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Sample records for lethality screen reveals

  1. Synthetic lethal screening reveals FGFR as one of the combinatorial targets to overcome resistance to Met-targeted therapy.

    PubMed

    Kim, B; Wang, S; Lee, J M; Jeong, Y; Ahn, T; Son, D-S; Park, H W; Yoo, H-s; Song, Y-J; Lee, E; Oh, Y M; Lee, S B; Choi, J; Murray, J C; Zhou, Y; Song, P H; Kim, K-A; Weiner, L M

    2015-02-26

    Met is a receptor tyrosine kinase that promotes cancer progression. In addition, Met has been implicated in resistance of tumors to various targeted therapies such as epidermal growth factor receptor inhibitors in lung cancers, and has been prioritized as a key molecular target for cancer therapy. However, the underlying mechanism of resistance to Met-targeting drugs is poorly understood. Here, we describe screening of 1310 genes to search for key regulators related to drug resistance to an anti-Met therapeutic antibody (SAIT301) by using a small interfering RNA-based synthetic lethal screening method. We found that knockdown of 69 genes in Met-amplified MKN45 cells sensitized the antitumor activity of SAIT301. Pathway analysis of these 69 genes implicated fibroblast growth factor receptor (FGFR) as a key regulator for antiproliferative effects of Met-targeting drugs. Inhibition of FGFR3 increased target cell apoptosis through the suppression of Bcl-xL expression, followed by reduced cancer cell growth in the presence of Met-targeting drugs. Treatment of cells with the FGFR inhibitors substantially restored the efficacy of SAIT301 in SAIT301-resistant cells and enhanced the efficacy in SAIT301-sensitive cells. In addition to FGFR3, integrin β3 is another potential target for combination treatment with SAIT301. Suppression of integrin β3 decreased AKT phosphorylation in SAIT301-resistant cells and restored SAIT301 responsiveness in HCC1954 cells, which are resistant to SAIT301. Gene expression analysis using CCLE database shows that cancer cells with high levels of FGFR and integrin β3 are resistant to crizotinib treatment, suggesting that FGFR and integrin β3 could be used as predictive markers for Met-targeted therapy and provide a potential therapeutic option to overcome acquired and innate resistance for the Met-targeting drugs. PMID:24662823

  2. Lifting Up the HAT: Synthetic Lethal Screening Reveals a Novel Vulnerability at the CBP-p300 Axis.

    PubMed

    Kadoch, Cigall

    2016-04-01

    Cancer genotype-specific synthetic lethal vulnerabilities represent promising therapeutic targets. In this issue of Cancer Discovery, Ogiwara and colleagues uncover a synthetic lethal relationship between two histone acetyl transferase paralogs, CBP and p300, highlighting that cancer cells deficient in CBP are uniquely sensitized to genetic and chemical inhibition of p300.

  3. A screen for F1 hybrid male rescue reveals no major-effect hybrid lethality loci in the Drosophila melanogaster autosomal genome.

    PubMed

    Cuykendall, Tawny N; Satyaki, P; Ji, Shuqing; Clay, Derek M; Edelman, Nathaniel B; Kimchy, Alexandra; Li, Ling-Hei; Nuzzo, Erin A; Parekh, Neil; Park, Suna; Barbash, Daniel A

    2014-12-01

    Hybrid sons between Drosophila melanogaster females and D. simulans males die as 3rd instar larvae. Two genes, D. melanogaster Hybrid male rescue (Hmr) on the X chromosome, and D. simulans Lethal hybrid rescue (Lhr) on chromosome II, interact to cause this lethality. Loss-of-function mutations in either gene suppress lethality, but several pieces of evidence suggest that additional factors are required for hybrid lethality. Here we screen the D. melanogaster autosomal genome by using the Bloomington Stock Center Deficiency kit to search for additional regions that can rescue hybrid male lethality. Our screen is designed to identify putative hybrid incompatibility (HI) genes similar to Hmr and Lhr which, when removed, are dominant suppressors of lethality. After screening 89% of the autosomal genome, we found no regions that rescue males to the adult stage. We did, however, identify several regions that rescue up to 13% of males to the pharate adult stage. This weak rescue suggests the presence of multiple minor-effect HI loci, but we were unable to map these loci to high resolution, presumably because weak rescue can be masked by genetic background effects. We attempted to test one candidate, the dosage compensation gene male specific lethal-3 (msl-3), by using RNA interference with short hairpin microRNA constructs targeted specifically against D. simulans msl-3 but failed to achieve knockdown, in part due to off-target effects. We conclude that the D. melanogaster autosomal genome likely does not contain additional major-effect HI loci. We also show that Hmr is insufficient to fully account for the lethality associated with the D. melanogaster X chromosome, suggesting that additional X-linked genes contribute to hybrid lethality.

  4. A Screen for F1 Hybrid Male Rescue Reveals No Major-Effect Hybrid Lethality Loci in the Drosophila melanogaster Autosomal Genome

    PubMed Central

    Cuykendall, Tawny N.; Satyaki, P.; Ji, Shuqing; Clay, Derek M.; Edelman, Nathaniel B.; Kimchy, Alexandra; Li, Ling-Hei; Nuzzo, Erin A.; Parekh, Neil; Park, Suna; Barbash, Daniel A.

    2014-01-01

    Hybrid sons between Drosophila melanogaster females and D. simulans males die as 3rd instar larvae. Two genes, D. melanogaster Hybrid male rescue (Hmr) on the X chromosome, and D. simulans Lethal hybrid rescue (Lhr) on chromosome II, interact to cause this lethality. Loss-of-function mutations in either gene suppress lethality, but several pieces of evidence suggest that additional factors are required for hybrid lethality. Here we screen the D. melanogaster autosomal genome by using the Bloomington Stock Center Deficiency kit to search for additional regions that can rescue hybrid male lethality. Our screen is designed to identify putative hybrid incompatibility (HI) genes similar to Hmr and Lhr which, when removed, are dominant suppressors of lethality. After screening 89% of the autosomal genome, we found no regions that rescue males to the adult stage. We did, however, identify several regions that rescue up to 13% of males to the pharate adult stage. This weak rescue suggests the presence of multiple minor-effect HI loci, but we were unable to map these loci to high resolution, presumably because weak rescue can be masked by genetic background effects. We attempted to test one candidate, the dosage compensation gene male specific lethal-3 (msl-3), by using RNA interference with short hairpin microRNA constructs targeted specifically against D. simulans msl-3 but failed to achieve knockdown, in part due to off-target effects. We conclude that the D. melanogaster autosomal genome likely does not contain additional major-effect HI loci. We also show that Hmr is insufficient to fully account for the lethality associated with the D. melanogaster X chromosome, suggesting that additional X-linked genes contribute to hybrid lethality. PMID:25352540

  5. Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

    PubMed Central

    Lun, Shichun; Miranda, David; Kubler, Andre; Guo, Haidan; Maiga, Mariama C.; Winglee, Kathryn; Pelly, Shaaretha

    2014-01-01

    ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. PMID:25227469

  6. RAS Synthetic Lethal Screens Revisited: Still Seeking the Elusive Prize?

    PubMed

    Downward, Julian

    2015-04-15

    The RAS genes are critical oncogenic drivers activated by point mutation in some 20% of human malignancies. However, no pharmacologic approaches to targeting RAS proteins directly have yet succeeded, leading to suggestions that these proteins may be "undruggable." This has led to two alternative indirect approaches to targeting RAS function in cancer. One has been to target RAS signaling pathways downstream at tractable enzymes such as kinases, particularly in combination. The other, which is the focus of this review, has been to seek targets that are essential in cells bearing an activated RAS oncogene, but not those without. This synthetic lethal approach, while rooted in ideas from invertebrate genetics, has been inspired most strongly by the successful use of PARP inhibitors, such as olaparib, in the clinic to treat BRCA defective cancers. Several large-scale screens have been carried out using RNA interference-mediated expression silencing to find genes that are uniquely essential to RAS-mutant but not wild-type cells. These screens have been notable for the low degree of overlap between their results, with the possible exception of proteasome components, and have yet to lead to successful new clinical approaches to the treatment of RAS-mutant cancers. Possible reasons for these disappointing results are discussed here, along with a reevaluation of the approaches taken. On the basis of experience to date, RAS synthetic lethality has so far fallen some way short of its original promise and remains unproven as an approach to finding effective new ways of tackling RAS-mutant cancers. Clin Cancer Res; 21(8); 1802-9. ©2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers." PMID:25878361

  7. RAS Synthetic Lethal Screens Revisited: Still Seeking the Elusive Prize?

    PubMed Central

    Downward, Julian

    2015-01-01

    The RAS genes are critical oncogenic drivers activated by point mutation in some 20% of human malignancies. However, no pharmacological approaches to targeting RAS proteins directly have yet succeeded, leading to suggestions that these proteins may be “undruggable.” This has led to two alternative indirect approaches to targeting RAS function in cancer. One has been to target RAS signaling pathways downstream at tractable enzymes such as kinases, particularly in combination. The other, which is the focus of this review, has been to seek targets that are essential in cells bearing an activated RAS oncogene, but not those without. This synthetic lethal approach, while rooted in ideas from invertebrate genetics, has been inspired most strongly by the successful use of PARP inhibitors, such as olaparib, in the clinic to treat BRCA defective cancers. Several large-scale screens have been carried out using RNA interference-mediated expression silencing to find genes that are uniquely essential to RAS mutant but not wild type cells. These screens have been notable for the low degree of overlap between their results, with the possible exception of proteasome components, and have yet to lead to successful new clinical approaches to the treatment of RAS mutant cancers. Possible reasons for these disappointing results are discussed here, along with a re-evaluation of the approaches taken. Based on experience to date, RAS synthetic lethality has so far fallen some way short of its original promise and remains unproven as an approach to finding effective new ways of tackling RAS mutant cancers. PMID:25878361

  8. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. We isolated 11 lethal lines that map...

  9. Evaluation of Caenorhabditis elegans as an acute lethality and a neurotoxicity screening model

    SciTech Connect

    Williams, P.L.

    1988-01-01

    This investigation evaluated C. elegans as a lethality and neurotoxicity screening model. The lethality experiments were performed in both agar and an aquatic medium. The salts of 8 metals (Hg, Be, Al, Cu, Zn, Pb, Cd, and Sr) were used in the agar studies and the salts of 14 metals (Ag, Hg, Cu, Be, Al, Pb, Cr, As, Tl, Zn, Cd, Ni, Sr, and Sb) were used in the aquatic tests. In each of these tests an LC50 value was determined. The data from the agar plates were compared to the published mammalian oral LD50 values for salts of the same metals. Within this set of chemicals C. elegans was found to be a predictor of mammalian acute lethality, generating LC50 values parallel to the rat and mouse LD50 values. The aquatic data were compared to data from EPA Ambient Water Quality Criteria documents. C. elegans was found to be less sensitive than Daphnia but generally more sensitive than the other invertebrate organisms that are presently used. The neurotoxicity testing also was performed in both agar and an aquatic media. The testing in agar was conducted with the salts of 4 metals (Cu, Be, Pb, and Hg) and 2 organophosphate pesticides (malathion and vapona). The studies in an aquatic medium tested the salts of 4 metals (Cu, Be, Pb, and Hg).

  10. A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis.

    PubMed Central

    Pultz, M A; Zimmerman, K K; Alto, N M; Kaeberlein, M; Lange, S K; Pitt, J N; Reeves, N L; Zehrung, D L

    2000-01-01

    We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila. PMID:10866651

  11. In Silico Screening Identifies a Novel Potential PARP1 Inhibitor Targeting Synthetic Lethality in Cancer Treatment

    PubMed Central

    Li, Jian; Zhou, Nan; Cai, Peiling; Bao, Jinku

    2016-01-01

    Synthetic lethality describes situations in which defects in two different genes or pathways together result in cell death. This concept has been applied to drug development for cancer treatment, as represented by Poly (ADP-ribose) polymerase (PARPs) inhibitors. In the current study, we performed a computational screening to discover new PARP inhibitors. Among the 11,247 compounds analyzed, one natural product, ZINC67913374, stood out by its superior performance in the simulation analyses. Compared with the FDA approved PARP1 inhibitor, olaparib, our results demonstrated that the ZINC67913374 compound achieved a better grid score (−86.8) and amber score (−51.42). Molecular dynamics simulations suggested that the PARP1-ZINC67913374 complex was more stable than olaparib. The binding free energy for ZINC67913374 was −177.28 kJ/mol while that of olaparib was −159.16 kJ/mol. These results indicated ZINC67913374 bound to PARP1 with a higher affinity, which suggest ZINC67913374 has promising potential for cancer drug development. PMID:26907257

  12. Lethality in PARP-1/Ku80 double mutant mice reveals physiologicalsynergy during early embryogenesis

    SciTech Connect

    Henrie, Melinda S.; Kurimasa, Akihiro; Burma, Sandeep; Menissier-de Murcia, Josiane; de Murcia, Gilbert; Li, Gloria C.; Chen,David J.

    2002-09-24

    Ku is an abundant heterodimeric nuclear protein, consisting of 70-kDa and 86-kDa tightly associated subunits that comprise the DNA binding component of DNA-dependent protein kinase. Poly(ADP)ribose polymerase-1 (PARP-1) is a 113-kDa protein that catalyzes the synthesis of poly(ADP-ribose) on target proteins. Both Ku and PARP-1 recognize and bind to DNA ends. Ku functions in the non-homologous end joining (NHEJ) repair pathway whereas PARP-1 functions in the single strand break repair and base excision repair (BER) pathways. Recent studies have revealed that PARP-1 and Ku80 interact in vitro. To determine whether the association of PARP-1 and Ku80 has any physiological significance or synergistic function in vivo, mice lacking both PARP-1 and Ku80 were generated. The resulting offspring died during embryonic development displaying abnormalities around the gastrulation stage. In addition, PARP-1-/-Ku80-/- cultured blastocysts had an increased level of apoptosis. These data suggest that the functions of both Ku80 and PARP-1 are essential for normal embryogenesis and that a loss of genomic integrity leading to cell death through apoptosis is likely the cause of the embryonic lethality observed in these mice.

