reported the same two findings attributed to {open_quotes}Leslie et al.; Elsas et al. and in press{close_quotes} and {open_quotes}Leslie et al.; Elsas et al.{close_quotes} The first finding on the prevalence of the Q188R galactosemia mutation in the G/G Caucasian population has also been described by Ng et al., and the second finding on the correlation of the N314D GALT mutation with the Duarte variant was reported by Lin et al. Second, Elsas et al. suggest that the E203K and N314D mutations may {open_quotes}produce intra-allelic complementation when in cis{close_quotes}. This speculation is supported by the activity data of individual III-2 but is inconsistent with the activities of three other individuals I-1, II-1, and III-1 of the same pedigree. The GALT activity measured in these three individuals suggests a dominant negative effect of E203K in E203K-N314D chromosomes, since they all have less than normal activity. Thus, the preponderance of the data in this paper is at odds with the authors speculation. It is worth recalling that Lin et al. also identified four N314D GALT mutations on 95 galactosemic chromosomes examined. A similar situation also appears to be the case in proband III-1 (with genotype E203K-N314D/IVSC) in the Elsas et al. paper. 9 refs.

  10. Mutation breeding newsletter. No. 45

    International Nuclear Information System (INIS)

    This issue of the Mutation Breeding newsletter contains 39 articles dealing with radiation induced mutations and chemical mutagenesis techniques in plant breeding programs with the aims of improving crop productivity and disease resistance as well as exploring genetic variabilities

  11. Mutation breeding newsletter. No. 33

    International Nuclear Information System (INIS)

    This issue of the newsletter reports a number of research news and research abstracts on application of radiation induced mutation techniques to increase mutagenesis and mutation frequency in plant breeding projects

  12. Hierarchical Cellular Structures in High-Capacity Cellular Communication Systems

    CERN Document Server

    Jain, R K; Agrawal, N K

    2011-01-01

    In the prevailing cellular environment, it is important to provide the resources for the fluctuating traffic demand exactly in the place and at the time where and when they are needed. In this paper, we explored the ability of hierarchical cellular structures with inter layer reuse to increase the capacity of mobile communication network by applying total frequency hopping (T-FH) and adaptive frequency allocation (AFA) as a strategy to reuse the macro and micro cell resources without frequency planning in indoor pico cells [11]. The practical aspects for designing macro- micro cellular overlays in the existing big urban areas are also explained [4]. Femto cells are inducted in macro / micro / pico cells hierarchical structure to achieve the required QoS cost effectively.

  13. High content cell-based assay for the inflammatory pathway

    Science.gov (United States)

    Mukherjee, Abhishek; Song, Joon Myong

    2015-07-01

    Cellular inflammation is a non-specific immune response to tissue injury that takes place via cytokine network orchestration to maintain normal tissue homeostasis. However chronic inflammation that lasts for a longer period, plays the key role in human diseases like neurodegenerative disorders and cancer development. Understanding the cellular and molecular mechanisms underlying the inflammatory pathways may be effective in targeting and modulating their outcome. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that effectively combines the pro-inflammatory features with the pro-apoptotic potential. Increased levels of TNF-α observed during acute and chronic inflammatory conditions are believed to induce adverse phenotypes like glucose intolerance and abnormal lipid profile. Natural products e. g., amygdalin, cinnamic acid, jasmonic acid and aspirin have proven efficacy in minimizing the TNF-α induced inflammation in vitro and in vivo. Cell lysis-free quantum dot (QDot) imaging is an emerging technique to identify the cellular mediators of a signaling cascade with a single assay in one run. In comparison to organic fluorophores, the inorganic QDots are bright, resistant to photobleaching and possess tunable optical properties that make them suitable for long term and multicolor imaging of various components in a cellular crosstalk. Hence we tested some components of the mitogen activated protein kinase (MAPK) pathway during TNF-α induced inflammation and the effects of aspirin in HepG2 cells by QDot multicolor imaging technique. Results demonstrated that aspirin showed significant protective effects against TNF-α induced cellular inflammation. The developed cell based assay paves the platform for the analysis of cellular components in a smooth and reliable way.

  14. Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

    Institute of Scientific and Technical Information of China (English)

    Lei GUO; Ying ZHANG; Yan-chun CHE; Wen-juan WU; Wei-zhong LI; Li-chun WANG; Yun LIAO; Long-ding LIU; Qi-han LI

    2008-01-01

    An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

  15. Oxidative stress action in cellular aging

    Directory of Open Access Journals (Sweden)

    Monique Cristine de Oliveira

    2010-12-01

    Full Text Available Various theories try to explain the biological aging by changing the functions and structure of organic systems and cells. During lifetime, free radicals in the oxidative stress lead to lipid peroxidation of cellular membranes, homeostasis imbalance, chemical residues formation, gene mutations in DNA, dysfunction of certain organelles, and the arise of diseases due to cell death and/or injury. This review describes the action of oxidative stress in the cells aging process, emphasizing the factors such as cellular oxidative damage, its consequences and the main protective measures taken to prevent or delay this process. Tests with antioxidants: vitamins A, E and C, flavonoids, carotenoids and minerals, the practice of caloric restriction and physical exercise, seeking the beneficial effects on human health, increasing longevity, reducing the level of oxidative stress, slowing the cellular senescence and origin of certain diseases, are discussed.Diferentes teorias tentam explicar o envelhecimento biológico através da alteração das funções e estrutura dos sistemas orgânicos e células. Ao longo da vida, os radicais livres presentes no estresse oxidativo conduzem à peroxidação dos lipídios das membranas celulares, desequilíbrio da homeostase, formação de resíduos químicos, mutações gênicas no DNA, disfunção de certas organelas, bem como ao surgimento de doenças devido à lesão e/ou morte celular. Nesta revisão descreve-se a ação do estresse oxidativo no processo de envelhecimento das células, enfatizando fatores como os danos oxidativos celulares, suas conseqüências e as principais medidas protetoras adotadas para se prevenir ou retardar este processo. Testes com antioxidantes: vitaminas A, E e C, flavonóides, carotenóides e minerais; a prática de restrição calórica e exercícios físicos, que buscam efeitos benéficos sobre a saúde humana, aumentando a longevidade, reduzindo o nível de estresse oxidativo

  16. Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies

    Directory of Open Access Journals (Sweden)

    Aniello Russo

    2010-02-01

    Full Text Available RNA interference (RNAi is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2 protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA. The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and/or B metal ion binding, doesn’t affect the enzymatic activity, suggesting that this residue doesn’t belong to the active site.

  17. ATM Couples Replication Stress and Metabolic Reprogramming during Cellular Senescence

    Directory of Open Access Journals (Sweden)

    Katherine M. Aird

    2015-05-01

    Full Text Available Replication stress induced by nucleotide deficiency plays an important role in cancer initiation. Replication stress in primary cells typically activates the cellular senescence tumor-suppression mechanism. Senescence bypass correlates with development of cancer, a disease characterized by metabolic reprogramming. However, the role of metabolic reprogramming in the cellular response to replication stress has been little explored. Here, we report that ataxia telangiectasia mutated (ATM plays a central role in regulating the cellular response to replication stress by shifting cellular metabolism. ATM inactivation bypasses senescence induced by replication stress triggered by nucleotide deficiency. This was due to restoration of deoxyribonucleotide triphosphate (dNTP levels through both upregulation of the pentose phosphate pathway via increased glucose-6-phosphate dehydrogenase (G6PD activity and enhanced glucose and glutamine consumption. These phenotypes were mediated by a coordinated suppression of p53 and upregulation of c-MYC downstream of ATM inactivation. Our data indicate that ATM status couples replication stress and metabolic reprogramming during senescence.

  18. Continuum representations of cellular solids

    Energy Technology Data Exchange (ETDEWEB)

    Neilsen, M.K.

    1993-09-01

    Cellular materials consist of interconnected struts or plates which form cells. The struts or plates are constructed from a variety of metals, polymers, ceramics and wood products. Cellular materials are often used in impact limiters for shipping containers to protect the contents from accidental impact events. These materials exhibit a variety of complex behavior when subjected to crushing loads. This research focuses on the development of continuum representations of cellular solids that can be used in the finite element analysis of shipping container accidents. A significant portion of this work is the development of a new methodology to relate localized deformations to appropriate constitutive descriptions. This methodology provides the insight needed to select constitutive descriptions for cellular solids that capture the localized deformations that are observed experimentally. Constitutive relations are developed for two different cellular materials, aluminum honeycomb and polyurethane foam. These constitutive relations are based on plasticity and continuum damage theories. Plasticity is used to describe the permanent deformation exhibited by both aluminum honeycomb and polyurethane foam. Continuum damage is needed to capture the change in elastic parameters due to cracking of the polyurethane cell wall materials. The new constitutive description of polyurethane foam is implemented in both static and dynamic finite element codes, and analytical and numerical predictions are compared with available experimental data.

  19. Prognosis of Different Cellular Generations

    Directory of Open Access Journals (Sweden)

    Preetish Ranjan

    2013-04-01

    Full Text Available Technological advancement in mobile telephony from 1G to 3G, 4G and 5G has a very axiomatic fact that made an entire world a global village. The cellular system employs a different design approach and technology that most commercial radio and television system use. In the cellular system, the service area is divided into cells and a transmitter is designed to serve an individual cell. The system seeks to make efficient use of available channels by using low-power transmitters to allow frequency reuse at a smaller distance. Maximizing the number of times each channel can be reused in a given geographical area is the key to an efficient cellular system design. During the past three decades, the world has seen significant changes in telecommunications industry. There have been some remarkable aspects to the rapid growth in wireless communications, as seen by the large expansion in mobile systems. This paper focuses on “Past, Present & Future of Cellular Telephony” and some light has been thrown upon the technologies of the cellular systems, namely 1G, 2G, 2.5G, 3G and future generations like 4G and 5G systems as well.

  20. Performance of the microscopic observation drug susceptibility assay in pyrazinamide susceptibility testing for Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    HUANG Zi-kun; LUO Qing; JIANG Bi-xia; LI Wei-ting; XU Xiao-meng; XIONG Guo-liang; LI Jun-ming

    2013-01-01

    Background Drug susceptibility assay is very important in tuberculosis therapy.Pyrazinamide is a first line antituberculosis drug and diagnosis of its resistance in Mycobacterium tuberculosis (M.tuberculosis) is difficult and time consuming by conventional methods.In this study,we aimed to evaluate the performance of the microscopic observation drug susceptibility (MODS) assay in the detection of pyrazinamide resistance in M.tuberculosis relative to the conventional Wayne assay and Lowenstein-Jensen (L J) proportion method.Methods M.tuberculosis clinical isolates (n=132) were tested by the MODS and the Wayne assay:the results were compared with those obtained by the LJ proportion method.Mutations in the gene were identified by direct sequencing of the pncA genes of all isolates in which pyrazinamide resistance was detected by any of the three methods.Results Compared to the LJ results,the sensitivity and specificity of the MODS assay were 97.8% and 96.5% respectively; the sensitivity and specificity of the Wayne assay were 87.0% and 97.7% respectively.Mutations in the pncA gene were found in 41 of 46 strains that were pyrazinamide resistant (3 tests),in 1 of the 4 strains (LJ only),in 42 of 48 strains (at least 1 test),but no mutations in 1 strain sensitive according to the MODS assay only.The MODS assay,Wayne assay and LJ proportion method provided results in a median time of 6,7 and 26 days respectively.Conclusions MODS assay offers a rapid,simple and reliable method for the detection of pyrazinamide resistance in M.tuberculosis and is an optimal alternative method in resource limited countries.

  1. Induced mutations for human welfare through agriculture

    International Nuclear Information System (INIS)

    Full text: Use of induced mutation for crop improvement started in 1920's. It gained momentum in 1960's when IAEA and FAO started training and guidance and funds were made available for undertaking mutation breeding. IARI established a Gamma Garden and a separate institution was carved by name 'Nuclear Research Laboratory' in 1970's. ICAR Institutes and State Agriculture Universities started using this facility for crop improvement. Similarly, BARC started extending its help for irradiating the seed material specially X-rays and it became one of the major source of generating variability for crop improvement. Induced mutation has resulted in development of more than 3000 varieties of different food, feed, fruit, vegetables and flowers. Apart from direct use of mutants as cultivars, mutants have played a vital role in creating useful variation for application in basic research and gene discovery. It has helped in increasing yield through use of heterosis by inducing male sterility. It has been used for creating useful variation for changing grain composition to improve nutrition and grain quality parameters, for tolerance against abiotic and biotic stresses. Gene sequencing and related technologies have opened up new application of induced mutations. In model organisms induced mutations provide new opportunities for identification of genes/bio-chemical, cellular, developmental or functional pathways. The use of stable isotopes in basic research is of fundamental use in crop improvement. Apart from crop improvement the nuclear technology has been used for numerous other applications in Agriculture such as soil fertility, plant nutrition, use of fertilizer and irrigation, control of insect pest and storage. In recent decades BARC has come in a big way through funding for projects to State Agricultural Universities and ICAR Institutes and has signed MoU's with few of the Agriculture Universities for testing and popularizing their identified field crop varieties in

  2. Cellular Antioxidant Effect of Four Bromophenols from the Red Algae, Vertebrata lanosa

    Directory of Open Access Journals (Sweden)

    Jeanette H. Andersen

    2013-08-01

    Full Text Available Three known bromophenols, 2,3-dibromo-4,5-dihydroxybenzylaldehyde (1, 2,2′,3-tribromo-3′,4,4′,5-tetrahydroxy-6′-hydroxymethyldiphenylmethane (2 and bis(2, 3-dibromo-4,5-dihydroxylbenzyl ether (3, and one new one, 5,5″-oxybis(methylenebis (3-bromo-4-(2′,3′-dibromo-4′,5′-dihydroxylbenzylbenzene-1,2-diol (4, were isolated from an extract of the red alga, Vertebrata lanosa. The antioxidant activity of these four bromophenols was examined using one biochemical and two cellular assays: Oxygen Radical Absorbance Capacity (ORAC, Cellular Antioxidant Activity (CAA and Cellular Lipid Peroxidation Antioxidant Activity (CLPAA assays. Compound 2 distinguished itself by showing potent activity, having a better antioxidant effect than luteolin in both the CAA and CLPAA assays and of quercetin in the CLPAA assay. Although several bromophenols are known to be potent antioxidants in biochemical assays, this is the first time their cellular antioxidant activity has been demonstrated.

  3. A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.

    Directory of Open Access Journals (Sweden)

    Chuan Yu

    Full Text Available Genome editing techniques such as the zinc-finger nucleases (ZFNs, transcription activator-like effecter nucleases (TALENs and clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas system Cas9 can induce efficient DNA double strand breaks (DSBs at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.

  4. A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.

    Science.gov (United States)

    Yu, Chuan; Zhang, Yaguang; Yao, Shaohua; Wei, Yuquan

    2014-01-01

    Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing. PMID:24901507

  5. Medullary carcinoma of breast with a novel germline mutation 1123>g in 11 of brca1

    International Nuclear Information System (INIS)

    Breast cancer, the most common malignancy in females, has an estimated 5-10% hereditary predisposition BRCAI is a tumor suppressor gene and is known to be responsible for breast cancer and breast-ovarian cancers running in families. In breast caner patients, several mutations in BRCAI have been reported throughout the gene. This report describes identification of a mutation in BRCAI gene using protein truncation (PTT) assay in a patient with medullary carcinoma of breast who also had a family history of breast cancer. Following DNA sequencing, the mutation was confirmed as substituation of thymine at position 1123 with guanine of exon 11 (1123 T>G). This mutation can be added to the pool of known BRCAI mutations in Pakistani population, which will help in developing a local screening panel of BRCAI mutations. (author)

  6. Lactate dehydrogenase assay for assessment of polycation cytotoxicity

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Andersen, Helene; Moghimi, Seyed Moien

    2013-01-01

    Cellular toxicity and/or cell death entail complex mechanisms that require detailed evaluation for proper characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay...... cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early...... event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late...

  7. Mutations in MECOM, Encoding Oncoprotein EVI1, Cause Radioulnar Synostosis with Amegakaryocytic Thrombocytopenia.

    Science.gov (United States)

    Niihori, Tetsuya; Ouchi-Uchiyama, Meri; Sasahara, Yoji; Kaneko, Takashi; Hashii, Yoshiko; Irie, Masahiro; Sato, Atsushi; Saito-Nanjo, Yuka; Funayama, Ryo; Nagashima, Takeshi; Inoue, Shin-Ichi; Nakayama, Keiko; Ozono, Keiichi; Kure, Shigeo; Matsubara, Yoichi; Imaizumi, Masue; Aoki, Yoko

    2015-12-01

    Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome, characterized by thrombocytopenia and congenital fusion of the radius and ulna. A heterozygous HOXA11 mutation has been identified in two unrelated families as a cause of RUSAT. However, HOXA11 mutations are absent in a number of individuals with RUSAT, which suggests that other genetic loci contribute to RUSAT. In the current study, we performed whole exome sequencing in an individual with RUSAT and her healthy parents and identified a de novo missense mutation in MECOM, encoding EVI1, in the individual with RUSAT. Subsequent analysis of MECOM in two other individuals with RUSAT revealed two additional missense mutations. These three mutations were clustered within the 8(th) zinc finger motif of the C-terminal zinc finger domain of EVI1. Chromatin immunoprecipitation and qPCR assays of the regions harboring the ETS-like motif that is known as an EVI1 binding site showed a reduction in immunoprecipitated DNA for two EVI1 mutants compared with wild-type EVI1. Furthermore, reporter assays showed that MECOM mutations led to alterations in both AP-1- and TGF-β-mediated transcriptional responses. These functional assays suggest that transcriptional dysregulation by mutant EVI1 could be associated with the development of RUSAT. We report missense mutations in MECOM resulting in a Mendelian disorder that provide compelling evidence for the critical role of EVI1 in normal hematopoiesis and in the development of forelimbs and fingers in humans. PMID:26581901