  13. Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

    PubMed

    Cardona-Correa, Albin; Rios-Velazquez, Carlos

    2016-05-01

    The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents. PMID

  14. Unraveling the disease pathogenesis behind lethal hydrolethalus syndrome revealed multiple changes in molecular and cellular level

    PubMed Central

    Honkala, Heli; Lahtela, Jenni; Fox, Heli; Gentile, Massimiliano; Pakkasjärvi, Niklas; Salonen, Riitta; Wartiovaara, Kirmo; Jauhiainen, Matti; Kestilä, Marjo

    2009-01-01

    Background Hydrolethalus syndrome (HLS) is a severe fetal malformation syndrome characterized by multiple developmental anomalies, including central nervous system (CNS) malformation such as hydrocephaly and absent midline structures of the brain, micrognathia, defective lobation of the lungs and polydactyly. Microscopically, immature cerebral cortex, abnormalities in radial glial cells and hypothalamic hamartoma are among key findings in the CNS of HLS fetuses. HLS is caused by a substitution of aspartic acid by glycine in the HYLS1 protein, whose function was previously unknown. Results To provide insight into the disease mechanism(s) of this lethal disorder we have studied different aspects of HLS and HYLS1. A genome-wide gene expression analysis indicated several upregulated genes in cell cycle regulatory cascades and in specific signal transduction pathways while many downregulated genes were associated with lipid metabolism. These changes were supported by findings in functional cell biology studies, which revealed an increased cell cycle rate and a decreased amount of apoptosis in HLS neuronal progenitor cells. Also, changes in lipid metabolism gene expression were reflected by a significant increase in the cholesterol levels of HLS liver tissues. In addition, based on our functional studies of HYLS1, we propose that HYLS1 is a transcriptional regulator that shuffles between the cytoplasm and the nucleus, and that when HYLS1 is mutated its function is significantly altered. Conclusion In this study, we have shown that the HYLS1 mutation has significant consequences in the cellular and tissue levels in HLS fetuses. Based on these results, it can be suggested that HYLS1 is part of the cellular transcriptional regulatory machinery and that the genetic defect has a widespread effect during embryonic and fetal development. These findings add a significant amount of new information to the pathogenesis of HLS and strongly suggest an essential role for HYLS1 in

  15. An Arrayed Genome-Scale Lentiviral-Enabled Short Hairpin RNA Screen Identifies Lethal and Rescuer Gene Candidates

    PubMed Central

    Bhinder, Bhavneet; Antczak, Christophe; Ramirez, Christina N.; Shum, David; Liu-Sullivan, Nancy; Radu, Constantin; Frattini, Mark G.

    2013-01-01

    Abstract RNA interference technology is becoming an integral tool for target discovery and validation.; With perhaps the exception of only few studies published using arrayed short hairpin RNA (shRNA) libraries, most of the reports have been either against pooled siRNA or shRNA, or arrayed siRNA libraries. For this purpose, we have developed a workflow and performed an arrayed genome-scale shRNA lethality screen against the TRC1 library in HeLa cells. The resulting targets would be a valuable resource of candidates toward a better understanding of cellular homeostasis. Using a high-stringency hit nomination method encompassing criteria of at least three active hairpins per gene and filtered for potential off-target effects (OTEs), referred to as the Bhinder–Djaballah analysis method, we identified 1,252 lethal and 6 rescuer gene candidates, knockdown of which resulted in severe cell death or enhanced growth, respectively. Cross referencing individual hairpins with the TRC1 validated clone database, 239 of the 1,252 candidates were deemed independently validated with at least three validated clones. Through our systematic OTE analysis, we have identified 31 microRNAs (miRNAs) in lethal and 2 in rescuer genes; all having a seed heptamer mimic in the corresponding shRNA hairpins and likely cause of the OTE observed in our screen, perhaps unraveling a previously unknown plausible essentiality of these miRNAs in cellular viability. Taken together, we report on a methodology for performing large-scale arrayed shRNA screens, a comprehensive analysis method to nominate high-confidence hits, and a performance assessment of the TRC1 library highlighting the intracellular inefficiencies of shRNA processing in general. PMID:23198867

  16. Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening

    PubMed Central

    Adams, David; Baldock, Richard; Bhattacharya, Shoumo; Copp, Andrew J.; Dickinson, Mary; Greene, Nicholas D. E.; Henkelman, Mark; Justice, Monica; Mohun, Timothy; Murray, Stephen A.; Pauws, Erwin; Raess, Michael; Rossant, Janet; Weaver, Tom; West, David

    2013-01-01

    Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data. PMID:23519032

  17. A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.

    PubMed Central

    Costigan, C; Gehrung, S; Snyder, M

    1992-01-01

    The Saccharomyces cerevisiae SPA2 protein localizes at sites involved in polarized cell growth in budding cells and mating cells. spa2 mutants have defects in projection formation during mating but are healthy during vegetative growth. A synthetic lethal screen was devised to identify mutants that require the SPA2 gene for vegetative growth. One mutant, called slk-1 (for synthetic lethal kinase), has been characterized extensively. The SLK1 gene has been cloned, and sequence analysis predicts that the SLK1 protein is 1,478 amino acid residues in length. Approximately 300 amino acids at the carboxy terminus exhibit sequence similarity with the catalytic domains of protein kinases. Disruption mutations have been constructed in the SLK1 gene. slk1 null mutants cannot grow at 37 degrees C, but many cells can grow at 30, 24, and 17 degrees C. Dead slk1 mutant cells usually have aberrant cell morphologies, and many cells are very small, approximately one-half the diameter of wild-type cells. Surviving slk1 cells also exhibit morphogenic defects; these cells are impaired in their ability to form projections upon exposure to mating pheromones. During vegetative growth, a higher fraction of slk1 cells are unbudded compared with wild-type cells, and under nutrient limiting conditions, slk1 cells exhibit defects in cell cycle arrest. The different slk1 mutant defects are partially rescued by an extra copy of the SSD1/SRK1 gene. SSD1/SRK1 has been independently isolated as a suppressor of mutations in genes involved in growth control, sit4, pde2, bcy1, and ins1 (A. Sutton, D. Immanuel, and K.T. Arnat, Mol. Cell. Biol. 11:2133-2148, 1991; R.B. Wilson, A.A. Brenner, T.B. White, M.J. Engler, J.P. Gaughran, and K. Tatchell, Mol. Cell. Biol. 11:3369-3373, 1991). These data suggest that SLK1 plays a role in both cell morphogenesis and the control of cell growth. We speculate that SLK1 may be a regulatory link for these two cellular processes. Images PMID:1545797

  18. High-throughput simultaneous screen and counterscreen identifies homoharringtonine as synthetic lethal with von Hippel-Lindau loss in renal cell carcinoma

    PubMed Central

    Wolff, Nicholas C.; Pavía-Jiménez, Andrea; Tcheuyap, Vanina T.; Alexander, Shane; Vishwanath, Mridula; Christie, Alana; Xie, Xian-Jin; Williams, Noelle S.; Kapur, Payal; Posner, Bruce; McKay, Renée M.; Brugarolas, James

    2015-01-01

    Renal cell carcinoma (RCC) accounts for 85% of primary renal neoplasms, and is rarely curable when metastatic. Approximately 70% of RCCs are clear-cell type (ccRCC), and in >80% the von Hippel-Lindau (VHL) gene is mutated or silenced. We developed a novel, high-content, screening strategy for the identification of small molecules that are synthetic lethal with genes mutated in cancer. In this strategy, the screen and counterscreen are conducted simultaneously by differentially labeling mutant and reconstituted isogenic tumor cell line pairs with different fluorochromes and using a highly sensitive high-throughput imaging-based platform. This approach minimizes confounding factors from sequential screening, and more accurately replicates the in vivo cancer setting where cancer cells are adjacent to normal cells. A screen of ~12,800 small molecules identified homoharringtonine (HHT), an FDA-approved drug for treating chronic myeloid leukemia, as a VHL-synthetic lethal agent in ccRCC. HHT induced apoptosis in VHL-mutant, but not VHL-reconstituted, ccRCC cells, and inhibited tumor growth in 30% of VHL-mutant patient-derived ccRCC tumorgraft lines tested. Building on a novel screening strategy and utilizing a validated RCC tumorgraft model recapitulating the genetics and drug responsiveness of human RCC, these studies identify HHT as a potential therapeutic agent for a subset of VHL-deficient ccRCCs. PMID:26219258

  19. Lethal course of meconium ileus in preterm twins revealing a novel cystic fibrosis mutation (p.Cys524Tyr)

    PubMed Central

    2014-01-01

    Background In term newborns meconium ileus is frequently associated with cystic fibrosis. Reports on meconium ileus in preterm infants being diagnosed with cystic fibrosis early after birth are very scarce. Associations between genotype and phenotype in cystic fibrosis and its particular comorbidities have been reported. Case presentation Two extremely preterm twin infants (26 weeks of gestation) born from a Malaysian mother and a Caucasian father were presented with typical signs of meconium ileus. Despite immediate surgery both displayed a unique and finally lethal course. Mutation analysis revealed a novel, probably pathogenic cystic fibrosis mutation, p.Cys524Tyr. The novel mutation might explain the severity of disease next to typical sequelae of prematurity. Conclusion Preterm neonates with meconium ileus have to be evaluated for cystic fibrosis beyond ethnical boundaries, but may take devastating clinical courses despite early treatment. The novel, potentially pathogenic CF mutation p.Cys524Tyr might be associated with severe meconium ileus in neonates. Disease-modifying loci are important targets for intestinal comorbidity of cystic fibrosis. PMID:24433235

  20. The genesis of an exceptionally lethal venom in the timber rattlesnake (Crotalus horridus) revealed through comparative venom-gland transcriptomics

    PubMed Central

    2013-01-01

    Background Snake venoms generally show sequence and quantitative variation within and between species, but some rattlesnakes have undergone exceptionally rapid, dramatic shifts in the composition, lethality, and pharmacological effects of their venoms. Such shifts have occurred within species, most notably in Mojave (Crotalus scutulatus), South American (C. durissus), and timber (C. horridus) rattlesnakes, resulting in some populations with extremely potent, neurotoxic venoms without the hemorrhagic effects typical of rattlesnake bites. Results To better understand the evolutionary changes that resulted in the potent venom of a population of C. horridus from northern Florida, we sequenced the venom-gland transcriptome of an animal from this population for comparison with the previously described transcriptome of the eastern diamondback rattlesnake (C. adamanteus), a congener with a more typical rattlesnake venom. Relative to the toxin transcription of C. adamanteus, which consisted primarily of snake-venom metalloproteinases, C-type lectins, snake-venom serine proteinases, and myotoxin-A, the toxin transcription of C. horridus was far simpler in composition and consisted almost entirely of snake-venom serine proteinases, phospholipases A2, and bradykinin-potentiating and C-type natriuretic peptides. Crotalus horridus lacked significant expression of the hemorrhagic snake-venom metalloproteinases and C-type lectins. Evolution of shared toxin families involved differential expansion and loss of toxin clades within each species and pronounced differences in the highly expressed toxin paralogs. Toxin genes showed significantly higher rates of nonsynonymous substitution than nontoxin genes. The expression patterns of nontoxin genes were conserved between species, despite the vast differences in toxin expression. Conclusions Our results represent the first complete, sequence-based comparison between the venoms of closely related snake species and reveal in unprecedented

  1. A synthetic lethal screen identifies a role for the cortical actin patch/endocytosis complex in the response to nutrient deprivation in Saccharomyces cerevisiae.

    PubMed Central

    Care, Alison; Vousden, Katherine A; Binley, Katie M; Radcliffe, Pippa; Trevethick, Janet; Mannazzu, Ilaria; Sudbery, Peter E

    2004-01-01

    Saccharomyces cerevisiae whi2Delta cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2Delta. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2Delta, siw14Delta, fen2Delta, and thr4Delta mutants. Furthermore, whi2Delta and siw14Delta mutants produce large actin clumps in stationary phase similar to those seen in prk1Delta ark1Delta mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1Delta strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161Delta, sla1Delta, sla2Delta, vrp1Delta, ypt51Delta, ypt52Delta, and end3Delta mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2Delta mutants. PMID:15020461

  2. Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis

    PubMed Central

    2009-01-01

    Background Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug. Results Using the example of the tumor cell invasion inhibitor dihydromotuporamine C, we show that a more complete picture of drug action can be obtained by combining different chemical genomics approaches – analysis of the sensitivity of ρ0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast by this chemical requires a functional mitochondrial electron-transport chain and cytochrome c heme lyase function. However, we find that it does not require genes associated with programmed cell death in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking and interferes with vacuolar acidification in yeast and in human cancer cells. These effects can all be ascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C. Conclusion Despite their similar conceptual basis, namely altering drug sensitivity by modifying gene dosage, each of the screening approaches provided a distinct set of information that, when integrated, revealed a more complete picture of the mechanism of action of a drug on cells. PMID:19144191

  3. Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

    PubMed

    Toledo, Chad M; Ding, Yu; Hoellerbauer, Pia; Davis, Ryan J; Basom, Ryan; Girard, Emily J; Lee, Eunjee; Corrin, Philip; Hart, Traver; Bolouri, Hamid; Davison, Jerry; Zhang, Qing; Hardcastle, Justin; Aronow, Bruce J; Plaisier, Christopher L; Baliga, Nitin S; Moffat, Jason; Lin, Qi; Li, Xiao-Nan; Nam, Do-Hyun; Lee, Jeongwu; Pollard, Steven M; Zhu, Jun; Delrow, Jeffery J; Clurman, Bruce E; Olson, James M; Paddison, Patrick J

    2015-12-22

    To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality. PMID:26673326

  4. Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

    PubMed

    Toledo, Chad M; Ding, Yu; Hoellerbauer, Pia; Davis, Ryan J; Basom, Ryan; Girard, Emily J; Lee, Eunjee; Corrin, Philip; Hart, Traver; Bolouri, Hamid; Davison, Jerry; Zhang, Qing; Hardcastle, Justin; Aronow, Bruce J; Plaisier, Christopher L; Baliga, Nitin S; Moffat, Jason; Lin, Qi; Li, Xiao-Nan; Nam, Do-Hyun; Lee, Jeongwu; Pollard, Steven M; Zhu, Jun; Delrow, Jeffery J; Clurman, Bruce E; Olson, James M; Paddison, Patrick J

    2015-12-22

    To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

  5. Functional metagenomic screen reveals new and diverse microbial rhodopsins.

    PubMed

    Pushkarev, Alina; Béjà, Oded

    2016-09-01

    Ion-translocating retinylidene rhodopsins are widely distributed among marine and freshwater microbes. The translocation is light-driven, contributing to the production of biochemical energy in diverse microbes. Until today, most microbial rhodopsins had been detected using bioinformatics based on homology to other rhodopsins. In the past decade, there has been increased interest in microbial rhodopsins in the field of optogenetics since microbial rhodopsins were found to be most useful in vertebrate neuronal systems. Here we report on a functional metagenomic assay for detecting microbial rhodopsins. Using an array of narrow pH electrodes and light-emitting diode illumination, we were able to screen a metagenomic fosmid library to detect diverse marine proteorhodopsins and an actinorhodopsin based solely on proton-pumping activity. Our assay therefore provides a rather simple phenotypic means to enrich our understanding of microbial rhodopsins without any prior knowledge of the genomic content of the environmental entities screened. PMID:26894445

  6. 2D NMR-spectroscopic screening reveals polyketides in ladybugs

    PubMed Central

    Deyrup, Stephen T.; Eckman, Laura E.; McCarthy, Patrick H.; Smedley, Scott R.; Meinwald, Jerrold; Schroeder, Frank C.