  8. The comet assay in testing the potential genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Amaya Azqueta

    2015-06-01

    Full Text Available In the last two decades the production and use of nanomaterials (NMs has impressively increased. Their small size, given a mass equal to that of the corresponding bulk material, implies an increase in the surface area and consequently in the number of atoms that can be reactive. They possess different physical, chemical and biological properties compared to bulk materials of the same composition, which makes them very interesting and valuable for many different applications in technology, energy, construction, electronics, agriculture, optics, paints, textiles, food, cosmetics, medicine... Toxicological assessment of NMs is crucial; the same properties that make them interesting also make them potentially harmful for health and the environment. However, the term NM covers many different kinds of particle , and so there is no simple, standard approach to assessing their toxicity. NMs can enter the cell, interact with cell components and even penetrate the nucleus and interfere with the genetic material. Among the different branches of toxicology, genotoxicity is a main area of concern since it is closely related with the carcinogenic potential of compounds. The Organisation for Economic Co-operation and Development (OECD has published internationally agreed in vitro and in vivo validated test methods to evaluate different genotoxic endpoints of chemicals, including chromosome and gene mutations, and DNA breaks. However not all the assays are suitable to study the genotoxic potential of NMs as has been shown by the OECD Working Party on Manufactured Nanomaterials (WPMN. Moreover, alterations to DNA bases, which are precursors to mutations and of great importance in elucidating the mechanism of action of NMs, are not covered by the OECD guidelines. The in vivo standard comet assay (which measures DNA breaks and alkali-labile sites was included in the OECD assays battery in September 2014 while the in vitro standard comet assay is currently under

  9. Aging, cellular senescence, and cancer.

    Science.gov (United States)

    Campisi, Judith

    2013-01-01

    For most species, aging promotes a host of degenerative pathologies that are characterized by debilitating losses of tissue or cellular function. However, especially among vertebrates, aging also promotes hyperplastic pathologies, the most deadly of which is cancer. In contrast to the loss of function that characterizes degenerating cells and tissues, malignant (cancerous) cells must acquire new (albeit aberrant) functions that allow them to develop into a lethal tumor. This review discusses the idea that, despite seemingly opposite characteristics, the degenerative and hyperplastic pathologies of aging are at least partly linked by a common biological phenomenon: a cellular stress response known as cellular senescence. The senescence response is widely recognized as a potent tumor suppressive mechanism. However, recent evidence strengthens the idea that it also drives both degenerative and hyperplastic pathologies, most likely by promoting chronic inflammation. Thus, the senescence response may be the result of antagonistically pleiotropic gene action. PMID:23140366

  10. Novel Materials for Cellular Nanosensors

    DEFF Research Database (Denmark)

    Sasso, Luigi

    The monitoring of cellular behavior is useful for the advancement of biomedical diagnostics, drug development and the understanding of a cell as the main unit of the human body. Micro- and nanotechnology allow for the creation of functional devices that enhance the study of cellular dynamics by...... modifications for electrochemical nanosensors for the detection of analytes released from cells. Two type of materials were investigated, each pertaining to the two different aspects of such devices: peptide nanostructures were studied for the creation of cellular sensing substrates that mimic in vivo surfaces...... and that offer advantages of functionalization, and conducting polymers were used as electrochemical sensor surface modifications for increasing the sensitivity towards relevant analytes, with focus on the detection of dopamine released from cells via exocytosis. Vertical peptide nanowires were...

  11. Cellular-based preemption system

    Science.gov (United States)

    Bachelder, Aaron D. (Inventor)

    2011-01-01

    A cellular-based preemption system that uses existing cellular infrastructure to transmit preemption related data to allow safe passage of emergency vehicles through one or more intersections. A cellular unit in an emergency vehicle is used to generate position reports that are transmitted to the one or more intersections during an emergency response. Based on this position data, the one or more intersections calculate an estimated time of arrival (ETA) of the emergency vehicle, and transmit preemption commands to traffic signals at the intersections based on the calculated ETA. Additional techniques may be used for refining the position reports, ETA calculations, and the like. Such techniques include, without limitation, statistical preemption, map-matching, dead-reckoning, augmented navigation, and/or preemption optimization techniques, all of which are described in further detail in the above-referenced patent applications.

  12. Mutation breeding in pepper

    International Nuclear Information System (INIS)

    Pepper (Capsicum sp.) is an important vegetable and spice crop widely grown in tropical as well as in temperate regions. Until recently the improvement programmes were based mainly on using natural sources of germ plasma, crossbreeding and exploiting the heterosis of F1 hybrids. However, interest in using induced mutations is growing. A great number of agronomically useful mutants as well as mutants valuable for genetic, cytological and physiological studies have been induced and described. In this review information is presented about suitable mutagen treatment procedures with radiation as well as chemicals, M1 effects, handling the treated material in M1, M2 and subsequent generations, and mutant screening procedures. This is supplemented by a description of reported useful mutants and released cultivars. Finally, general advice is given on when and how to incorporate mutation induction in Capsicum improvement programmes. (author)

  13. Kin Selection - Mutation Balance

    DEFF Research Database (Denmark)

    Dyken, J. David Van; Linksvayer, Timothy Arnold; Wade, Michael J.

    2011-01-01

    Abstract Social conflict, in the form of intraspecific selfish "cheating" has been observed in a number of natural systems. However, a formal, evolutionary genetic theory of social cheating that provides an explanatory, predictive framework for these observations is lacking. Here we derive the kin...... selection-mutation balance, which provides an evolutionary null hypothesis for the statics and dynamics of cheating. When social interactions have linear fitness effects and Hamilton´s rule is satisfied, selection is never strong enough to eliminate recurrent cheater mutants from a population, but cheater...... lineages are transient and do not invade. Instead, cheating lineages are eliminated by kin selection but are constantly reintroduced by mutation, maintaining a stable equilibrium frequency of cheaters. The presence of cheaters at equilibrium creates a "cheater load" that selects for mechanisms of cheater...

  14. Cellular function and pathological role of ATP13A2 and related P-type transport ATPases in Parkinson's disease and other neurological disorders

    DEFF Research Database (Denmark)

    van Veen, Sarah; Sørensen, Danny M.; Holemans, Tine;

    2014-01-01

    Mutations in ATP13A2 lead to Kufor-Rakeb syndrome, a parkinsonism with dementia. ATP13A2 belongs to the P-type transport ATPases, a large family of primary active transporters that exert vital cellular functions. However, the cellular function and transported substrate of ATP13A2 remain unknown. ...

  15. Mutation selection of strawberries

    International Nuclear Information System (INIS)

    A brief account is given of the preliminary results of selection work carried out with the aim of deriving a variety of strawberry suitable for mechanized picking. Mutation selection based on irradiation by gamma rays, fast neutrons and a laser beam has been used. The irradiation was performed on strawberry seedlings grown under field conditions and on in vitro cultures at different stages of development. The studies are continuing. (author)

  16. Mutate my software

    OpenAIRE

    Micallef, Mark; Colombo, Christian; Duca, Edward

    2015-01-01

    Computer systems run the world and are found in fridges to hospitals. Every application needs testing, which is expensive and time-consuming. Dr Mark Micallef and Dr Christian Colombo from the PEST research group (Faculty of ICT, University of Malta) tells THINK about a new technique which could make testing easier and more consistent. Illustrations by NO MAD. http://www.um.edu.mt/think/mutate-my-software/

  17. Adaptive stochastic cellular automata: Applications

    Science.gov (United States)

    Qian, S.; Lee, Y. C.; Jones, R. D.; Barnes, C. W.; Flake, G. W.; O'Rourke, M. K.; Lee, K.; Chen, H. H.; Sun, G. Z.; Zhang, Y. Q.; Chen, D.; Giles, C. L.

    1990-09-01

    The stochastic learning cellular automata model has been applied to the problem of controlling unstable systems. Two example unstable systems studied are controlled by an adaptive stochastic cellular automata algorithm with an adaptive critic. The reinforcement learning algorithm and the architecture of the stochastic CA controller are presented. Learning to balance a single pole is discussed in detail. Balancing an inverted double pendulum highlights the power of the stochastic CA approach. The stochastic CA model is compared to conventional adaptive control and artificial neural network approaches.

  18. Cellular automaton for chimera states

    Science.gov (United States)

    García-Morales, Vladimir

    2016-04-01

    A minimalistic model for chimera states is presented. The model is a cellular automaton (CA) which depends on only one adjustable parameter, the range of the nonlocal coupling, and is built from elementary cellular automata and the majority (voting) rule. This suggests the universality of chimera-like behavior from a new point of view: Already simple CA rules based on the majority rule exhibit this behavior. After a short transient, we find chimera states for arbitrary initial conditions, the system spontaneously splitting into stable domains separated by static boundaries, some synchronously oscillating and the others incoherent. When the coupling range is local, nontrivial coherent structures with different periodicities are formed.

  19. Prognosis of Different Cellular Generations

    OpenAIRE

    Preetish Ranjan; Prabhat Kumar

    2013-01-01

    Technological advancement in mobile telephony from 1G to 3G, 4G and 5G has a very axiomatic fact that made an entire world a global village. The cellular system employs a different design approach and technology that most commercial radio and television system use. In the cellular system, the service area is divided into cells and a transmitter is designed to serve an individual cell. The system seeks to make efficient use of available channels by using low-power transmitters to allow frequen...

  20. Oncogenic JAK1 and JAK2-activating mutations resistant to ATP-competitive inhibitors

    OpenAIRE

    Hornakova, Tekla; Springuel, Lorraine; Devreux, Julien; Dusa, Alexandra; Constantinescu, Stefan,; Knoops, Laurent; Renauld, Jean-Christophe

    2011-01-01

    BACKGROUND: Activating mutations in JAK1 and JAK2 have been described in patients with various hematologic malignancies including acute lymphoblastic leukemia and myeloproliferative neoplasms, leading to clinical trials with JAK inhibitors. While there has been a tremendous effort towards the development of specific JAK inhibitors, mutations conferring resistance to such drugs have not yet been observed. DESIGN AND METHODS: Taking advantage of a model of spontaneous cellular transformation...

  1. Improved discrimination of melanotic schwannoma from melanocytic lesions by combined morphological and GNAQ mutational analysis

    OpenAIRE

    Küsters-Vandevelde, Heidi V. N.; van Engen-van Grunsven, Ilse A. C. H.; Küsters, Benno; van Dijk, Marcory R. C. F.; Groenen, Patricia J. T. A.; Wesseling, Pieter; Blokx, Willeke A. M.

    2010-01-01

    The histological differential diagnosis between melanotic schwannoma, primary leptomeningeal melanocytic lesions and cellular blue nevus can be challenging. Correct diagnosis of melanotic schwannoma is important to select patients who need clinical evaluation for possible association with Carney complex. Recently, we described the presence of activating codon 209 mutations in the GNAQ gene in primary leptomeningeal melanocytic lesions. Identical codon 209 mutations have been described in blue...

  2. Heterogeneity of Leucine-Rich Repeat Kinase 2 Mutations: Genetics, Mechanisms and Therapeutic Implications

    OpenAIRE

    Rudenko, Iakov N; Cookson, Mark R.

    2014-01-01

    Variation within and around the leucine-rich repeat kinase 2 (LRRK2) gene is associated with familial and sporadic Parkinson’s disease (PD). Here, we discuss the prevalence of LRRK2 substitutions in different populations and their association with PD, as well as molecular and cellular mechanisms of pathologically relevant LRRK2 mutations. Kinase activation was proposed as a universal molecular mechanism for all pathogenic LRRK2 mutations, but later reports revealed heterogeneity in the effect...

  3. Identification of Mutations and Evaluation of Cardiomyopathy in Turkish Patients with Primary Carnitine Deficiency

    OpenAIRE

    Kilic, M.; Özgül, R. K.; Coşkun, T.; Yücel, D.; Karaca, M.; Sivri, H. S.; Tokatli, A.; Şahin, M.; Karagöz, T.; Dursun, A.

    2011-01-01

    Primary systemic carnitine deficiency (SCD) is an autosomal recessive disorder caused by defective cellular carnitine transport. Patients usually present with predominant metabolic or cardiac manifestations. SCD is caused by mutations in the organic cation/carnitine transporter OCTN2 (SLC22A5) gene. Mutation analysis of SLC22A5 gene was carried out in eight Turkish patients from six families. Six patients presented with signs and symptoms of heart failure, cardiomyopathy, and low plasma carni...

  4. Validation of the cell cycle G2 delay assay in assessing ionizing radiation sensitivity and breast cancer risk

    Directory of Open Access Journals (Sweden)

    Jeff W Hill

    2009-04-01

    Full Text Available Jeff W Hill1, Kristina Tansavatdi4, Kristin L Lockett4, Glenn O Allen1, Cristiane Takita2, Alan Pollack2, Jennifer J Hu1,31Sylvester Comprehensive Cancer Center, 2Department of Radiation Oncology, 3Department of Epidemiology and Public Health, University of Miami Miller School of Medicine, Miami, FL, USA; 4Department of Cancer Biology, Wake Forest University Health Sciences, Winston-Salem, NC, USAAbstract: Genetic variations in cell cycle checkpoints and DNA repair genes are associated with prolonged cell cycle G2 delay following ionizing radiation (IR treatment and breast cancer risk. However, different studies reported conflicting results examining the association between post-IR cell cycle delay and breast cancer risk utilizing four different parameters: cell cycle G2 delay index, %G2–M, G2/G0–G1, and (G2/G0–G1/S. Therefore, we evaluated whether different parameters may influence study results using a data set from 118 breast cancer cases and 225 controls as well as lymphoblastoid and breast cancer cell lines with different genetic defects. Our results suggest that cell cycle G2 delay index may serve as the best parameter in assessing breast cancer risk, genetic regulation of IR-sensitivity, and mutations of ataxia telangiectasia mutated (ATM and TP53. Cell cycle delay in 21 lymphoblastoid cell lines derived from BRCA1 mutation carriers was not different from that in controls. We also showed that IR-induced DNA-damage signaling, as measured by phosphorylation of H2AX on serine 139 (γ-H2AX was inversely associated with cell cycle G2 delay index. In summary, the cellular responses to IR are extremely complex; mutations or genetic variations in DNA damage signaling, cell cycle checkpoints, and DNA repair contribute to cell cycle G2 delay and breast cancer risk. The cell cycle G2 delay assay characterized in this study may help identify subpopulations with elevated risk of breast cancer or susceptibility to adverse effects in normal tissue

  5. Calreticulin Mutations in Myeloproliferative Neoplasms

    Directory of Open Access Journals (Sweden)

    Noa Lavi

    2014-10-01

    Full Text Available With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph− myeloproliferative neoplasms (MPNs in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (ET and primary myelofibrosis (PMF. At the end of 2013, two studies identified recurrent mutations in the gene encoding calreticulin (CALR using whole-exome sequencing. These mutations were revealed in the majority of ET and PMF patients with non-mutated JAK2 or MPL but not in polycythemia vera patients. Somatic 52-bp deletions (type 1 mutations and recurrent 5-bp insertions (type 2 mutations in exon 9 of the CALR gene (the last exon encoding the C-terminal amino acids of the protein calreticulin were detected and found always to generate frameshift mutations. All detected mutant calreticulin proteins shared a novel amino acid sequence at the C-terminal. Mutations in CALR are acquired early in the clonal history of the disease, and they cause activation of JAK/STAT signaling. The CALR mutations are the second most frequent mutations in Ph− MPN patients after the JAK2V617F mutation, and their detection has significantly improved the diagnostic approach for ET and PMF. The characteristics of the CALR mutations as well as their diagnostic, clinical, and pathogenesis implications are discussed in this review.

  6. A Cellular GWAS Approach to Define Human Variation in Cellular Pathways Important to Inflammation.

    Science.gov (United States)

    Miller, Samuel I; Chaudhary, Anu

    2016-01-01

    An understanding of common human diversity in innate immune pathways should be beneficial in understanding autoimmune diseases, susceptibility to infection, and choices of anti-inflammatory treatment. Such understanding could also result in definition of currently unknown components of human inflammation pathways. A cellular genome-wide association studies (GWAS) platform, termed Hi-HOST (High-throughput human in vitro susceptibility testing), was developed to assay in vitro cellular phenotypes of infection in genotyped lymphoblastoid cells from genetically diverse human populations. Hi-HOST allows for measurement of multiple host and pathogen parameters of infection/inflammation including: bacterial invasion and intracellular replication, host cell death, and cytokine production. Hi-HOST has been used to successfully define a significant portion of the heritable human diversity in inflammatory cell death in response to Salmonella typhimurium. It also led to the discovery of genetic variants important to protection against systemic inflammatory response syndrome (SIRS) and protection against death and bacteremia in individuals with SIRS. Our laboratory is currently using this platform to define human diversity in autophagy and the NLPR3 inflammasome pathways, and to define new components that can impact the expression of phenotypes related to these pathways. PMID:27128945

  7. Functional characterization of human cancer-derived TRKB mutations.

    Directory of Open Access Journals (Sweden)

    Thomas R Geiger

    Full Text Available Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKB(T695I and TRKB(D751N. Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKB(L138F. Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKB(T695I and TRKB(D751N, displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKB(L138F and TRKB(P507L were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

  8. Functional Assays for Neurotoxicity Testing

    Science.gov (United States)

    Neurobehavioral and pathological evaluations of the nervous system are complementary components of basic research and toxicity testing of pharmaceutical and environmental chemicals. While neuropathological assessments provide insight as to cellular changes in neurons, behavioral ...

  9. Functional Assays for Neurotoxicity Testing*

    Science.gov (United States)

    Neurobehavioral and pathological evaluations of the nervous system are complementary components of basic research and toxicity testing of pharmaceutical and environmental chemicals. While neuropathological assessments provide insight as to cellular changes in neurons, behavioral ...

  10. Somatic mutation in peripheral blood lymphocytes among Metro Manila residents: indicator of exposure to environmental pollution

    International Nuclear Information System (INIS)

    Metro Manila is ranked as one of the world's most polluted cities where air quality levels are 2-3 times higher than the levels set by WHO. Development of diseases could be alleviated if early warning signs as occurrence of gene mutations are detected early enough. The adapted hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutation assay measures the degree of mutation on the HGPRT gene and allows rapid evaluation of the occurrence of mutation in an individual exposed to radiation or mutagens within six months after exposure. The objective of the project is to (1) assay exposure of Metro Manila residents exposed to environmental pollution, (2) determine population groups significantly affected by pollutants and (3) construct an environmental baseline HGPRT mutation data bank specific to area in Metro Manila. A composite table of personal information of donors against mutation index in two barangays in Venezuela is presented. About 30% of the total samples are shown to have mutation index greater than 0.5. So far, the data show a slightly higher mutation rate among donors who are smokers with more than 5 hours outdoor exposure to pollutants per day than the corresponding class of non-smokers. (author). 5 refs.; 5 tabs

  11. Repaglinide at a cellular level

    DEFF Research Database (Denmark)

    Krogsgaard Thomsen, M; Bokvist, K; Høy, M;

    2002-01-01

    To investigate the hormonal and cellular selectivity of the prandial glucose regulators, we have undertaken a series of experiments, in which we characterised the effects of repaglinide and nateglinide on ATP-sensitive potassium ion (KATP) channel activity, membrane potential and exocytosis in ra...