    2011-01-01

    Small molecules of biological origin continue to yield the most promising leads for drug design, but systematic approaches for exploring nature’s cache of structural diversity are lacking. Here, we demonstrate the use of 2D NMR spectroscopy to screen a library of biorationally selected insect metabolite samples for partial structures indicating the presence of new chemical entities. This NMR-spectroscopic survey enabled detection of novel compounds in complex metabolite mixtures without prior fractionation or isolation. Our screen led to discovery and subsequent isolation of two families of tricyclic pyrones in Delphastus catalinae, a tiny ladybird beetle that is employed commercially as a biological pest control agent. The D. catalinae pyrones are based on 23-carbon polyketide chains forming 1,11-dioxo-2,6,10-trioxaanthracene and 4,8-dioxo-1,9,13-trioxaanthracene derivatives, representing ring systems not previously found in nature. This study highlights the utility of 2D NMR-spectroscopic screening for exploring nature’s structure space and suggests that insect metabolomes remain vastly underexplored. PMID:21646540

  7. 2D NMR-spectroscopic screening reveals polyketides in ladybugs.

    PubMed

    Deyrup, Stephen T; Eckman, Laura E; McCarthy, Patrick H; Smedley, Scott R; Meinwald, Jerrold; Schroeder, Frank C

    2011-06-14

    Small molecules of biological origin continue to yield the most promising leads for drug design, but systematic approaches for exploring nature's cache of structural diversity are lacking. Here, we demonstrate the use of 2D NMR spectroscopy to screen a library of biorationally selected insect metabolite samples for partial structures indicating the presence of new chemical entities. This NMR-spectroscopic survey enabled detection of novel compounds in complex metabolite mixtures without prior fractionation or isolation. Our screen led to discovery and subsequent isolation of two families of tricyclic pyrones in Delphastus catalinae, a tiny ladybird beetle that is employed commercially as a biological pest control agent. The D. catalinae pyrones are based on 23-carbon polyketide chains forming 1,11-dioxo-2,6,10-trioxaanthracene and 4,8-dioxo-1,9,13-trioxaanthracene derivatives, representing ring systems not previously found in nature. This study highlights the utility of 2D NMR-spectroscopic screening for exploring nature's structure space and suggests that insect metabolomes remain vastly underexplored. PMID:21646540

  8. Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors.

    PubMed

    Vafai, Scott B; Mevers, Emily; Higgins, Kathleen W; Fomina, Yevgenia; Zhang, Jianming; Mandinova, Anna; Newman, David; Shaw, Stanley Y; Clardy, Jon; Mootha, Vamsi K

    2016-01-01

    Deficiency of mitochondrial complex I is encountered in both rare and common diseases, but we have limited therapeutic options to treat this lesion to the oxidative phosphorylation system (OXPHOS). Idebenone and menadione are redox-active molecules capable of rescuing OXPHOS activity by engaging complex I-independent pathways of entry, often referred to as "complex I bypass." In the present study, we created a cellular model of complex I deficiency by using CRISPR genome editing to knock out Ndufa9 in mouse myoblasts, and utilized this cell line to develop a high-throughput screening platform for novel complex I bypass factors. We screened a library of ~40,000 natural product extracts and performed bioassay-guided fractionation on a subset of the top scoring hits. We isolated four plant-derived 1,4-naphthoquinone complex I bypass factors with structural similarity to menadione: chimaphilin and 3-chloro-chimaphilin from Chimaphila umbellata and dehydro-α-lapachone and dehydroiso-α-lapachone from Stereospermum euphoroides. We also tested a small number of structurally related naphthoquinones from commercial sources and identified two additional compounds with complex I bypass activity: 2-methoxy-1,4-naphthoquinone and 2-methoxy-3-methyl-1,4,-naphthoquinone. The six novel complex I bypass factors reported here expand this class of molecules and will be useful as tool compounds for investigating complex I disease biology. PMID:27622560

  9. Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors

    PubMed Central

    Mevers, Emily; Higgins, Kathleen W.; Fomina, Yevgenia; Zhang, Jianming; Mandinova, Anna; Newman, David; Shaw, Stanley Y.; Clardy, Jon; Mootha, Vamsi K.

    2016-01-01

    Deficiency of mitochondrial complex I is encountered in both rare and common diseases, but we have limited therapeutic options to treat this lesion to the oxidative phosphorylation system (OXPHOS). Idebenone and menadione are redox-active molecules capable of rescuing OXPHOS activity by engaging complex I-independent pathways of entry, often referred to as “complex I bypass.” In the present study, we created a cellular model of complex I deficiency by using CRISPR genome editing to knock out Ndufa9 in mouse myoblasts, and utilized this cell line to develop a high-throughput screening platform for novel complex I bypass factors. We screened a library of ~40,000 natural product extracts and performed bioassay-guided fractionation on a subset of the top scoring hits. We isolated four plant-derived 1,4-naphthoquinone complex I bypass factors with structural similarity to menadione: chimaphilin and 3-chloro-chimaphilin from Chimaphila umbellata and dehydro-α-lapachone and dehydroiso-α-lapachone from Stereospermum euphoroides. We also tested a small number of structurally related naphthoquinones from commercial sources and identified two additional compounds with complex I bypass activity: 2-methoxy-1,4-naphthoquinone and 2-methoxy-3-methyl-1,4,-naphthoquinone. The six novel complex I bypass factors reported here expand this class of molecules and will be useful as tool compounds for investigating complex I disease biology. PMID:27622560

  10. Identification of unique sensitizing targets for anti-inflammatory CDDO-Me in metastatic melanoma by a large-scale synthetic lethal RNAi screening

    PubMed Central

    Ekmekcioglu, Suhendan; Grimm, Elizabeth A.

    2013-01-01

    Summary CDDO-Me has been shown to exert potent anti-inflammatory activity for chronic kidney disease and antitumor activity for several tumors, including melanoma, in early clinical trials. To improve CDDO-Me response in melanoma, we utilized a large-scale synthetic lethal RNAi screen targeting 6,000 human druggable genes to identify targets that would sensitize melanoma cells to CDDO-Me. Based on screening results, five unique genes (GNPAT, SUMO1, SPINT2, FLI1, and SSX1) significantly potentiated the growth-inhibitory effects of CDDO-Me and induced apoptosis in A375, a BRAF mutated melanoma line (P<0.001). These five genes were then individually validated as targets to potentiate CDDO-Me activity, and related downstream signaling pathways of these genes were analyzed. In addition, the levels of phosphorylated Erk1/2, Akt, GSK-2, and PRAS40 were dramatically decreased by downregulating each of these five genes separately, suggesting a set of common mediators. Our findings indicate that GNPAT, SUMO1, SPINT2, FLI1, and SSX1 play critical roles in synergy with inflammation pathways in modulating melanoma cell survival, and could serve as sensitizing targets to enhance CDDO-Me efficacy in melanoma growth control. PMID:23020131

  11. Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer.

    PubMed

    Ahmad, Imran; Mui, Ernest; Galbraith, Laura; Patel, Rachana; Tan, Ee Hong; Salji, Mark; Rust, Alistair G; Repiscak, Peter; Hedley, Ann; Markert, Elke; Loveridge, Carolyn; van der Weyden, Louise; Edwards, Joanne; Sansom, Owen J; Adams, David J; Leung, Hing Y

    2016-07-19

    Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition. PMID:27357679

  12. Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer

    PubMed Central

    Ahmad, Imran; Mui, Ernest; Galbraith, Laura; Patel, Rachana; Tan, Ee Hong; Salji, Mark; Rust, Alistair G.; Repiscak, Peter; Hedley, Ann; Markert, Elke; Loveridge, Carolyn; van der Weyden, Louise; Edwards, Joanne; Sansom, Owen J.; Adams, David J.; Leung, Hing Y.

    2016-01-01

    Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition. PMID:27357679

  13. Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer.

    PubMed

    Ahmad, Imran; Mui, Ernest; Galbraith, Laura; Patel, Rachana; Tan, Ee Hong; Salji, Mark; Rust, Alistair G; Repiscak, Peter; Hedley, Ann; Markert, Elke; Loveridge, Carolyn; van der Weyden, Louise; Edwards, Joanne; Sansom, Owen J; Adams, David J; Leung, Hing Y

    2016-07-19

    Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition.

  14. Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

    PubMed Central

    Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

  15. A Translational Murine Model of Sub-Lethal Intoxication with Shiga Toxin 2 Reveals Novel Ultrastructural Findings in the Brain Striatum

    PubMed Central

    Tironi-Farinati, Carla; Geoghegan, Patricia A.; Cangelosi, Adriana; Pinto, Alipio; Loidl, C. Fabian; Goldstein, Jorge

    2013-01-01

    Infection by Shiga toxin-producing Escherichia coli causes hemorrhagic colitis, hemolytic uremic syndrome (HUS), acute renal failure, and also central nervous system complications in around 30% of the children affected. Besides, neurological deficits are one of the most unrepairable and untreatable outcomes of HUS. Study of the striatum is relevant because basal ganglia are one of the brain areas most commonly affected in patients that have suffered from HUS and since the deleterious effects of a sub-lethal dose of Shiga toxin have never been studied in the striatum, the purpose of this study was to attempt to simulate an infection by Shiga toxin-producing E. coli in a murine model. To this end, intravenous administration of a sub-lethal dose of Shiga toxin 2 (0.5 ηg per mouse) was used and the correlation between neurological manifestations and ultrastructural changes in striatal brain cells was studied in detail. Neurological manifestations included significant motor behavior abnormalities in spontaneous motor activity, gait, pelvic elevation and hind limb activity eight days after administration of the toxin. Transmission electron microscopy revealed that the toxin caused early perivascular edema two days after administration, as well as significant damage in astrocytes four days after administration and significant damage in neurons and oligodendrocytes eight days after administration. Interrupted synapses and mast cell extravasation were also found eight days after administration of the toxin. We thus conclude that the chronological order of events observed in the striatum could explain the neurological disorders found eight days after administration of the toxin. PMID:23383285

  16. Kinome RNAi Screens Reveal Synergistic Targeting of MTOR and FGFR1 Pathways for Treatment of Lung Cancer and HNSCC.

    PubMed

    Singleton, Katherine R; Hinz, Trista K; Kleczko, Emily K; Marek, Lindsay A; Kwak, Jeff; Harp, Taylor; Kim, Jihye; Tan, Aik Choon; Heasley, Lynn E

    2015-10-15

    The FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with a single TKI represents a logical step toward personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multikinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between MTOR and ponatinib in non-small cell lung carcinoma cells. In addition, treatment with MTOR-targeting shRNAs and pharmacologic inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Furthermore, they suggest that clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs. PMID:26359452

  17. A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

    PubMed

    Jen, Freda E-C; Djoko, Karrera Y; Bent, Stephen J; Day, Christopher J; McEwan, Alastair G; Jennings, Michael P

    2015-09-01

    Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species.

  18. Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen.

    PubMed

    Adissu, Hibret A; Estabel, Jeanne; Sunter, David; Tuck, Elizabeth; Hooks, Yvette; Carragher, Damian M; Clarke, Kay; Karp, Natasha A; Newbigging, Susan; Jones, Nora; Morikawa, Lily; White, Jacqueline K; McKerlie, Colin

    2014-05-01

    The Mouse Genetics Project (MGP) at the Wellcome Trust Sanger Institute aims to generate and phenotype over 800 genetically modified mouse lines over the next 5 years to gain a better understanding of mammalian gene function and provide an invaluable resource to the scientific community for follow-up studies. Phenotyping includes the generation of a standardized biobank of paraffin-embedded tissues for each mouse line, but histopathology is not routinely performed. In collaboration with the Pathology Core of the Centre for Modeling Human Disease (CMHD) we report the utility of histopathology in a high-throughput primary phenotyping screen. Histopathology was assessed in an unbiased selection of 50 mouse lines with (n=30) or without (n=20) clinical phenotypes detected by the standard MGP primary phenotyping screen. Our findings revealed that histopathology added correlating morphological data in 19 of 30 lines (63.3%) in which the primary screen detected a phenotype. In addition, seven of the 50 lines (14%) presented significant histopathology findings that were not associated with or predicted by the standard primary screen. Three of these seven lines had no clinical phenotype detected by the standard primary screen. Incidental and strain-associated background lesions were present in all mutant lines with good concordance to wild-type controls. These findings demonstrate the complementary and unique contribution of histopathology to high-throughput primary phenotyping of mutant mice.