  12. Cellular signalling properties in microcircuits

    DEFF Research Database (Denmark)

    Toledo-Rodriguez, Maria; El Manira, Abdeljabbar; Wallén, Peter; Svirskis, Gytis; Hounsgaard, Jørn

    2005-01-01

    Molecules and cells are the signalling elements in microcircuits. Recent studies have uncovered bewildering diversity in postsynaptic signalling properties in all areas of the vertebrate nervous system. Major effort is now being invested in establishing the specialized signalling properties at th...... cellular and molecular levels in microcircuits in specific brain regions. This review is part of the TINS Microcircuits Special Feature....

  13. Quantum Cloning by Cellular Automata

    OpenAIRE

    D'Ariano, G. M.; Macchiavello, C.; M. Rossi

    2012-01-01

    We introduce a quantum cellular automaton that achieves approximate phase-covariant cloning of qubits. The automaton is optimized for 1-to-2N economical cloning. The use of the automaton for cloning allows us to exploit different foliations for improving the performance with given resources.

  14. Analysis of cellular manufacturing systems

    NARCIS (Netherlands)

    Heragu, Sunderesh; Meng, Gang; Zijm, Henk; Ommeren, van Jan-Kees

    2001-01-01

    In this paper, we present an open queuing network modeling approach to estimate performance measures of a cellular manufacturing layout. It is assumed a layout and production data for a planning period of specified length are available. The production data takes into account, processing and handli

  15. From Antenna to Assay

    Science.gov (United States)

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  16. Functional impact of HIV coreceptor-binding site mutations

    International Nuclear Information System (INIS)

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner

  17. Alternative mechanisms of telomere lengthening: permissive mutations, DNA repair proteins and tumorigenic progression

    OpenAIRE

    Sandy Gocha1, April Renee; Harris, Julia; Groden, Joanna

    2012-01-01

    Telomeres protect chromosome termini to maintain genomic stability and regulate cellular lifespan. Maintenance of telomere length is required for neoplastic cells after the acquisition of mutations that deregulate cell cycle control and increase cellular proliferation, and can occur through expression of the enzyme telomerase or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor ...

  18. Three families with 'de novo' m.3243A > G mutation.

    Science.gov (United States)

    de Laat, Paul; Janssen, Mirian C H; Alston, Charlotte L; Taylor, Robert W; Rodenburg, Richard J T; Smeitink, Jan A M

    2016-12-01

    The m.3243A > G mutation is the most prevalent, disease-causing mitochondrial DNA (mtDNA) mutation. In a national cohort study of 48 families harbouring the m.3243A > G mutation, we identified three families in which the mutation appeared to occur sporadically within these families. In this report we describe these three families. Based on detailed mtDNA analysis of three different tissues using two different quantitative pyrosequencing assays with sensitivity to a level of 1% mutated mtDNA, we conclude that the m.3243A > G mutation has arisen de novo in each of these families. The symptomatic carriers presented with a variety of symptoms frequently observed in patients harbouring the m.3243A > G mutation. A more severe phenotype is seen in the de novo families compared to recent cohort studies, which might be due to reporting bias. The observation that de novo m.3243A > G mutations exist is of relevance for both diagnostic investigations and genetic counselling. Firstly, even where there is no significant (maternal) family history in patients with stroke-like episodes, diabetes and deafness or other unexplained organ dysfunction, the m.3243A > G mutation should be screened as a possible cause of the disease. Second, analysis of maternally-related family members is highly recommended to provide reliable counselling for these families, given that the m.3243A > G mutation may have arisen de novo. PMID:27331024

  19. New Application of the Comet Assay: Chromosome–Comet Assay

    OpenAIRE

    Cortés-Gutiérrez, Elva I.; DÁVILA-RODRÍGUEZ, MARTHA I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be use...

  20. Calorimetric assay of minor actinides

    Energy Technology Data Exchange (ETDEWEB)

    Rudy, C.; Bracken, D.; Cremers, T.; Foster, L.A.; Ensslin, N.

    1996-12-31

    This paper reviews the principles of calorimetric assay and evaluates its potential application to the minor actinides (U-232-4, Am-241, Am- 243, Cm-245, Np-237). We conclude that calorimetry and high- resolution gamma-ray isotopic analysis can be used for the assay of minor actinides by adapting existing methodologies for Pu/Am-241 mixtures. In some cases, mixtures of special nuclear materials and minor actinides may require the development of new methodologies that involve a combination of destructive and nondestructive assay techniques.

  1. Calorimetric assay of minor actinides

    International Nuclear Information System (INIS)

    This paper reviews the principles of calorimetric assay and evaluates its potential application to the minor actinides (U-232-4, Am-241, Am- 243, Cm-245, Np-237). We conclude that calorimetry and high- resolution gamma-ray isotopic analysis can be used for the assay of minor actinides by adapting existing methodologies for Pu/Am-241 mixtures. In some cases, mixtures of special nuclear materials and minor actinides may require the development of new methodologies that involve a combination of destructive and nondestructive assay techniques

  2. Somatic mutation profiles in primary colorectal cancers and matching ovarian metastases: Identification of driver and passenger mutations

    Science.gov (United States)

    Crobach, Stijn; Ruano, Dina; van Eijk, Ronald; Schrumpf, Melanie; Fleuren, Gertjan; van Wezel, Tom

    2016-01-01

    Abstract The mutational profiles of primary colorectal cancers (CRCs) and corresponding ovarian metastases were compared. Using a custom‐made next generation sequencing panel, 115 cancer‐driving genes were analyzed in a cohort of 26 primary CRCs and 30 matching ovarian metastases (four with bilateral metastases). To obtain a complete overview of the mutational profile, low thresholds were used in bioinformatics analysis to prevent low frequency passenger mutations from being filtered out. A subset of variants was validated using Sanger and/or hydrolysis probe assays. The mutational landscape of CRC that metastasized to the ovary was not strikingly different from CRC in consecutive series. When comparing primary CRCs and their matching ovarian metastases, there was considerable overlap in the mutations of early affected genes. A subset of mutations demonstrated less overlap, presumably being passenger mutations. In particular, primary CRCs showed a substantially high number of passenger mutations. We also compared the primary CRCs and matching metastases for stratifying variants of six genes (KRAS, NRAS, BRAF, FBXW7, PTEN and PIK3CA) that select for established (EGFR directed) or future targeted therapies. In a total of 31 variants 12 were not found in either of the two locations. Tumours thus differed in the number of discordant variants between the primary tumours and matching metastases. Half of these discordant variants were definitive class 4/5 pathogenic variants. However, in terms of temporal heterogeneity, no clear relationship was observed between the number of discordant variants and the time interval between primary CRCs and the detection of ovarian metastases. This suggests that dormant metastases may be present from the early days of the primary tumours. PMID:27499925

  3. Evaluation of the AID TB Resistance Line Probe Assay for Rapid Detection of Genetic Alterations Associated with Drug Resistance in Mycobacterium tuberculosis Strains

    OpenAIRE

    C. Ritter; Lucke, K.; F. A. Sirgel; Warren, R. W.; van Helden, P. D.; Böttger, E C; Bloemberg, G.V.

    2014-01-01

    The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected r...

  4. Mutation breeding in chickpea

    International Nuclear Information System (INIS)

    Chickpea is an important food legume in Turkey. Turkey is one of the most important gene centers in the world for legumes. The most widely known characteristic of chickpea is that it is an important vegetable protein source used in human and animal nutrition. However, the dry grains of chickpea, has 2-3 times more protein than our traditional food of wheat. In addition, cheakpea is also energy source because of its high carbohydrate content. It is very rich in some vitamin and mineral basis. In the plant breeding, mutation induction has become an effective way of supplementing existing germplasm and improving cultivars. Many successful examples of mutation induction have proved that mutation breeding is an effective and important approach to food legume improvement. The induced mutation technique in chickpea has proved successful and good results have been attained. Realizing the potential of induced mutations, a mutation breeding programme was initiated at the Nuclear Agriculture Section of the Saraykoey Nuclear Research and Training Center in 1994. The purpose of the study was to obtain high yielding chickpea mutants with large seeds, good cooking quality and high protein content. Beside this some characters such as higher adaptation ability, tolerant to cold and drought, increased machinery harvest type, higher yield, resistant to diseases especially to antracnose and pest were investigated too. Parents varieties were ILC-482, AK-7114 and AKCIN-91 (9 % seed moisture content and germination percentage 98 %) in these experiments. The irradiation doses were 0 (control), 50, 100, 150, 200, 250, 300, 350, 400, 500 ve 600 Gy for greenhouse experiments and 0 (control), 50, 100, 150, 200, 250, 300, 350 ve 400 Gy for field experiments, respectively. One thousand seeds for per treatment were sown in the field for the M1. At maturity, 3500 single plants were harvested and 20 seeds were taken from each M1 plant and planted in the following season. During plant growth

  5. Development of ARMS PCR tests for detection of common CFTR gene mutations

    Directory of Open Access Journals (Sweden)

    Livshits L. A.

    2010-09-01

    Full Text Available Aim. To develop diagnostic assays, based on the amplification refractory mutation system (ARMS principle, for detection of common mutations in the CFTR gene using two approaches: standard PCR with further gel-electrophoresis and Real-Time PCR with SYBR Green. Materials. For this study we have chosen the following mutations: dF508, W1282X, R117H, 621 + 1G > T, 2143delT with the frequencies in Ukraine: dF508 – 43.3 %; 2143delT – 1.38 %; W1282X – 1.1 %; R117H, 621 + 1G > T – T, 2143delT mutations. To validate the developed assays we have analyzed control DNA samples with the following mutations: W1282X (n = 3, R117H (n = 2, 621 + 1G > T (n = 1, 2143delT (n = = 1. For validation of the dF508 assay we have analyzed 100 heterozygous carriers and 50 homozygous carriers. We have analyzed 48 patients with cystic fibrosis, in which only one mutation was previously detected in combination with unknown mutant variant, using the developed ARMS assay for the 2143delT mutation, and detected 4 heterozygous carriers. No differences were observed in comparison with the standard protocols. Conclusions. It was shown that ARMS is a reliable, rapid and inexpensive method, and the developed assays can be applied in the standard PCR protocol with further gel-electrophoresis as well as using Real-Time PCR with SYBR Green for the molecular genetic diagnostics of cystic fibrosis.

  6. Multistructural biomimetic substrates for controlled cellular differentiation

    International Nuclear Information System (INIS)

    Multidimensional scaffolds are considered to be ideal candidates for regenerative medicine and tissue engineering based on their potential to provide an excellent microenvironment and direct the fate of the cultured cells. More recently, the use of stem cells in medicine has opened a new technological opportunity for controlled tissue formation. However, the mechanism through which the substrate directs the differentiation of stem cells is still rather unclear. Data concerning its specific surface chemistry, topology, and its signaling ability need to be further understood and analyzed. In our study, atomic force microscopy was used to study the stiffness, roughness, and topology of the collagen (Coll) and metallized collagen (MC) substrates, proposed as an excellent substrate for regenerative medicine. The importance of signaling molecules was studied by constructing a new hybrid signaling substrate that contains both collagen and laminin extracellular matrix (ECM) proteins. The cellular response—such as attachment capability, proliferation and cardiac and neuronal phenotype expression on the metallized and non-metallized hybrid substrates (collagen + laminin)—was studied using MTT viability assay and immunohistochemistry studies. Our findings indicate that such hybrid materials could play an important role in the regeneration of complex tissues. (paper)

  7. Cellular indicators of preclinical phases of leukemia

    International Nuclear Information System (INIS)

    The objectives of this project are to: (1) define the stages of pathological progression of myelogenous leukemia occurring in dogs undergoing continuous radiation exposure; (2) develop sensitive predictors of the pathological responses based on early cellular events; and (3) utilize cell cloning methods to clarify mechanisms of early hemopoietic dysfunctions. Based on previous work which showed that myelogenous leukemia occurs with a 50% incidence in beagles exposed for protracted periods to 60Co gamma radiation, beagles are irradiated continuously (i.e., 22 hours/day at the dose rate of 10 R/day) at the dose rate of 10 R/day. The experimental approach is then to assess serially the phase related changes within the granulopoietic system of the exposed animals. Assessment employs both in vitro and in vivo assays aimed at compartmentalizing the granulopoietic system. The marrow compartments presently being examined include: (1) the granulocyte-monocyte committed stem cells (GM-CFned by two-dimensional electrophoresis to detect disease and pollutant related changes. Assessment of human risk associated with nuclearing collective dose commitment will result in more attention being paid to potential releases of radionuclides at relatively short times after disposal

  8. Cellular pathways controlling integron cassette site folding.

    Science.gov (United States)

    Loot, Céline; Bikard, David; Rachlin, Anna; Mazel, Didier

    2010-08-01

    By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single-stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double-stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non-recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination. PMID:20628355

  9. Induced mutations in citrus

    International Nuclear Information System (INIS)

    Full text: Parthenocarpic tendency is an important prerequisite for successful induction of seedlessness in breeding and especially in mutation breeding. A gene for asynapsis and accompanying seedless fruit has been found by us in inbred progeny of cv. 'Wilking'. Using budwood irradiation by gamma rays, seedless mutants of 'Eureka' and 'Villafranca' lemon (original clone of the latter has 25 seeds) and 'Minneola' tangelo have been obtained. Ovule sterility of the three mutants is nearly complete, with some pollen fertility still remaining. A semi-compact mutant of Shamouti orange has been obtained by irradiation. A programme for inducing seedlessness in easy peeling citrus varieties and selections has been initiated. (author)

  10. Induced skeletal mutations

    International Nuclear Information System (INIS)

    This paper describes a large-scale experiment that, by means of breeding tests, confirmed that many dominant skeletal mutations are induced by large-dose radiation exposure. The author also discusses: (1) the major advantages and disadvantages of the skeletal method in improving estimates of genetic hazard to man; (2) future uses of the skeletal method; (3) direct estimation of risk beyond the first generation using the skeletal method; and (4) the possibility of using the skeletal method as a quick and easy screen for chemical mutagens

  11. R31C GNRH1 Mutation and Congenital Hypogonadotropic Hypogonadism

    Science.gov (United States)

    Maione, Luigi; Albarel, Frederique; Bouchard, Philippe; Gallant, Megan; Flanagan, Colleen A.; Bobe, Regis; Cohen-Tannoudji, Joelle; Pivonello, Rosario; Colao, Annamaria; Brue, Thierry; Millar, Robert P.; Lombes, Marc; Young, Jacques; Guiochon-Mantel, Anne; Bouligand, Jerome

    2013-01-01

    Normosmic congenital hypogonadotropic hypogonadism (nCHH) is a rare reproductive disease leading to lack of puberty and infertility. Loss-of-function mutations of GNRH1 gene are a very rare cause of autosomal recessive nCHH. R31C GNRH1 is the only missense mutation that affects the conserved GnRH decapeptide sequence. This mutation was identified in a CpG islet in nine nCHH subjects from four unrelated families, giving evidence for a putative “hot spot”. Interestingly, all the nCHH patients carry this mutation in heterozygosis that strikingly contrasts with the recessive inheritance associated with frame shift and non-sense mutations. Therefore, after exclusion of a second genetic event, a comprehensive functional characterization of the mutant R31C GnRH was undertaken. Using different cellular models, we clearly demonstrate a dramatic reduction of the mutant decapeptide capacity to bind GnRH-receptor, to activate MAPK pathway and to trigger inositol phosphate accumulation and intracellular calcium mobilization. In addition it is less able than wild type to induce lh-beta transcription and LH secretion in gonadotrope cells. Finally, the absence of a negative dominance in vitro offers a unique opportunity to discuss the complex in vivo patho-physiology of this form of nCHH. PMID:23936060

  12. Biotechnological approach in crop improvement by mutation breeding in Indonesia

    International Nuclear Information System (INIS)

    Mutation breeding has become a proven method of improving crop varieties. Most research on plant mutation breeding in Indonesia is carried out at the Center for Research and Development of Isotope and Radiation Technology, National Nuclear Energy Agency (BATAN). Nowadays, a biotechnological approach has been incorporated in some mutation breeding researches in order to improve crop cultivars. This approach is simply based on cellular totipotency, or the ability to regenerate whole, flowering plants from isolated organs, pieces of tissue, individual cells, and protoplasts. Tissue culture technique has bee extensively used for micro propagation of disease-free plants. Other usage of this technique involves in various steps of the breeding process such as germplasm preservation, clonal propagation, and distant hybridization. Mutation breeding combined with tissue culture technique has made a significant contribution in inducing plant genetic variation, by improving selection technology, and by accelerating breeding time as for that by using anther or pollen culture. In Indonesia, research on mutation breeding combined with tissue culture techniques has been practiced in different crop species including rice, ginger, banana, sorghum etc. Specially in rice, a research on identification of DNA markers linked to blast disease resistance is now still progressing. A compiled report from some research activities is presented in this paper. (author)

  13. The use of comet assay in plant toxicology: recent advances

    Directory of Open Access Journals (Sweden)

    Conceição LV Santos

    2015-06-01

    Full Text Available The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g. Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

  14. Mutation Breeding Newsletter. No. 39

    International Nuclear Information System (INIS)

    This newsletter contains brief articles on the use of radiation to induce mutations in plants; radiation-induced mutants in Chrysanthemum; disrupting the association between oil and protein content in soybean seeds; mutation studies on bougainvillea; a new pepper cultivar; and the use of mutation induction to improve the quality of yam beans. A short review of the seminar on the use of mutation and related biotechnology for crop improvement in the Middle East and Mediterranean regions, and a description of a Co-ordinated Research Programme on the application of DNA-based marker mutations for the improvement of cereals and other sexually reproduced crop species are also included. Two tables are given: these are based on the ''FAO/IAEA Mutant Varieties Database'' and show the number of mutated varieties and the number of officially released mutant varieties in particular crops/species. Refs and tabs

  15. Chromosome aberration assays in Pisum for the study of environmental mutagens.

    Science.gov (United States)

    Grant, W F; Owens, E T

    2001-05-01

    From a literature survey, 117 chemicals are tabulated that have been assayed in 179 assays for their clastogenic effects in Pisum. Of the 117 chemicals that have been assayed, 65 are reported at giving a positive reaction (i.e. causing chromosome aberrations), 30 positive with a dose response, five borderline positive. Seventeen chemicals gave a negative response. Eighty-one percent of the chemicals gave a definite positive response. A c-mitotic effect was detected from treatment with 17 chemicals. In addition to the above tabulation of chemicals, 39 chemicals have been reported with an antimitotic effect. Thirteen assays have been recorded for five types of radiation, which with the exception of ultrasound reacted positively. The results of assays with 38 chemicals and/or radiations in combined treatments, as well as 15 chemicals and three types of radiations that induce somatic mutations are tabulated. The Pisum sativum (2n=14) bioassay has been shown to be a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis for somatic mutations induced by chemicals, radiations, and environmental pollutants. For some chemicals, the Pisum assay is not as sensitive in assessing clastogenicity as the Allium assay, although this should be considered in relative terms. Pisum fulvum (2n=14) has been used in clastogenic studies also, but to a much lesser extent. PMID:11344039

  16. Calreticulin Mutations in Myeloproliferative Neoplasms

    OpenAIRE

    Noa Lavi

    2014-01-01

    With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph−) myeloproliferative neoplasms (MPNs) in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (...