  19. A functional genomics screen in planarians reveals regulators of whole-brain regeneration

    PubMed Central

    Roberts-Galbraith, Rachel H; Brubacher, John L; Newmark, Phillip A

    2016-01-01

    Planarians regenerate all body parts after injury, including the central nervous system (CNS). We capitalized on this distinctive trait and completed a gene expression-guided functional screen to identify factors that regulate diverse aspects of neural regeneration in Schmidtea mediterranea. Our screen revealed molecules that influence neural cell fates, support the formation of a major connective hub, and promote reestablishment of chemosensory behavior. We also identified genes that encode signaling molecules with roles in head regeneration, including some that are produced in a previously uncharacterized parenchymal population of cells. Finally, we explored genes downregulated during planarian regeneration and characterized, for the first time, glial cells in the planarian CNS that respond to injury by repressing several transcripts. Collectively, our studies revealed diverse molecules and cell types that underlie an animal’s ability to regenerate its brain. DOI: http://dx.doi.org/10.7554/eLife.17002.001 PMID:27612384

  20. Kinome RNAi Screens Reveal Synergistic Targeting of MTOR and FGFR1 Pathways for Treatment of Lung Cancer and HNSCC

    PubMed Central

    Singleton, Katherine R.; Hinz, Trista K.; Kleczko, Emily K.; Marek, Lindsay A.; Kwak, Jeff; Harp, Taylor; Kim, Jihye; Tan, Aik Choon; Heasley, Lynn E.

    2015-01-01

    The fibroblast growth factor receptor FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with single TKI represents a logical step towards personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multi-kinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between Mammalian Target of Rapamycin (MTOR) and ponatinib in non-small cell lung carcinoma cells. Additionally, treatment with MTOR-targeting shRNAs and pharmacological inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Further, they suggest clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs. PMID:26359452

  1. RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection.

    PubMed

    Mercer, Jason; Snijder, Berend; Sacher, Raphael; Burkard, Christine; Bleck, Christopher Karl Ernst; Stahlberg, Henning; Pelkmans, Lucas; Helenius, Ari

    2012-10-25

    A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry, including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinated during virus assembly. After entering the cytosol of an uninfected cell, the viral DNA was released from the core through the activity of the cell's proteasomes. Next, a Cullin3-based ubiquitin ligase mediated a further round of ubiquitination and proteasome action. This was needed in order to initiate viral DNA replication. The results accentuate the value of large-scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways. The list of cell functions required during poxvirus infection will, moreover, provide a resource for future virus-host cell interaction studies and for the discovery of antivirals. PMID:23084750

  2. Development of synthetic lethality anticancer therapeutics.

    PubMed

    Fang, Bingliang

    2014-10-01

    The concept of synthetic lethality (the creation of a lethal phenotype from the combined effects of mutations in two or more genes) has recently been exploited in various efforts to develop new genotype-selective anticancer therapeutics. These efforts include screening for novel anticancer agents, identifying novel therapeutic targets, characterizing mechanisms of resistance to targeted therapy, and improving efficacies through the rational design of combination therapy. This review discusses recent developments in synthetic lethality anticancer therapeutics, including poly ADP-ribose polymerase inhibitors for BRCA1- and BRCA2-mutant cancers, checkpoint inhibitors for p53 mutant cancers, and small molecule agents targeting RAS gene mutant cancers. Because cancers are caused by mutations in multiple genes and abnormalities in multiple signaling pathways, synthetic lethality for a specific tumor suppressor gene or oncogene is likely cell context-dependent. Delineation of the mechanisms underlying synthetic lethality and identification of treatment response biomarkers will be critical for the success of synthetic lethality anticancer therapy.

  3. Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces Cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na(+) Tolerance

    PubMed Central

    White, W. H.; Johnson, D. I.

    1997-01-01

    Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4(ts) at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet(-) vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4(ts) cells were elongated or had misshapen buds. A cdc24-4(ts) Δvma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4(ts) synthetic-lethality was not simply due to altered vacuole function. The cdc24-4(ts) mutant, like Δvma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca(2+) as well as Na(+) in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na(+) tolerance. PMID:9286667

  4. Rapid ester biosynthesis screening reveals a high activity alcohol-O-acyltransferase (AATase) from tomato fruit.

    PubMed

    Lin, Jyun-Liang; Zhu, Jie; Wheeldon, Ian

    2016-05-01

    Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl-CoA with an alcohol by alcohol-O-acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short- and medium-chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf-S.l). Atf1-S.l exhibited broad specificity towards acyl-CoAs with chain length from C4 to C10 and was specific towards 1-pentanol. The AATase screen also revealed new acyl-CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf-C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester-based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels. PMID:26814045

  5. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

    PubMed

    Schmitt-Engel, Christian; Schultheis, Dorothea; Schwirz, Jonas; Ströhlein, Nadi; Troelenberg, Nicole; Majumdar, Upalparna; Dao, Van Anh; Grossmann, Daniela; Richter, Tobias; Tech, Maike; Dönitz, Jürgen; Gerischer, Lizzy; Theis, Mirko; Schild, Inga; Trauner, Jochen; Koniszewski, Nikolaus D B; Küster, Elke; Kittelmann, Sebastian; Hu, Yonggang; Lehmann, Sabrina; Siemanowski, Janna; Ulrich, Julia; Panfilio, Kristen A; Schröder, Reinhard; Morgenstern, Burkhard; Stanke, Mario; Buchhholz, Frank; Frasch, Manfred; Roth, Siegfried; Wimmer, Ernst A; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-01-01

    Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila. PMID:26215380

  6. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

    PubMed

    Schmitt-Engel, Christian; Schultheis, Dorothea; Schwirz, Jonas; Ströhlein, Nadi; Troelenberg, Nicole; Majumdar, Upalparna; Dao, Van Anh; Grossmann, Daniela; Richter, Tobias; Tech, Maike; Dönitz, Jürgen; Gerischer, Lizzy; Theis, Mirko; Schild, Inga; Trauner, Jochen; Koniszewski, Nikolaus D B; Küster, Elke; Kittelmann, Sebastian; Hu, Yonggang; Lehmann, Sabrina; Siemanowski, Janna; Ulrich, Julia; Panfilio, Kristen A; Schröder, Reinhard; Morgenstern, Burkhard; Stanke, Mario; Buchhholz, Frank; Frasch, Manfred; Roth, Siegfried; Wimmer, Ernst A; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-07-28

    Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

  7. Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide.

    PubMed

    Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; Dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

    2014-03-01

    β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63-116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20-52.54% and -0.95-62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide.

  8. Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide

    PubMed Central

    Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

    2014-01-01

    β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63–116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20–52.54% and −0.95–62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide. PMID:24688298

  9. Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide.

    PubMed

    Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; Dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

    2014-03-01

    β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63-116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20-52.54% and -0.95-62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide. PMID:24688298

  10. RNAi Screen Reveals an Abl Kinase-Dependent Host Cell Pathway Involved in Pseudomonas aeruginosa Internalization

    PubMed Central

    Pielage, Julia F.; Powell, Kimberly R.; Kalman, Daniel; Engel, Joanne N.

    2008-01-01

    Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of ∼80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa–induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections. PMID:18369477

  11. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators

    PubMed Central

    Dopie, Joseph; Rajakylä, Eeva K.; Joensuu, Merja S.; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K.

    2015-01-01

    ABSTRACT Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes. PMID:26021350

  12. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

    PubMed Central

    Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  13. A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia

    PubMed Central

    Marker, Simone; Carradec, Quentin; Tanty, Véronique; Arnaiz, Olivier; Meyer, Eric

    2014-01-01

    In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia. PMID:24860163

  14. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators.

    PubMed

    Dopie, Joseph; Rajakylä, Eeva K; Joensuu, Merja S; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K

    2015-07-01

    Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes.

  15. A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia.

    PubMed

    Marker, Simone; Carradec, Quentin; Tanty, Véronique; Arnaiz, Olivier; Meyer, Eric

    2014-06-01

    In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia. PMID:24860163

  16. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

    PubMed

    Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

    2016-08-18

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

  17. Genetic Screen Reveals the Role of Purine Metabolism in Staphylococcus aureus Persistence to Rifampicin

    PubMed Central

    Yee, Rebecca; Cui, Peng; Shi, Wanliang; Feng, Jie; Zhang, Ying

    2015-01-01

    Chronic infections with Staphylococcus aureus such as septicemia, osteomyelitis, endocarditis, and biofilm infections are difficult to treat because of persisters. Despite many efforts in understanding bacterial persistence, the mechanisms of persister formation in S. aureus remain elusive. Here, we performed a genome-wide screen of a transposon mutant library to study the molecular mechanisms involved in persistence of community-acquired S. aureus. Screening of the library for mutants defective in persistence or tolerance to rifampicin revealed many genes involved in metabolic pathways that are important for antibiotic persistence. In particular, the identified mutants belonged to metabolic pathways involved in carbohydrate, amino acid, lipid, vitamin and purine biosynthesis. Five mutants played a role in purine biosynthesis and two mutants, purB, an adenylosuccinate lyase, and purM, a phosphoribosylaminoimidazole synthetase, were selected for further confirmation. Mutants purB and purM showed defective persistence compared to the parental strain USA300 in multiple stress conditions including various antibiotics, low pH, and heat stress. The defect in persistence was restored by complementation with the wildtype purB and purM gene in the respective mutants. These findings provide new insights into the mechanisms of persistence in S. aureus and provide novel therapeutic targets for developing more effective treatment for persistent infections due to S. aureus. PMID:27025643

  18. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

    PubMed

    Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  19. Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication

    PubMed Central

    Wu, Kan Xing; Phuektes, Patchara; Kumar, Pankaj; Goh, Germaine Yen Lin; Moreau, Dimitri; Chow, Vincent Tak Kwong; Bard, Frederic; Chu, Justin Jang Hann

    2016-01-01

    Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets. PMID:27748395

  20. Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon

    PubMed Central

    Leis, Benedikt; Heinze, Simon; Angelov, Angel; Pham, Vu Thuy Trang; Thürmer, Andrea; Jebbar, Mohamed; Golyshin, Peter N.; Streit, Wolfgang R.; Daniel, Rolf; Liebl, Wolfgang

    2015-01-01

    Extreme habitats serve as a source of enzymes that are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8–70°C). Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70°C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12) endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45% sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92°C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential. PMID:26191525

  1. Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila.

    PubMed

    Harrison, Christopher F; Chiriano, Gianpaolo; Finsel, Ivo; Manske, Christian; Hoffmann, Christine; Steiner, Bernhard; Kranjc, Agata; Patthey-Vuadens, Ophelie; Kicka, Sébastien; Trofimov, Valentin; Ouertatani-Sakouhi, Hajer; Soldati, Thierry; Scapozza, Leonardo; Hilbi, Hubert

    2015-07-10

    The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s). PMID:27622823

  2. Multiparameter screening reveals a role for Na+ channels in cytokine-induced β-cell death.

    PubMed

    Yang, Yu Hsuan Carol; Vilin, Yury Y; Roberge, Michel; Kurata, Harley T; Johnson, James D

    2014-03-01

    Pancreatic β-cell death plays a role in both type 1 and type 2 diabetes, but clinical treatments that specifically target β-cell survival have not yet been developed. We have recently developed live-cell imaging-based, high-throughput screening methods capable of identifying factors that modulate pancreatic β-cell death, with the hope of finding drugs that can intervene in this process. In the present study, we used a high-content screen and the Prestwick Chemical Library of small molecules to identify drugs that block cell death resulting from exposure to a cocktail of cytotoxic cytokines (25 ng/mL TNF-α, 10 ng/mL IL-1β, and 10 ng/mL IFN-γ). Data analysis with self-organizing maps revealed that 19 drugs had profiles similar to that of the no cytokine condition, indicating protection. Carbamazepine, an antiepileptic Na(+) channel inhibitor, was particularly interesting because Na(+) channels are not generally considered targets for antiapoptotic therapy in diabetes and because the function of these channels in β-cells has not been well studied. We analyzed the expression and characteristics of Na(+) currents in mature β-cells from MIP-GFP mice. We confirmed the dose-dependent protective effects of carbamazepine and another use-dependent Na(+) channel blocker in cytokine-treated mouse islet cells. Carbamazepine down-regulated the proapoptotic and endoplasmic reticulum stress signaling induced by cytokines. Together, these studies point to Na(+) channels as a novel therapeutic target in diabetes. PMID:24438339

  3. Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin–dockerin interactions

    PubMed Central

    Hamberg, Yuval; Ruimy-Israeli, Vered; Dassa, Bareket; Barak, Yoav; Lamed, Raphael; Cameron, Kate; Fontes, Carlos M.G.A.

    2014-01-01

    s total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs. PMID:25374780

  4. Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin-dockerin interactions.

    PubMed

    Hamberg, Yuval; Ruimy-Israeli, Vered; Dassa, Bareket; Barak, Yoav; Lamed, Raphael; Cameron, Kate; Fontes, Carlos M G A; Bayer, Edward A; Fried, Daniel B

    2014-01-01

    and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

  5. Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex

    PubMed Central

    Peters, Bettina; Kirfel, Jutta; Büssow, Heinrich; Vidal, Miguel; Magin, Thomas M.