  17. Cellular solidification of transparent monotectics

    Science.gov (United States)

    Kaulker, W. F.

    1986-01-01

    Understanding how liquid phase particles are engulfed or pushed during freezing of a monotectic is addressed. The additional complication is that the solid-liquid interface is nonplanar due to constitutional undercooling. Some evidence of particle pushing where the particles are the liquid phase of the montectic was already observed. Cellular freezing of the succinonitrile-glycerol system also occurred. Only a few compositions were tested at that time. The starting materials were not especially pure so that cellular interface observed was likely due to the presence of unkown impurities, the major portion of which was water. Topics addressed include: the effort of modeling the particle pushing process using the computer, establishing an apparatus for the determination of phase diagrams, and the measurement of the temperature gradients with a specimen which will solidify on the temperature gradient microscope stage.

  18. Cellular ceramics in combustion environments

    Energy Technology Data Exchange (ETDEWEB)

    Fuessel, Alexander; Boettge, Daniela; Adler, Joerg; Marschallek, Felix; Michaelis, Alexander [Fraunhofer Institute for Ceramic Technologies and Systems IKTS, Dresden (Germany)

    2011-11-15

    Cellular materials have become increasingly interesting for applications in combustion environments. Improvements like high power efficiency and low emissions are the main targets of technological development in combustion processes. However, despite scientific and technical success in developing new or improved burner concepts over recent years, a lot of problems remain to be solved in the field of materials science: due to the high power density of the burners the materials are subjected to high loads in terms of thermal shock, temperature and corrosion, especially in so-called porous burner technology. This article shows some examples of research and development strategies and results in developing improved cellular ceramics. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  19. Designing Underwater Cellular Networks Parameters

    Directory of Open Access Journals (Sweden)

    Pejman Khadivi

    2008-09-01

    Full Text Available Oceanographic data collection, pollution monitoring, offshore exploration, disaster prevention, assisted navigation and tactical surveillance are some of the applications of underwater networks. Underwater networks should send the gathered information to other users or an offshore station via a base station in the sea. Since the available bandwidth in underwater is severely limited, frequency reuse and cellular networks concepts are very important. In this paper, after driving the ratio of signal to interference for underwater acoustic channels, the constraints for the cell radius are determined. One of the important results of this work is that, for special parameters like bandwidth, it may be impossible to provide the required signal to interference ratio and bandwidth for the network users. Furthermore, in this paper, number of supportable users, per-user bandwidth, and the user capacity for a cellular underwater network are determined.

  20. Frequency of mutations and polymorphisms in borderline ovarian tumors of known cancer genes.

    Science.gov (United States)

    Stemke-Hale, Katherine; Shipman, Kristy; Kitsou-Mylona, Isidora; de Castro, David G; Hird, Vicky; Brown, Robert; Flanagan, James; Gabra, Hani; Mills, Gordon B; Agarwal, Roshan; El-Bahrawy, Mona

    2013-04-01

    Borderline ovarian tumors represent an understudied subset of ovarian tumors. Most studies investigating aberrations in borderline tumors have focused on KRAS/BRAF mutations. In this study, we conducted an extensive analysis of mutations and single-nucleotide polymorphisms (SNPs) in borderline ovarian tumors. Using the Sequenom MassArray platform, we investigated 160 mutations/polymorphisms in 33 genes involved in cell signaling, apoptosis, angiogenesis, cell cycle regulation and cellular senescence. Of 52 tumors analyzed, 33 were serous, 18 mucinous and 1 endometrioid. KRAS c.35G>A p.Gly12Asp mutations were detected in eight tumors (six serous and two mucinous), BRAF V600E mutations in two serous tumors, and PIK3CA H1047Y and PIK3CA E542K mutations in a serous and an endometrioid BOT, respectively. CTNNB1 mutation was detected in a serous tumor. Potentially functional polymorphisms were found in vascular endothelial growth factor (VEGF), ABCB1, FGFR2 and PHLPP2. VEGF polymorphisms were the most common and detected at four loci. PHLPP2 polymorphisms were more frequent in mucinous as compared with serous tumors (P=0.04), with allelic imbalance in one case. This study represents the largest and most comprehensive analysis of mutations and functional SNPs in borderline ovarian tumors to date. At least 25% of borderline ovarian tumors harbor somatic mutations associated with potential response to targeted therapeutics. PMID:23174937

  1. Mutator suppression and escape from replication error-induced extinction in yeast.

    Directory of Open Access Journals (Sweden)

    Alan J Herr

    2011-10-01

    Full Text Available Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10⁻³ inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer.

  2. Mutation breeding in mangosteen

    International Nuclear Information System (INIS)

    Mangosteen the queen of the tropical fruits is apomitic and only a cultivar is reported and it reproduces asexually. Conventional breeding is not possible and the other methods to create variabilities are through genetic engineering and mutation breeding. The former technique is still in the infantry stage in mangosteen research while the latter has been an established tool in breeding to improve cultivars. In this mutation breeding seeds of mangosteen were irradiated using gamma rays and the LD 50 for mangosteen was determined and noted to be very low at 10 Gy. After sowing in the seedbed, the seedlings were transplanted in polybags and observed in the nursery bed for about one year before planted in the field under old oil palm trees in Station MARDI, Kluang. After evaluation and screening, about 120 mutant mangosteen plants were selected and planted in Kluang. The plants were observed and some growth data taken. There were some mutant plants that have good growth vigour and more vigorous that the control plants. The trial are now in the fourth year and the plants are still in the juvenile stage. (Author)

  3. Mutation breeding in chickpea

    International Nuclear Information System (INIS)

    Chickpea is an important food legume in Turkey. Turkey is one of the most important gene centers in the world for legumes. Realizing the potential of induced mutations, a mutation breeding programme was initiated at the Nuclear Agriculture Section of the Saraykoy Nuclear Research and Training Center in 1994. The purpose of the study was to obtain high yielding chickpea mutants with large seeds, good cooking quality and high protein content. Beside this some characters such as higher adaptation ability, tolerant to cold and drought, increased machinery harvest type, higher yield, resistant to diseases especially to antracnose and pest were investigated too. Parent varieties were ILC-482, AK-7114 and AKCIN-91 had been used in these experiments. The irradiation doses were 0 (control), 50, 100, 150, 200, 250, 300, 350 and 400 Gy for field experiments, respectively. As a result of these experiments, two promising mutant lines were chosen and given to the Seed Registration and Certification Center for official registration These two promising mutants were tested at five different locations of Turkey, in 2004 and 2005 years. After 2 years of registration experiments one of outstanding mutants was officially released as mutant chickpea variety under the name TAEK-SAGEL, in 2006. Some basic characteristics of this mutant are; earliness (95-100 day), high yield capacity (180-220 kg/da), high seed protein (22-25 %), first pot height (20-25 cm), 100 seeds weight (42-48 g), cooking time (35-40 min) and resistance to Ascochyta blight.

  4. Stochastic Nature in Cellular Processes

    Institute of Scientific and Technical Information of China (English)

    刘波; 刘圣君; 王祺; 晏世伟; 耿轶钊; SAKATA Fumihiko; GAO Xing-Fa

    2011-01-01

    The importance of stochasticity in cellular processes is increasingly recognized in both theoretical and experimental studies. General features of stochasticity in gene regulation and expression are briefly reviewed in this article, which include the main experimental phenomena, classification, quantization and regulation of noises. The correlation and transmission of noise in cascade networks are analyzed further and the stochastic simulation methods that can capture effects of intrinsic and extrinsic noise are described.

  5. Xtoys cellular automata on xwindows

    CERN Document Server

    Creutz, M

    1995-01-01

    Xtoys is a collection of xwindow programs for demonstrating simulations of various statistical models. Included are xising, for the two dimensional Ising model, xpotts, for the q-state Potts model, xautomalab, for a fairly general class of totalistic cellular automata, xsand, for the Bak-Tang-Wiesenfield model of self organized criticality, and xfires, a simple forest fire simulation. The programs should compile on any machine supporting xwindows.

  6. Cellular reactions to patterned biointerfaces

    OpenAIRE

    Schulte, Vera Antonie

    2012-01-01

    The subject of this thesis is to study cellular reactions to topographically, mechanically and biochemically tunable polymeric biomaterials. Different aspects of in vitro cell-biomaterial interactions were systematically studied with the murine fibroblast cell line NIH L929 and primary human dermal fibroblasts (HDFs). Besides a general cytocompatibility assessment of the applied materials and the quantification of cell adhesion per se, cell morphological changes (e.g. cell spreading) and intr...

  7. Signal processing in cellular clocks

    OpenAIRE

    Forger, Daniel B.

    2011-01-01

    Many biochemical events within a cell need to be timed properly to occur at specific times of day, after other events have happened within the cell or in response to environmental signals. The cellular biochemical feedback loops that time these events have already received much recent attention in the experimental and modeling communities. Here, we show how ideas from signal processing can be applied to understand the function of these clocks. Consider two signals from the network s(t) and r(...