    2001-01-01

    In human patients, a wide range of mutations in keratin (K) 5 or K14 lead to the blistering skin disorder epidermolysis bullosa simplex. Given that K14 deficiency does not lead to the ablation of a basal cell cytoskeleton because of a compensatory role of K15, we have investigated the requirement for the keratin cytoskeleton in basal cells by inactivating the K5 gene in mice. We report that the K5−/− mice die shortly after birth, lack keratin filaments in the basal epidermis, and are more severely affected than K14−/− mice. In contrast to the K14−/− mice, we detected a strong induction of the wound-healing keratin K6 in the suprabasal epidermis of cytolyzed areas of postnatal K5−/− mice. In addition, K5 and K14 mice differed with respect to tongue lesions. Moreover, we show that in the absence of K5 and other type II keratins, residual K14 and K15 aggregated along hemidesmosomes, demonstrating that individual keratins without a partner are stable in vivo. Our data indicate that K5 may be the natural partner of K15 and K17. We suggest that K5 null mutations may be lethal in human epidermolysis bullosa simplex patients. PMID:11408584

  6. A genetic screen and transcript profiling reveal a shared regulatory program for Drosophila linker histone H1 and chromatin remodeler CHD1.

    PubMed

    Kavi, Harsh; Lu, Xingwu; Xu, Na; Bartholdy, Boris A; Vershilova, Elena; Skoultchi, Arthur I; Fyodorov, Dmitry V

    2015-01-27

    Chromatin structure and activity can be modified through ATP-dependent repositioning of nucleosomes and posttranslational modifications of core histone tails within nucleosome core particles and by deposition of linker histones into the oligonucleosome fiber. The linker histone H1 is essential in metazoans. It has a profound effect on organization of chromatin into higher-order structures and on recruitment of histone-modifying enzymes to chromatin. Here, we describe a genetic screen for modifiers of the lethal phenotype caused by depletion of H1 in Drosophila melanogaster. We identify 41 mis-expression alleles that enhance and 20 that suppress the effect of His1 depletion in vivo. Most of them are important for chromosome organization, transcriptional regulation, and cell signaling. Specifically, the reduced viability of H1-depleted animals is strongly suppressed by ubiquitous mis-expression of the ATP-dependent chromatin remodeling enzyme CHD1. Comparison of transcript profiles in H1-depleted and Chd1 null mutant larvae revealed that H1 and CHD1 have common transcriptional regulatory programs in vivo. H1 and CHD1 share roles in repression of numerous developmentally regulated and extracellular stimulus-responsive transcripts, including immunity-related and stress response-related genes. Thus, linker histone H1 participates in various regulatory programs in chromatin to alter gene expression.

  7. A potentially exhaustive screening strategy reveals two novel divergent myosins in Dictyostelium.

    PubMed

    Schwarz, E C; Geissler, H; Soldati, T

    1999-01-01

    In recent years, the myosin superfamily has kept expanding at an explosive rate, but the understanding of their complex functions has been lagging. Therefore, Dictyostelium discoideum, a genetically and biochemically tractable eukaryotic amoeba, appears as a powerful model organism to investigate the involvement of the actomyosin cytoskeleton in a variety of cellular tasks. Because of the relatively high degree of functional redundancy, such studies would be greatly facilitated by the prior knowledge of the whole myosin repertoire in this organism. Here, we present a strategy based on PCR amplification using degenerate primers and followed by negative hybridization screening which led to the potentially exhaustive identification of members of the myosin family in D. discoideum. Two novel myosins were identified and their genetic loci mapped by hybridization to an ordered YAC library. Preliminary inspection of myoK and myoM sequences revealed that, despite carrying most of the hallmarks of myosin motors, both molecules harbor features surprisingly divergent from most known myosins. PMID:10403059

  8. Mutation screen reveals novel variants and expands the phenotypes associated with DYNC1H1

    PubMed Central

    Strickland, Alleene V.; Schabhüttl, Maria; Offenbacher, Hans; Synofzik, Matthis; Hauser, Natalie S.; Brunner-Krainz, Michaela; Gruber-Sedlmayr, Ursula; Moore, Steven A.; Windhager, Reinhard; Bender, Benjamin; Harms, Matthew; Klebe, Stephan; Young, Peter; Kennerson, Marina; Garcia, Avencia Sanchez Mejias; Gonzalez, Michael A.; Züchner, Stephan; Schule, Rebecca; Shy, Michael E.; Auer-Grumbach, Michaela

    2015-01-01

    Dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) encodes a necessary subunit of the cytoplasmic dynein complex, which traffics cargo along microtubules. Dominant DYNC1H1 mutations are implicated in neural diseases, including spinal muscular atrophy with lower extremity dominance (SMA-LED), intellectual disability with neuronal migration defects, malformations of cortical development (MCD), and Charcot-Marie-Tooth disease, type 2O (CMT2O). We hypothesized that additional variants could be found in these and novel motoneuron and related diseases. Therefore we analysed our database of 1,024 whole exome sequencing samples of motoneuron and related diseases for novel single nucleotide variations. We filtered these results for significant variants, which were further screened using segregation analysis in available family members. Analysis revealed six novel, rare, and highly conserved variants. Three of these are likely pathogenic and encompass a broad phenotypic spectrum with distinct disease clusters. Our findings suggest that DYNC1H1 variants can cause not only lower, but also upper motor neuron disease. It thus adds DYNC1H1 to the growing list of spastic paraplegia related genes in microtubule-dependent motor protein pathways. PMID:26100331

  9. Early Embryonic Lethality of Mice Lacking ZO-2, but Not ZO-3, Reveals Critical and Nonredundant Roles for Individual Zonula Occludens Proteins in Mammalian Development▿ †

    PubMed Central

    Xu, Jianliang; Kausalya, P. Jaya; Phua, Dominic C. Y.; Ali, Safiah Mohamed; Hossain, Zakir; Hunziker, Walter

    2008-01-01

    ZO-1, ZO-2, and ZO-3 are closely related scaffolding proteins that link tight junction (TJ) transmembrane proteins such as claudins, junctional adhesion molecules, and occludin to the actin cytoskeleton. Even though the zonula occludens (ZO) proteins are among the first TJ proteins to have been identified and have undergone extensive biochemical analysis, little is known about the physiological roles of individual ZO proteins in different tissues or during vertebrate development. Here, we show that ZO-3 knockout mice lack an obvious phenotype. In contrast, embryos deficient for ZO-2 die shortly after implantation due to an arrest in early gastrulation. ZO-2−/− embryos show decreased proliferation at embryonic day 6.5 (E6.5) and increased apoptosis at E7.5 compared to wild-type embryos. The asymmetric distribution of prominin and E-cadherin to the apical and lateral plasma membrane domains, respectively, is maintained in cells of ZO-2−/− embryos. However, the architecture of the apical junctional complex is altered, and paracellular permeability of a low-molecular-weight tracer is increased in ZO-2−/− embryos. Leaky TJs and, given the association of ZO-2 with connexins and several transcription factors, effects on gap junctions and gene expression, respectively, are likely causes for embryonic lethality. Thus, ZO-2 is required for mouse embryonic development, but ZO-3 is dispensable. This is to our knowledge the first report showing that an individual ZO protein plays a nonredundant and critical role in mammalian development. PMID:18172007

  10. Comprehensive Bee Pathogen Screening in Belgium Reveals Crithidia mellificae as a New Contributory Factor to Winter Mortality

    PubMed Central

    Ravoet, Jorgen; Maharramov, Jafar; Meeus, Ivan; De Smet, Lina; Wenseleers, Tom; Smagghe, Guy; de Graaf, Dirk C.

    2013-01-01

    Since the last decade, unusually high honey bee colony losses have been reported mainly in North-America and Europe. Here, we report on a comprehensive bee pathogen screening in Belgium covering 363 bee colonies that were screened for 18 known disease-causing pathogens and correlate their incidence in summer with subsequent winter mortality. Our analyses demonstrate that, in addition to Varroa destructor, the presence of the trypanosomatid parasite Crithidia mellificae and the microsporidian parasite Nosema ceranae in summer are also predictive markers of winter mortality, with a negative synergy being observed between the two in terms of their effects on colony mortality. Furthermore, we document the first occurrence of a parasitizing phorid fly in Europe, identify a new fourth strain of Lake Sinai Virus (LSV), and confirm the presence of other little reported pathogens such as Apicystis bombi, Aphid Lethal Paralysis Virus (ALPV), Spiroplasma apis, Spiroplasma melliferum and Varroa destructor Macula-like Virus (VdMLV). Finally, we provide evidence that ALPV and VdMLV replicate in honey bees and show that viruses of the LSV complex and Black Queen Cell Virus tend to non-randomly co-occur together. We also noticed a significant correlation between the number of pathogen species and colony losses. Overall, our results contribute significantly to our understanding of honey bee diseases and the likely causes of their current decline in Europe. PMID:23991113

  11. A Genomewide Screen Reveals a Role of Mitochondria in Anaerobic Uptake of Sterols in Yeast

    PubMed Central

    Reiner, Sonja; Micolod, Delphine; Zellnig, Günther; Schneiter, Roger

    2006-01-01

    The mechanisms that govern intracellular transport of sterols in eukaryotic cells are not well understood. Saccharomyces cerevisiae is a facultative anaerobic organism that becomes auxotroph for sterols and unsaturated fatty acids in the absence of oxygen. To identify pathways that are required for uptake and transport of sterols, we performed a systematic screen of the yeast deletion mutant collection for genes that are required for growth under anaerobic conditions. Of the ∼4800 nonessential genes represented in the deletion collection, 37 were essential for growth under anaerobic conditions. These affect a wide range of cellular functions, including biosynthetic pathways for certain amino acids and cofactors, reprogramming of transcription and translation, mitochondrial function and biogenesis, and membrane trafficking. Thirty-three of these mutants failed to grow on lipid-supplemented media when combined with a mutation in HEM1, which mimics anaerobic conditions in the presence of oxygen. Uptake assays with radio- and fluorescently labeled cholesterol revealed that 17 of the 33 mutants strongly affect uptake and/or esterification of exogenously supplied cholesterol. Examination of the subcellular distribution of sterols in these uptake mutants by cell fractionation and fluorescence microscopy indicates that some of the mutants block incorporation of cholesterol into the plasma membrane, a presumably early step in sterol uptake. Unexpectedly, the largest class of uptake mutants is affected in mitochondrial functions, and many of the uptake mutants show electron-dense mitochondrial inclusions. These results indicate that a hitherto uncharacterized mitochondrial function is required for sterol uptake and/or transport under anaerobic conditions and are discussed in light of the fact that mitochondrial import of cholesterol is required for steroidogenesis in vertebrate cells. PMID:16251356

  12. A genome scale overexpression screen to reveal drug activity in human cells

    PubMed Central

    2014-01-01

    Target identification is a critical step in the lengthy and expensive process of drug development. Here, we describe a genome-wide screening platform that uses systematic overexpression of pooled human ORFs to understand drug mode-of-action and resistance mechanisms. We first calibrated our screen with the well-characterized drug methotrexate. We then identified new genes involved in the bioactivity of diverse drugs including antineoplastic agents and biologically active molecules. Finally, we focused on the transcription factor RHOXF2 whose overexpression conferred resistance to DNA damaging agents. This approach represents an orthogonal method for functional screening and, to our knowledge, has never been reported before. PMID:24944581

  13. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction.

    PubMed

    Tiwari, Prabhat; Kumar, Arun; Das, Rudra Nayan; Malhotra, Vivek; VijayRaghavan, K

    2015-01-01

    Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells. PMID:26488612

  14. MicroSCALE Screening Reveals Genetic Modifiers of Therapeutic Response in Melanoma

    PubMed Central

    Wood, Kris C.; Konieczkowski, David J.; Johannessen, Cory M.; Boehm, Jesse S.; Tamayo, Pablo; Botvinnik, Olga B.; Mesirov, Jill P.; Hahn, William C.; Root, David E.; Garraway, Levi A.; Sabatini, David M.

    2012-01-01

    Cell microarrays are a promising tool for performing large-scale functional genomic screening in mammalian cells at reasonable cost, but due to technical limitations, have been restricted for use with a narrow range of cell lines and short-term assays. Here, we describe MicroSCALE (Microarrays of Spatially Confined Adhesive Lentiviral Features), a cell microarray-based platform that enables application of this technology to a wide range of cell types and longer term assays. We used MicroSCALE to uncover kinases that when overexpressed partially desensitized B-RAFV600E-mutant melanoma cells to inhibitors of the mitogen-activated protein kinase kinase kinase (MAPKKK) RAF, the MAPKKs MEK1 and 2, mTOR (mammalian target of rapamycin), or PI3K (phosphatidylinositol 3-kinase). These screens indicated that cells treated with inhibitors acting through common mechanisms were affected by a similar profile of overexpressed proteins. In contrast, screens involving inhibitors acting through distinct mechanisms yielded unique profiles, a finding that has potential relevance for small molecule target identification and combination drugging studies. Further, by integrating large-scale functional screening results with cancer cell line gene expression and pharmacological sensitivity data, we validated the nuclear factor κB (NF-κB) pathway as a potential mediator of resistance to MAPK pathway inhibitors. The MicroSCALE platform described here may enable new classes of large-scale, resource-efficient screens that were not previously feasible, including those involving combinations of cell lines, perturbations, and assay outputs or those involving limited numbers of cells and limited or expensive reagents. PMID:22589389

  15. Systematic analysis of RNAi reports identifies dismal commonality at gene-level & reveals an unprecedented enrichment in pooled shRNA screens

    PubMed Central

    Bhinder, Bhavneet; Djaballah, Hakim

    2013-01-01

    RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (~80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes. PMID:23848309

  16. Genome-wide enrichment screening reveals multiple targets and resistance genes for triclosan in Escherichia coli.

    PubMed

    Yu, Byung Jo; Kim, Jung Ae; Ju, Hyun Mok; Choi, Soo-Kyung; Hwang, Seung Jin; Park, Sungyoo; Kim, Euijoong; Pan, Jae-Gu

    2012-10-01

    Triclosan is a widely used biocide effective against different microorganisms. At bactericidal concentrations, triclosan appears to affect multiple targets, while at bacteriostatic concentrations, triclosan targets FabI. The site-specific antibiotic-like mode-of-action and a widespread use of triclosan in household products claimed to possibly induce cross-resistance to other antibiotics. Thus, we set out to define more systematically the genes conferring resistance to triclosan; A genomic library of Escherichia coli strain W3110 was constructed and enriched in a selective medium containing a lethal concentration of triclosan. The genes enabling growth in the presence of triclosan were identified by using a DNA microarray and confirmed consequently by ASKA clones overexpressing the selected 62 candidate genes. Among these, forty-seven genes were further confirmed to enhance the resistance to triclosan; these genes, including the FabI target, were involved in inner or outer membrane synthesis, cell-surface material synthesis, transcriptional activation, sugar phosphotransferase (PTS) systems, various transporter systems, cell division, and ATPase and reductase/dehydrogenase reactions. In particular, overexpression of pgsA, rcsA, or gapC conferred to E. coli cells a similar level of triclosan resistance induced by fabI overexpression. These results indicate that triclosan may have multiple targets other than well-known FabI and that there are several undefined novel mechanisms for the resistance development to triclosan, thus probably inducing cross antibiotic resistance.

  17. A Chemical-Genomic Screen of Neglected Antibiotics Reveals Illicit Transport of Kasugamycin and Blasticidin S

    PubMed Central

    Shiver, Anthony L.; Kritikos, George; Li, Bo; Krogan, Nevan; Typas, Athanasios

    2016-01-01

    Fighting antibiotic resistance requires a deeper understanding of the genetic factors that determine the antibiotic susceptibility of bacteria. Here we describe a chemical-genomic screen in Escherichia coli K-12 that was designed to discover new aspects of antibiotic resistance by focusing on a set of 26 antibiotics and other stresses with poorly characterized mode-of-action and determinants of resistance. We show that the screen identifies new resistance determinants for these antibiotics including a common signature from two antimicrobials, kasugamycin and blasticidin S, used to treat crop diseases like rice blast and fire blight. Following this signature, we further investigated the mechanistic basis for susceptibility to kasugamycin and blasticidin S in E. coli using both genetic and biochemical approaches. We provide evidence that these compounds hijack an overlapping set of peptide ABC-importers to enter the bacterial cell. Loss of uptake may be an underappreciated mechanism for the development of kasugamycin resistance in bacterial plant pathogens. PMID:27355376

  18. A Chemical-Genomic Screen of Neglected Antibiotics Reveals Illicit Transport of Kasugamycin and Blasticidin S.

    PubMed

    Shiver, Anthony L; Osadnik, Hendrik; Kritikos, George; Li, Bo; Krogan, Nevan; Typas, Athanasios; Gross, Carol A

    2016-06-01

    Fighting antibiotic resistance requires a deeper understanding of the genetic factors that determine the antibiotic susceptibility of bacteria. Here we describe a chemical-genomic screen in Escherichia coli K-12 that was designed to discover new aspects of antibiotic resistance by focusing on a set of 26 antibiotics and other stresses with poorly characterized mode-of-action and determinants of resistance. We show that the screen identifies new resistance determinants for these antibiotics including a common signature from two antimicrobials, kasugamycin and blasticidin S, used to treat crop diseases like rice blast and fire blight. Following this signature, we further investigated the mechanistic basis for susceptibility to kasugamycin and blasticidin S in E. coli using both genetic and biochemical approaches. We provide evidence that these compounds hijack an overlapping set of peptide ABC-importers to enter the bacterial cell. Loss of uptake may be an underappreciated mechanism for the development of kasugamycin resistance in bacterial plant pathogens. PMID:27355376

  19. Large pathogen screening reveals first report of Megaselia scalaris (Diptera: Phoridae) parasitizing Apis mellifera intermissa (Hymenoptera: Apidae).

    PubMed

    Menail, Ahmed Hichem; Piot, Niels; Meeus, Ivan; Smagghe, Guy; Loucif-Ayad, Wahida

    2016-06-01

    As it is most likely that global warming will also lead to a shift in pollinator-habitats northwards, the study of southern species becomes more and more important. Pathogen screenings in subspecies of Apis mellifera capable of withstanding higher temperatures, provide an insight into future pathogen host interactions. Screenings in different climate regions also provide a global perspective on the prevalence of certain pathogens. In this project, we performed a pathogen screening in Apis mellifera intermissa, a native subspecies of Algeria in northern Africa. Colonies were sampled from different areas in the region of Annaba over a period of two years. Several pathogens were detected, among them Apicystis bombi, Crithidia mellificae, Nosema ceranae, Paenibacillus larvae, Lake Sinai Virus, Sacbrood Virus and Deformed Wing Virus (DWV). Our screening also revealed a phoroid fly, Megaselia scalaris, parasitizing honey bee colonies, which we report here for the first time. In addition, we found DWV to be present in the adult flies and replicating virus in the larval stages of the fly, which could indicate that M. scalaris acts as a vector of DWV. PMID:27130035

  20. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes

    PubMed Central

    Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-01-01

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone1,2, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down’s syndrome3-5. Which genes safeguard accurate progression through meiosis is largely unclear. Here, we developed high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNAi within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated dataset of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and now allows systematic studies of meiosis in mammals. PMID:26147080

  1. Intoxication of a Young Girl Reveals the Pitfalls of GHB Rapid Screening.

    PubMed

    Franken, Linda G; Andrews, Louise M; Slooff, Valerie D; de Wildt, Saskia N; Koch, Birgit C P

    2016-02-01

    The authors discuss the case of a 14-year-old girl who was transferred to the ICU of our hospital with ethanol intoxication (3.3 g/L), loss of consciousness (E5M3V1), and severe amnesia on recovery that was suspected of gamma-hydroxybutyric acid (GHB) intoxication. STAT toxicology screening may be necessary, when sexual assault under GHB intoxication is suspected. Therefore, the initial analysis of a urine sample was performed with a new enzymatic assay analysis for GHB. The enzymatic assay reported a GHB concentration of 26 mg/L, which is above the cut-off value of 10 mg/L. This cut-off value is to differentiate endogenous and exogenous levels because low levels of GHB occur naturally in the body. However, confirmation of these results by gas chromatography, which is common practice to confirm a positive GHB, gave a negative result. This discrepancy is probably contributed to interference of ethanol with the assay. This is a substantial downside of the GHB rapid screening, since the combination of GHB and ethanol is common. It is therefore advised to confirm that the positive GHB results are lower than 50 mg/L by gas chromatography, when using the rapid screening. This way the false-positive results and consequent inappropriate social and legal actions may be avoided.

  2. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

    PubMed

    Pfender, Sybille; Kuznetsov, Vitaliy; Pasternak, Michał; Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-08-13

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

  3. Copper Complexation Screen Reveals Compounds with Potent Antibiotic Properties against Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Haeili, Mehri; Moore, Casey; Davis, Christopher J. C.; Cochran, James B.; Shah, Santosh; Shrestha, Tej B.; Zhang, Yaofang; Bossmann, Stefan H.; Benjamin, William H.

    2014-01-01

    Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface. PMID:24752262

  4. Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

    PubMed Central

    Servienė, Elena; Lukša, Juliana; Orentaitė, Irma

    2012-01-01

    Background Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. Principal Findings We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Significance Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action. PMID:23227207

  5. Misexpression screen in Drosophila melanogaster aiming to reveal novel factors involved in formation of body parts.

    PubMed

    Grieder, Nicole C; Charlafti, Ilias; Kloter, Urs; Jäckle, Herbert; Schäfer, Ulrich; Gehring, Walter J

    2007-04-01

    To identify novel factors that lead a fly imaginal disc to adopt its developmental fate, we carried out a modular dominant misexpression screen in imaginal discs. We have identified two factors that appear to change the fate of the respective body structure and appear to lead to the transformation of a body part. In one mutant line, notum tissue, normally derived from wing imaginal tissue, formed close to the site of the sternopleural bristles, which are leg disc derivatives. In the other line, the arista is transformed into a tubular structure, resembling an abnormal leg. We found that ectopic expression of abrupt was responsible for this potential transformation of the arista.

  6. A genetic screen reveals Foxa3 and TNFR1 as key regulators of liver repopulation

    PubMed Central

    Wangensteen, Kirk J.; Zhang, Sophia; Greenbaum, Linda E.

    2015-01-01

    The fundamental question of which genes are most important in controlling liver regeneration remains unanswered. We employed a parallel screen to test the impact of 43 selected genes on liver repopulation in the Fah−/− mouse model of hereditary tyrosinemia. We discovered that the transcription factor Foxa3 was a strong promoter of liver regeneration, while tumor necrosis factor receptor 1 (TNFR1) was the most significant suppressor of repopulation among all of the genes tested. Our approach enabled the identification of these factors as important regulators of liver repopulation and potential drug targets for the promotion of liver repopulation. PMID:25934503

  7. Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen

    PubMed Central

    Liakath-Ali, Kifayathullah; Vancollie, Valerie E.; Heath, Emma; Smedley, Damian P.; Estabel, Jeanne; Sunter, David; DiTommaso, Tia; White, Jacqueline K.; Ramirez-Solis, Ramiro; Smyth, Ian; Steel, Karen P.; Watt, Fiona M.

    2014-01-01

    Permanent stop-and-shop large-scale mouse mutant resources provide an excellent platform to decipher tissue phenogenomics. Here we analyse skin from 538 knockout mouse mutants generated by the Sanger Institute Mouse Genetics Project. We optimize immunolabelling of tail epidermal wholemounts to allow systematic annotation of hair follicle, sebaceous gland and interfollicular epidermal abnormalities using ontology terms from the Mammalian Phenotype Ontology. Of the 50 mutants with an epidermal phenotype, 9 map to human genetic conditions with skin abnormalities. Some mutant genes are expressed in the skin, whereas others are not, indicating systemic effects. One phenotype is affected by diet and several are incompletely penetrant. In-depth analysis of three mutants, Krt76, Myo5a (a model of human Griscelli syndrome) and Mysm1, provides validation of the screen. Our study is the first large-scale genome-wide tissue phenotype screen from the International Knockout Mouse Consortium and provides an open access resource for the scientific community. PMID:24721909

  8. A Germline Clone Screen on the X Chromosome Reveals Novel Meiotic Mutants in Drosophila melanogaster

    PubMed Central

    Collins, Kimberly A.; Callicoat, Jonathon G.; Lake, Cathleen M.; McClurken, Cailey M.; Kohl, Kathryn P.; Hawley, R. Scott

    2012-01-01

    In an effort to isolate novel meiotic mutants that are severely defective in chromosome segregation and/or exchange, we employed a germline clone screen of the X chromosome of Drosophila melanogaster. We screened over 120,000 EMS-mutagenized chromosomes and isolated 19 mutants, which comprised nine complementation groups. Four of these complementation groups mapped to known meiotic genes, including mei-217, mei-218, mei-9, and nod. Importantly, we have identified two novel complementation groups with strong meiotic phenotypes, as assayed by X chromosome nondisjunction. One complementation group is defined by three alleles, and the second novel complementation group is defined by a single allele. All 19 mutants are homozygous viable, fertile, and fully recessive. Of the 9 mutants that have been molecularly characterized, 5 are canonical EMS-induced transitions, and the remaining 4 are transversions. In sum, we have identified two new genes that are defined by novel meiotic mutants, in addition to isolating new alleles of mei-217, mei-218, mei-9, and nod. PMID:23173088

  9. A large scale screen reveals genes that mediate electrotaxis in Dictyostelium discoideum**

    PubMed Central

    Gao, Runchi; Zhao, Siwei; Jiang, Xupin; Sun, Yaohui; Zhao, Sanjun; Gao, Jing; Borleis, Jane; Willard, Stacey; Tang, Ming; Cai, Huaqing; Kamimura, Yoichiro; Huang, Yuesheng; Jiang, Jianxin; Huang, Zunxi; Mogilner, Alex; Pan, Tingrui; Devreotes, Peter N; Zhao, Min

    2015-01-01

    Directional cell migration in an electric field, a phenomenon called galvanotaxis or electrotaxis, occurs in many types of cells, and may play an important role in wound healing and development. Small extracellular electric fields can guide the migration of amoeboid cells, and here, we established a large-scale screening approach to search for mutants with electrotaxis phenotypes from a collection of 563 Dictyostelium discoideum strains with morphological defects. We identified 28 strains that were defective in electrotaxis and 10 strains with a slightly higher directional response. Using plasmid rescue followed by gene disruption, we identified some of the mutated genes, including some previously implicated in chemotaxis. Amongst these we studied PiaA, which encodes a critical component of TORC2, a kinase protein complex that transduces changes in motility by activating the kinase PKB (also known as Akt). Furthermore, we found that electrotaxis was decreased in mutants lacking gefA, rasC, rip3, lst8 or pkbR1, genes that encode other components of the TORC2-PKB pathway. Thus, we have developed a high-throughput screening technique that will be a useful tool to elucidate the molecular mechanisms of electrotaxis. PMID:26012633

  10. A Forward Genetic Screen Reveals that Calcium-dependent Protein Kinase 3 Regulates Egress in Toxoplasma

    PubMed Central

    Ehret, Emma; Butz, Heidi; Garbuz, Tamila; Oswald, Benji P.; Settles, Matt; Boothroyd, John; Arrizabalaga, Gustavo

    2012-01-01

    Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle. PMID:23209419

  11. A Sleeping Beauty screen reveals NF-kB activation in CLL mouse model

    PubMed Central

    Zanesi, Nicola; Balatti, Veronica; Riordan, Jesse; Burch, Aaron; Rizzotto, Lara; Palamarchuk, Alexey; Cascione, Luciano; Lagana, Alessandro; Dupuy, Adam J.; Croce, Carlo M.