  8. Analysis of cellular manufacturing systems

    OpenAIRE

    Heragu, Sunderesh; Meng, Gang; Zijm, Henk; Ommeren, van, J.C.

    2001-01-01

    In this paper, we present an open queuing network modeling approach to estimate performance measures of a cellular manufacturing layout. It is assumed a layout and production data for a planning period of specified length are available. The production data takes into account, processing and handling set-up times as well as transfer and process batch size information of multiple products that flow through the system. It is assumed that two sets of discrete material handling devices are used fo...

  9. Cellular Dynamics of RNA Modification

    OpenAIRE

    Yi, Chengqi; Pan, Tao

    2011-01-01

    Decades of research have identified over 100 types of ribonucleosides that are post-transcriptionally modified. Many modified nucleosides are conserved in bacteria, archeae and eukaryotes, while some modified nucleosides are unique to each branch of life. However, the cellular and functional dynamics of RNA modifications remains largely unexplored, mostly due to the lack of functional hypotheses and experimental methods for quantification and large scale analysis. Just as many well characteri...

  10. Cellular Dynamics of RNA Modification

    Science.gov (United States)

    Yi, Chengqi; Pan, Tao

    2011-01-01

    Conspectus Decades of research have identified over 100 types of ribonucleosides that are post-transcriptionally modified. Many modified nucleosides are conserved in bacteria, archeae and eukaryotes, while some modified nucleosides are unique to each branch of life. However, the cellular and functional dynamics of RNA modifications remains largely unexplored, mostly due to the lack of functional hypotheses and experimental methods for quantification and large scale analysis. Just as many well characterized protein and DNA modifications, many RNA modifications are not essential for life. Instead, increasingly more evidence indicates that RNA modifications can play regulatory roles in cells, especially in response to stress conditions. In this Account, we review some known examples of RNA modifications that are dynamically controlled in cells and introduce some contemporary technologies and methods that enhance the studies of cellular dynamics of RNA modifications. Examples of RNA modifications discussed in this Account include (Figure 1): (1) 4-thio uridine (s4U) which can act as a cellular sensor of near UV-light; (2) queuosine (Q) which is a potential biomarker for malignancy; (3) N6-methyl adenine (m6A) which is the prevalent modification in eukaryotic mRNAs; and (4) pseudouridine (ψ) which are inducible by nutrient deprivation. Two recent technical advances that stimulated the studies of cellular dynamics of modified ribonucleosides are also described. First, a genome-wide method combines primer extension and microarray to study N1-methyl adenine (m1A) hypomodification in human tRNA. Second, a quantitative mass spectrometric method investigates dynamic changes of a wide range of tRNA modifications under stress conditions in yeast. In addition, we discuss potential mechanisms that control dynamic regulation of RNA modifications, and hypotheses for discovering potential RNA de-modification enzymes. We conclude the Account by highlighting the need to develop new

  11. CELLULAR FETAL MICROCHIMERISM IN PREECLAMPSIA

    OpenAIRE

    Gammill, Hilary S; Aydelotte, Tessa M.; Guthrie, Katherine A.; Nkwopara, Evangelyn C.; Nelson, J. Lee

    2013-01-01

    Previous studies have shown elevated concentrations of free fetal deoxyribonucleic acid and erythroblasts in maternal circulation in preeclampsia compared with normal pregnancy. Pluripotent and immunocompetent fetal cells also transfer to the maternal circulation during pregnancy, but whether concentrations of fetal mononuclear cells also differed in preeclampsia was unknown. We sought to quantify cellular fetal microchimerism in maternal circulation in women with preeclampsia and healthy con...

  12. The Origins of Cellular Life

    OpenAIRE

    Schrum, Jason P.; Zhu, Ting F.; SZOSTAK, JACK W.

    2010-01-01

    Understanding the origin of cellular life on Earth requires the discovery of plausible pathways for the transition from complex prebiotic chemistry to simple biology, defined as the emergence of chemical assemblies capable of Darwinian evolution. We have proposed that a simple primitive cell, or protocell, would consist of two key components: a protocell membrane that defines a spatially localized compartment, and an informational polymer that allows for the replication and inheritance of fun...

  13. Mutation breeding newsletter. No. 43

    International Nuclear Information System (INIS)

    This issue of the Newsletter includes articles dealing with radiation induced mutation based plant breeding research findings aimed at improving productivity, disease resistance and tolerance of stress conditions

  14. Progress of cellular dedifferentiation research

    Institute of Scientific and Technical Information of China (English)

    LIU Hu-xian; HU Da-hai; JIA Chi-yu; FU Xiao-bing

    2006-01-01

    Differentiation, the stepwise specialization of cells, and transdifferentiation, the apparent switching of one cell type into another, capture much of the stem cell spotlight. But dedifferentiation, the developmental reversal of a cell before it reinvents itself, is an important process too. In multicellular organisms, cellular dedifferentiation is the major process underlying totipotency, regeneration and formation of new stem cell lineages. In humans,dedifferentiation is often associated with carcinogenesis.The study of cellular dedifferentiation in animals,particularly early events related to cell fate-switch and determination, is limited by the lack of a suitable,convenient experimental system. The classic example of dedifferentiation is limb and tail regeneration in urodele amphibians, such as salamanders. Recently, several investigators have shown that certain mammalian cell types can be induced to dedifferentiate to progenitor cells when stimulated with the appropriate signals or materials. These discoveries open the possibility that researchers might enhance the endogenous regenerative capacity of mammals by inducing cellular dedifferentiation in vivo.

  15. CtIP Mutations Cause Seckel and Jawad Syndromes.

    Directory of Open Access Journals (Sweden)

    Per Qvist

    2011-10-01

    Full Text Available Seckel syndrome is a recessively inherited dwarfism disorder characterized by microcephaly and a unique head profile. Genetically, it constitutes a heterogeneous condition, with several loci mapped (SCKL1-5 but only three disease genes identified: the ATR, CENPJ, and CEP152 genes that control cellular responses to DNA damage. We previously mapped a Seckel syndrome locus to chromosome 18p11.31-q11.2 (SCKL2. Here, we report two mutations in the CtIP (RBBP8 gene within this locus that result in expression of C-terminally truncated forms of CtIP. We propose that these mutations are the molecular cause of the disease observed in the previously described SCKL2 family and in an additional unrelated family diagnosed with a similar form of congenital microcephaly termed Jawad syndrome. While an exonic frameshift mutation was found in the Jawad family, the SCKL2 family carries a splicing mutation that yields a dominant-negative form of CtIP. Further characterization of cell lines derived from the SCKL2 family revealed defective DNA damage induced formation of single-stranded DNA, a critical co-factor for ATR activation. Accordingly, SCKL2 cells present a lowered apoptopic threshold and hypersensitivity to DNA damage. Notably, over-expression of a comparable truncated CtIP variant in non-Seckel cells recapitulates SCKL2 cellular phenotypes in a dose-dependent manner. This work thus identifies CtIP as a disease gene for Seckel and Jawad syndromes and defines a new type of genetic disease mechanism in which a dominant negative mutation yields a recessively inherited disorder.

  16. Dynamic properties of cellular neural networks

    Directory of Open Access Journals (Sweden)

    Angela Slavova

    1993-01-01

    Full Text Available Dynamic behavior of a new class of information-processing systems called Cellular Neural Networks is investigated. In this paper we introduce a small parameter in the state equation of a cellular neural network and we seek for periodic phenomena. New approach is used for proving stability of a cellular neural network by constructing Lyapunov's majorizing equations. This algorithm is helpful for finding a map from initial continuous state space of a cellular neural network into discrete output. A comparison between cellular neural networks and cellular automata is made.

  17. Cellular communications a comprehensive and practical guide

    CERN Document Server

    Tripathi, Nishith

    2014-01-01

    Even as newer cellular technologies and standards emerge, many of the fundamental principles and the components of the cellular network remain the same. Presenting a simple yet comprehensive view of cellular communications technologies, Cellular Communications provides an end-to-end perspective of cellular operations, ranging from physical layer details to call set-up and from the radio network to the core network. This self-contained source forpractitioners and students represents a comprehensive survey of the fundamentals of cellular communications and the landscape of commercially deployed

  18. Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans.

    Science.gov (United States)

    Dudiuk, Catiana; Gamarra, Soledad; Jimenez-Ortigosa, Cristina; Leonardelli, Florencia; Macedo, Daiana; Perlin, David S; Garcia-Effron, Guillermo

    2015-07-01

    A rapid molecular-based assay for the detection of the Candida albicans FKS1 gene mutations responsible for resistance to echinocandin drugs was designed and evaluated. The assay consisted of a multiplexed PCR set of 5 tubes able to detect the most commonly described resistance mechanism, including FKS1 hot spot 1 and hot spot 2 mutations. The performance and specificity of the assay was evaluated using a double-blinded panel of 50 C. albicans strains. The assay showed a sensitivity of 96% and was able to detect all homozygous mutants included in the collection of strains, demonstrating that it is a robust, quick, and labor-saving method that is suitable for a routine clinical diagnostic laboratory. PMID:25878347

  19. HIGH-THROUGHPUT CELLULAR ASSAYS FOR MODELING TOXICITY IN THE FISH REPRODUCTIVE SYSTEM

    Science.gov (United States)

    The most important benefit of this project is the experimental evaluation of all essential steps in the development and testing of adverse outcome pathways (AOP) for a diverse set of reproductive and non-reproductive toxicants. In contrast to human testing and the toxicity pat...

  20. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-06-01

    The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.