    2013-01-01

    TCL1 oncogene is overexpressed in aggressive form of human chronic lymphocytic leukemia (CLL) and its dysregulation in mouse B cells causes a CD5-positive leukemia similar to the aggressive form of human CLLs. To identify oncogenes that cooperate with Tcl1, we performed genetic screen in Eμ−TCL1 mice using Sleeping Beauty transposon-mediated mutagenesis. Analysis of transposon common insertion sites identified 7 genes activated by transposon insertions. Overexpression of these genes in mouse CLL was confirmed by real time reverse transcription-polymerase chain reaction. Interestingly, the main known function of 4 of 7 genes (Nfkb1, Tab2, Map3K14, and Nfkbid) is participation in or activation of the nuclear factor-kB (NF-kB) pathway. In addition, activation of the NF-kB is 1 of main functions of Akt2, also identified in the screen. These findings demonstrate cooperation of Tcl1 and the NF-kB pathway in the pathogenesis of aggressive CLL. Identification cooperating cancer genes will result in the development of combinatorial therapies to treat CLL. PMID:23591791

  12. A Sleeping Beauty screen reveals NF-kB activation in CLL mouse model.

    PubMed

    Zanesi, Nicola; Balatti, Veronica; Riordan, Jesse; Burch, Aaron; Rizzotto, Lara; Palamarchuk, Alexey; Cascione, Luciano; Lagana, Alessandro; Dupuy, Adam J; Croce, Carlo M; Pekarsky, Yuri

    2013-05-23

    TCL1 oncogene is overexpressed in aggressive form of human chronic lymphocytic leukemia (CLL) and its dysregulation in mouse B cells causes a CD5-positive leukemia similar to the aggressive form of human CLLs. To identify oncogenes that cooperate with Tcl1, we performed genetic screen in Eμ-TCL1 mice using Sleeping Beauty transposon-mediated mutagenesis. Analysis of transposon common insertion sites identified 7 genes activated by transposon insertions. Overexpression of these genes in mouse CLL was confirmed by real time reverse transcription-polymerase chain reaction. Interestingly, the main known function of 4 of 7 genes (Nfkb1, Tab2, Map3K14, and Nfkbid) is participation in or activation of the nuclear factor-kB (NF-kB) pathway. In addition, activation of the NF-kB is 1 of main functions of Akt2, also identified in the screen. These findings demonstrate cooperation of Tcl1 and the NF-kB pathway in the pathogenesis of aggressive CLL. Identification cooperating cancer genes will result in the development of combinatorial therapies to treat CLL. PMID:23591791

  13. A cell-based screen reveals that the albendazole metabolite, albendazole sulfone, targets Wolbachia.

    PubMed

    Serbus, Laura R; Landmann, Frederic; Bray, Walter M; White, Pamela M; Ruybal, Jordan; Lokey, R Scott; Debec, Alain; Sullivan, William

    2012-09-01

    Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts. PMID:23028321

  14. Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability.

    PubMed

    Forster, J I; Köglsberger, S; Trefois, C; Boyd, O; Baumuratov, A S; Buck, L; Balling, R; Antony, P M A

    2016-06-01

    The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels.

  15. A directed mutagenesis screen in Drosophila melanogaster reveals new mutants that influence hedgehog signaling.

    PubMed Central

    Haines, N; van den Heuvel, M

    2000-01-01

    The Hedgehog signaling pathway has been recognized as essential for patterning processes in development of metazoan animal species. The signaling pathway is, however, not entirely understood. To start to address this problem, we set out to isolate new mutations that influence Hedgehog signaling. We performed a mutagenesis screen for mutations that dominantly suppress Hedgehog overexpression phenotypes in the Drosophila melanogaster wing. We isolated four mutations that influence Hedgehog signaling. These were analyzed in the amenable wing system using genetic and molecular techniques. One of these four mutations affects the stability of the Hedgehog expression domain boundary, also known as the organizer in the developing wing. Another mutation affects a possible Hedgehog autoregulation mechanism, which stabilizes the same boundary. PMID:11102373

  16. Screening for Dimethylarginine Dimethylaminohydrolase Inhibitors Reveals Ebselen as a Bioavailable Inactivator

    PubMed Central

    2011-01-01

    Dimethylarginine dimethylaminohydrolase (DDAH) is an endogenous regulator of nitric oxide production and represents a potential therapeutic target. However, only a small number of biologically useful inhibitors have been reported, and many of these are substrate analogues. To seek more diverse scaffolds, we developed a high-throughput screening (HTS) assay and queried two small libraries totaling 2446 compounds. The HTS assay proved to be robust, reproducible, and scalable, with Z′ factors ≥ 0.78. One inhibitor, ebselen, is structurally divergent from substrate and was characterized in detail. This selenazole covalently inactivates DDAH in vitro and in cultured cells. The rate constant for inactivation of DDAH (44000 ± 2400 M–1 s–1) is greater than those reported for any other target, suggesting that this pathway is an important aspect of ebselen's total pharmacological effects. PMID:21927644

  17. What RNAi screens in model organisms revealed about microbicidal response in mammals?

    PubMed Central

    Abnave, Prasad; Conti, Filippo; Torre, Cedric; Ghigo, Eric

    2015-01-01

    The strategies evolved by pathogens to infect hosts and the mechanisms used by the host to eliminate intruders are highly complex. Because several biological pathways and processes are conserved across model organisms, these organisms have been used for many years to elucidate and understand the mechanisms of the host-pathogen relationship and particularly to unravel the molecular processes enacted by the host to kill pathogens. The emergence of RNA interference (RNAi) and the ability to apply it toward studies in model organisms have allowed a breakthrough in the elucidation of host-pathogen interactions. The aim of this mini-review is to highlight and describe recent breakthroughs in the field of host-pathogen interactions using RNAi screens of model organisms. We will focus specifically on the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio. Moreover, a recent study examining the immune system of planarian will be discussed. PMID:25629007

  18. High-Copy Overexpression Screening Reveals PDR5 as the Main Doxorubicin Resistance Gene in Yeast

    PubMed Central

    Demir, Ayse Banu; Koc, Ahmet

    2015-01-01

    Doxorubicin is one of the most potent anticancer drugs used in the treatment of various cancer types. The efficacy of doxorubicin is influenced by the drug resistance mechanisms and its cytotoxicity. In this study, we performed a high-copy screening analysis to find genes that play a role in doxorubicin resistance and found several genes (CUE5, AKL1, CAN1, YHR177W and PDR5) that provide resistance. Among these genes, overexpression of PDR5 provided a remarkable resistance, and deletion of it significantly rendered the tolerance level for the drug. Q-PCR analyses suggested that transcriptional regulation of these genes was not dependent on doxorubicin treatment. Additionally, we profiled the global expression pattern of cells in response to doxorubicin treatment and highlighted the genes and pathways that are important in doxorubicin tolerance/toxicity. Our results suggest that many efflux pumps and DNA metabolism genes are upregulated by the drug and required for doxorubicin tolerance. PMID:26690737

  19. Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability

    PubMed Central

    Forster, J. I.; Köglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A.

    2016-01-01

    The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels. PMID:26738520

  20. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry.

    PubMed

    Silva-Ayala, Daniela; López, Tomás; Gutiérrez, Michelle; Perrimon, Norbert; López, Susana; Arias, Carlos F

    2013-06-18

    Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells.

  1. Population Screening Using Sewage Reveals Pan-Resistant Bacteria in Hospital and Community Samples

    PubMed Central

    Mileguir, Fernando; Azar, Roberto; Smollan, Gill; Belausov, Natasha; Rahav, Galia; Shamiss, Ari; Mendelson, Ella; Keller, Nathan

    2016-01-01

    The presence of pan-resistant bacteria worldwide possesses a threat to global health. It is difficult to evaluate the extent of carriage of resistant bacteria in the population. Sewage sampling is a possible way to monitor populations. We evaluated the presence of pan-resistant bacteria in Israeli sewage collected from all over Israel, by modifying the pour plate method for heterotrophic plate count technique using commercial selective agar plates. This method enables convenient and fast sewage sampling and detection. We found that sewage in Israel contains multiple pan-resistant bacteria including carbapenemase resistant Enterobacteriacae carrying blaKPC and blaNDM-1, MRSA and VRE. blaKPC carrying Klebsiella pneumonia and Enterobacter cloacae were the most common Enterobacteriacae drug resistant bacteria found in the sewage locations we sampled. Klebsiella pneumonia, Enterobacter spp., Escherichia coli and Citrobacter spp. were the 4 main CRE isolated from Israeli sewage and also from clinical samples in our clinical microbiology laboratory. Hospitals and Community sewage had similar percentage of positive samplings for blaKPC and blaNDM-1. VRE was found to be more abundant in sewage in Israel than MRSA but there were more locations positive for MRSA and VRE bacteria in Hospital sewage than in the Community. Therefore, our upgrade of the pour plate method for heterotrophic plate count technique using commercial selective agar plates can be a useful tool for routine screening and monitoring of the population for pan-resistant bacteria using sewage. PMID:27780222

  2. Extensive screen for bacterial endosymbionts reveals taxon-specific distribution patterns among bees (Hymenoptera, Anthophila).

    PubMed

    Gerth, Michael; Saeed, Abiya; White, Jennifer A; Bleidorn, Christoph

    2015-06-01

    Bacterial endosymbionts play key roles in arthropod biology, ranging from beneficial mutualists to parasitic sex ratio manipulators. The number of described endosymbiotic bacterial taxa has accumulated continuously in recent years. While the understanding of arthropod-microbe interactions has advanced significantly, especially in model organisms, relatively little is known about symbiont distribution and effects in non-model organisms. As a first step to alleviate this gap in understanding, we performed an endosymbiont survey in bees (Anthophila), an ecologically and economically important group of hymenopterans. To this end, we sampled 170 bee species and screened by PCR for the presence of Wolbachia, Rickettsia, Arsenophonus and Cardinium. Detected strains were then further diagnosed by additional markers. Additionally, we tested if certain ecological traits, bee phylogeny or geographic origin of bees explain endosymbiont distribution. Our results indicate that supergroup A Wolbachia are very common in bees and that their distribution can be significantly correlated to both host ecology and phylogeny, although a distinction of these factors is not possible. Furthermore, bees from the same region (Old World or New World) are more likely to harbour identical Wolbachia strains than expected by chance. Other endosymbionts (Rickettsia, Arsenophonus) were less common, and specific to particular host taxa, suggesting that host phylogeny is a major predictor for endosymbiont distribution in bees.

  3. Metagenome-based screening reveals worldwide distribution of LOV-domain proteins.

    PubMed

    Pathak, Gopal P; Losi, Aba; Gärtner, Wolfgang

    2012-01-01

    Metagenomes from various environments were screened for sequences homologous to light, oxygen, voltage (LOV)-domain proteins. LOV domains are flavin binding, blue-light (BL)-sensitive photoreceptors present in 10-15% of deposited prokaryotic genomes. The LOV domain has been selected, since BL is an ever present and sometimes harmful environmental factor for microbial communities. The majority of the metagenome material originated from the Sargasso Sea Project and from open-ocean sampling. In total, more than 40 million open reading frames were investigated for LOV-domain sequences. Most sequences were identified from aquatic material, but they were also found in metagenomes from soil and extreme environments, e.g. hypersaline ponds, acidic mine drainage or wastewater treatment facilities. A total of 578 LOV domains was assigned by three criteria: (1) the highly conserved core region, (2) the presence of minimally 14 essential amino acids and (3) a minimal length of 80 amino acids. More than three quarters of these identified genes showed a sequence divergence of more than 20% from database-deposited LOV domains from known organisms, indicating the large variation of this photoreceptor motif. The broad occurrence of LOV domains in metagenomes emphasizes their important physiological role for light-induced signal transduction, stress adaptation and survival mechanisms. PMID:22044076

  4. Functional mutagenesis screens reveal the 'cap structure' formation in disulfide-bridge free TASK channels.

    PubMed

    Goldstein, Matthias; Rinné, Susanne; Kiper, Aytug K; Ramírez, David; Netter, Michael F; Bustos, Daniel; Ortiz-Bonnin, Beatriz; González, Wendy; Decher, Niels

    2016-01-22

    Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels.

  5. Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice

    PubMed Central

    Been, Raha A.; Linden, Michael A.; Hager, Courtney J.; DeCoursin, Krista J.; Abrahante, Juan E.; Landman, Sean R.; Steinbach, Michael; Sarver, Aaron L.; Largaespada, David A.; Starr, Timothy K.

    2014-01-01

    Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients. PMID:24827933

  6. Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations.

    PubMed

    Sampieri, Katia; Hadjistilianou, Theodora; Mari, Francesca; Speciale, Caterina; Mencarelli, Maria Antonietta; Cetta, Francesco; Manoukian, Siranoush; Peissel, Bernard; Giachino, Daniela; Pasini, Barbara; Acquaviva, Antonio; Caporossi, Aldo; Frezzotti, Renato; Renieri, Alessandra; Bruttini, Mirella

    2006-01-01

    Retinoblastoma (RB, OMIM#180200) is the most common intraocular tumour in infancy and early childhood. Constituent mutations in the RB1 gene predispose individuals to RB development. We performed a mutational screening of the RB1 gene in Italian patients affected by RB referred to the Medical Genetics of the University of Siena. In 35 unrelated patients, we identified germline RB1 mutations in 6 out of 9 familial cases (66%) and in 7 out of 26 with no family history of RB (27%). Using the single-strand conformational polymorphism (SSCP) technique, 11 novel mutations were detected, including 3 nonsense, 5 frameshift and 4 splice-site mutations. Only two of these mutations (1 splice site and 1 missense) were previously reported. The mutation spectrum reflects the published literature, encompassing predominately nonsense or frameshift and splicing mutations. RB1 germline mutation was detected in 37% of our cases. Gross rearrangements outside the investigated region, altered DNA methylation, or mutations in non-coding regions, may be the cause of disease in the remainder of the patients. Some cases, e.g. a case of incomplete penetrance, or variable expressivity ranging from retinoma to multiple tumours, are discussed in detail. In addition, a case of pre-conception genetic counselling resolved by rescue of banked cordonal blood of the affected deceased child is described.

  7. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

    PubMed Central

    Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

    2016-01-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

  8. A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis.

    PubMed

    Ables, Elizabeth T; Hwang, Grace H; Finger, Danielle S; Hinnant, Taylor D; Drummond-Barbosa, Daniela

    2016-01-01

    Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies. PMID:27226164

  9. Screening of Escherichia coli Species Biodiversity Reveals New Biofilm-Associated Antiadhesion Polysaccharides

    PubMed Central

    Rendueles, Olaya; Travier, Laetitia; Latour-Lambert, Patricia; Fontaine, Thierry; Magnus, Julie; Denamur, Erick; Ghigo, Jean-Marc

    2011-01-01

    ABSTRACT Bacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted from in vitro mature biofilms formed by 122 natural Escherichia coli isolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animal E. coli strain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion of Staphylococcus aureus from mixed E. coli and S. aureus biofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices. PMID:21558434

  10. A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis

    PubMed Central

    Ables, Elizabeth T.; Hwang, Grace H.; Finger, Danielle S.; Hinnant, Taylor D.; Drummond-Barbosa, Daniela

    2016-01-01

    Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies. PMID:27226164

  11. Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

    PubMed

    Kok, Fatma O; Shin, Masahiro; Ni, Chih-Wen; Gupta, Ankit; Grosse, Ann S; van Impel, Andreas; Kirchmaier, Bettina C; Peterson-Maduro, Josi; Kourkoulis, George; Male, Ira; DeSantis, Dana F; Sheppard-Tindell, Sarah; Ebarasi, Lwaki; Betsholtz, Christer; Schulte-Merker, Stefan; Wolfe, Scot A; Lawson, Nathan D

    2015-01-12

    The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

  12. Reverse genetic screening reveals poor correlation between Morpholino-induced and mutant phenotypes in zebrafish

    PubMed Central

    Gupta, A.; Grosse, A. S.; van Impel, A.; Kirchmaier, B. C.; Peterson-Maduro, J.; Kourkoulis, G.; Male, I.; DeSantis, D.F.; Sheppard-Tindell, S.; Ebarasi, L.; Betsholtz, C.; Schulte-Merker, S.; Wolfe, S. A.; Lawson, N. D.

    2014-01-01

    SUMMARY The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than twenty genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately eighty percent of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses. PMID:25533206

  13. An integrated systems genetics screen reveals the transcriptional structure of inherited predisposition to metastatic disease

    PubMed Central

    Faraji, Farhoud; Hu, Ying; Wu, Gang; Goldberger, Natalie E.; Walker, Renard C.; Zhang, Jinghui; Hunter, Kent W.

    2014-01-01

    Metastasis is the result of stochastic genomic and epigenetic events leading to gene expression profiles that drive tumor dissemination. Here we exploit the principle that metastatic propensity is modified by the genetic background to generate prognostic gene expression signatures that illuminate regulators of metastasis. We also identify multiple microRNAs whose germline variation is causally linked to tumor progression and metastasis. We employ network analysis of global gene expression profiles in tumors derived from a panel of recombinant inbred mice to identify a network of co-expressed genes centered on Cnot2 that predicts metastasis-free survival. Modulating Cnot2 expression changes tumor cell metastatic potential in vivo, supporting a functional role for Cnot2 in metastasis. Small RNA sequencing of the same tumor set revealed a negative correlation between expression of the Mir216/217 cluster and tumor progression. Expression quantitative trait locus analysis (eQTL) identified cis-eQTLs at the Mir216/217 locus, indicating that differences in expression may be inherited. Ectopic expression of Mir216/217 in tumor cells suppressed metastasis in vivo. Finally, small RNA sequencing and mRNA expression profiling data were integrated to reveal that miR-3470a/b target a high proportion of network transcripts. In vivo analysis of Mir3470a/b demonstrated that both promote metastasis. Moreover, Mir3470b is a likely regulator of the Cnot2 network as its overexpression down-regulated expression of network hub genes and enhanced metastasis in vivo, phenocopying Cnot2 knockdown. The resulting data from this strategy identify Cnot2 as a novel regulator of metastasis and demonstrate the power of our systems-level approach in identifying modifiers of metastasis. PMID:24322557

  14. In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis.

    PubMed

    Li, Ling; Bonneton, François; Tohme, Marie; Bernard, Laure; Chen, Xiao Yong; Laudet, Vincent

    2016-01-01

    The effects of endocrine disrupting chemicals (EDCs) on reproduction are well known, whereas their developmental effects are much less characterized. However, exposure to endocrine disruptors during organogenesis may lead to deleterious and permanent problems later in life. Zebrafish (Danio rerio) transgenic lines expressing the green fluorescent protein (GFP) in specific organs and tissues are powerful tools to uncover developmental defects elicited by EDCs. Here, we used seven transgenic lines to visualize in vivo whether a series of EDCs and other pharmaceutical compounds can alter organogenesis in zebrafish. We used transgenic lines expressing GFP in pancreas, liver, blood vessels, inner ear, nervous system, pharyngeal tooth and pectoral fins. This screen revealed that four of the tested chemicals have detectable effects on different organs, which shows that the range of effects elicited by EDCs is wider than anticipated. The endocrine disruptor tetrabromobisphenol-A (TBBPA), as well as the three drugs diclofenac, trichostatin A (TSA) and valproic acid (VPA) induced abnormalities in the embryonic vascular system of zebrafish. Moreover, TSA and VPA induced specific alterations during the development of pancreas, an observation that was confirmed by in situ hybridization with specific markers. Developmental delays were also induced by TSA and VPA in the liver and in pharyngeal teeth, resulting in smaller organ size. Our results show that EDCs can induce a large range of developmental alterations during embryogenesis of zebrafish and establish GFP transgenic lines as powerful tools to screen for EDCs effects in vivo.

  15. In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis

    PubMed Central

    Li, Ling; Bonneton, François; Tohme, Marie; Bernard, Laure; Chen, Xiao Yong; Laudet, Vincent

    2016-01-01

    The effects of endocrine disrupting chemicals (EDCs) on reproduction are well known, whereas their developmental effects are much less characterized. However, exposure to endocrine disruptors during organogenesis may lead to deleterious and permanent problems later in life. Zebrafish (Danio rerio) transgenic lines expressing the green fluorescent protein (GFP) in specific organs and tissues are powerful tools to uncover developmental defects elicited by EDCs. Here, we used seven transgenic lines to visualize in vivo whether a series of EDCs and other pharmaceutical compounds can alter organogenesis in zebrafish. We used transgenic lines expressing GFP in pancreas, liver, blood vessels, inner ear, nervous system, pharyngeal tooth and pectoral fins. This screen revealed that four of the tested chemicals have detectable effects on different organs, which shows that the range of effects elicited by EDCs is wider than anticipated. The endocrine disruptor tetrabromobisphenol-A (TBBPA), as well as the three drugs diclofenac, trichostatin A (TSA) and valproic acid (VPA) induced abnormalities in the embryonic vascular system of zebrafish. Moreover, TSA and VPA induced specific alterations during the development of pancreas, an observation that was confirmed by in situ hybridization with specific markers. Developmental delays were also induced by TSA and VPA in the liver and in pharyngeal teeth, resulting in smaller organ size. Our results show that EDCs can induce a large range of developmental alterations during embryogenesis of zebrafish and establish GFP transgenic lines as powerful tools to screen for EDCs effects in vivo. PMID:26900852

  16. A transcriptome-wide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway.

    PubMed

    Czech, Benjamin; Preall, Jonathan B; McGinn, Jon; Hannon, Gregory J

    2013-06-01

    The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two interrelated branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborate pathway centered on the three gonad-specific Argonaute proteins (Piwi, Aubergine, and Argonaute 3). While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals key factors of piRNA-mediated transposon silencing, including the piRNA biogenesis factors CG2183 (GASZ) and Deadlock. Our data uncover a previously unanticipated set of factors preferentially required for repression of different transposon types. PMID:23665227

  17. A Genome-wide Screen Reveals a Role for microRNA-1 in Modulating Cardiac Cell Polarity

    PubMed Central

    King, Isabelle N.; Qian, Li; Liang, Jianping; Huang, Yu; Shieh, Joseph T.C.; Kwon, Chulan; Srivastava, Deepak

    2011-01-01

    Summary Many molecular pathways involved in heart disease have their roots in evolutionarily ancient developmental programs that depend critically on gene dosage and timing. MicroRNAs (miRNAs) modulate gene dosage post-transcriptionally, and among these, the muscle-specific miR-1 is particularly important for developing and maintaining somatic/skeletal and cardiac muscle. To identify pathways regulated by miR-1, we performed a forward genetic screen in Drosophila using wing-vein patterning as a biological assay. We identified several unexpected genes that genetically interacted with dmiR-1, one of which was kayak, encoding a developmentally-regulated transcription factor. Additional studies directed at this genetic relationship revealed a previously unappreciated function of dmiR-1 in regulating the polarity of cardiac progenitor cells. The mammalian orthologue of kay, c-Fos, was dysregulated in hearts of gain- or loss-of-function miR-1 mutant mice in a stress-dependent manner. These findings illustrate the power of Drosophila-based screens to find points of intersection between miRNAs and conserved pathways in mammals. PMID:21497762

  18. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

    PubMed Central

    Rallis, Charalampos; López-Maury, Luis; Georgescu, Teodora; Pancaldi, Vera; Bähler, Jürg

    2014-01-01

    Summary Target of rapamycin complex 1 (TORC1), which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not. PMID:24463365

  19. In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis.

    PubMed

    Li, Ling; Bonneton, François; Tohme, Marie; Bernard, Laure; Chen, Xiao Yong; Laudet, Vincent

    2016-01-01

    The effects of endocrine disrupting chemicals (EDCs) on reproduction are well known, whereas their developmental effects are much less characterized. However, exposure to endocrine disruptors during organogenesis may lead to deleterious and permanent problems later in life. Zebrafish (Danio rerio) transgenic lines expressing the green fluorescent protein (GFP) in specific organs and tissues are powerful tools to uncover developmental defects elicited by EDCs. Here, we used seven transgenic lines to visualize in vivo whether a series of EDCs and other pharmaceutical compounds can alter organogenesis in zebrafish. We used transgenic lines expressing GFP in pancreas, liver, blood vessels, inner ear, nervous system, pharyngeal tooth and pectoral fins. This screen revealed that four of the tested chemicals have detectable effects on different organs, which shows that the range of effects elicited by EDCs is wider than anticipated. The endocrine disruptor tetrabromobisphenol-A (TBBPA), as well as the three drugs diclofenac, trichostatin A (TSA) and valproic acid (VPA) induced abnormalities in the embryonic vascular system of zebrafish. Moreover, TSA and VPA induced specific alterations during the development of pancreas, an observation that was confirmed by in situ hybridization with specific markers. Developmental delays were also induced by TSA and VPA in the liver and in pharyngeal teeth, resulting in smaller organ size. Our results show that EDCs can induce a large range of developmental alterations during embryogenesis of zebrafish and establish GFP transgenic lines as powerful tools to screen for EDCs effects in vivo. PMID:26900852

  20. New aspects of the phosphatase VHZ revealed by a high-resolution structure with vanadate and substrate screening

    PubMed Central

    Kuznetsov, Vyacheslav I.; Hengge, Alvan C.; Johnson, Sean J.

    2013-01-01

    The recently discovered 150-residue human VHZ (VH1 related protein, Z member) is one of the smallest protein tyrosine phosphatases (PTPs) known, and contains only the minimal structural elements common to all PTPs. We report a substrate screening analysis and a crystal structure of the VHZ complex with vanadate at 1.1 Å resolution, with a detailed structural comparison with other members of the protein tyrosine phosphatase family, including classical tyrosine-specific protein tyrosine phosphatases (PTPs) and dual specific phosphatases (DSPs). A screen with 360 phosphorylated peptides shows VHZ efficiently catalyzes the hydrolysis of phospho-tyrosine(pY)-containing peptides, but exhibits no activity toward phospho-serine (pS) or phospho-threonine (pT) peptides. The new structure reveals a deep and narrow active site more typical of the classical tyrosine specific PTPs. Despite the high structural and sequence similarities between VHZ and classical PTPs, its general acid IPD-loop is most likely conformationally rigid, in contrast to the flexible WPD counterpart of classical PTPs. VHZ also lacks substrate recognition domains and other domains typically found on classical PTPs. It is therefore proposed that VHZ is more properly classified as an atypical PTP rather than an atypical DSP, as has been suggested. PMID:23145819

  1. A Genome-Wide Screen Reveals that the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Modulates Virulence Gene Expression

    PubMed Central

    Millet, Yves A.; Chao, Michael C.; Sasabe, Jumpei; Davis, Brigid M.

    2015-01-01

    Diverse environmental stimuli and a complex network of regulatory factors are known to modulate expression of Vibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression of tcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor. Besides identifying most of the genes known to modulate tcpA expression, the screen yielded ptsI and ptsH, which encode the enzyme I (EI) and Hpr components of the V. cholerae phosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIAGlc protein produced less cholera toxin (CT) and had a diminished capacity to colonize the infant mouse intestine. The PTS modulates virulence gene expression by regulating expression of tcpPH and aphAB, which themselves control expression of toxT, the central activator of virulence gene expression. One mechanism by which PTS promotes virulence gene expression appears to be by modulating the amounts of intracellular cyclic AMP (cAMP). Our findings reveal that the V. cholerae PTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks. PMID:26056384

  2. Quantitative Genome-Wide Genetic Interaction Screens Reveal Global Epistatic Relationships of Protein Complexes in Escherichia coli

    PubMed Central

    Kumar, Ashwani; Stewart, Geordie; Samanfar, Bahram; Aoki, Hiroyuki; Wagih, Omar; Vlasblom, James; Phanse, Sadhna; Lad, Krunal; Yeou Hsiung Yu, Angela; Graham, Christopher; Jin, Ke; Brown, Eric; Golshani, Ashkan; Kim, Philip; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack; Houry, Walid A.; Parkinson, John; Emili, Andrew

    2014-01-01

    Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems. PMID:24586182

  3. Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

    PubMed Central

    Shindo, Takayuki; Kaschani, Farnusch; Kovács, Judit; Tian, Fang; Kourelis, Jiorgos; Hong, Tram Ngoc; Colby, Tom; Shabab, Mohammed; Chawla, Rohini; Kumari, Selva; Ilyas, Muhammad; Hörger, Anja C.; Alfano, James R.; van der Hoorn, Renier A. L.

    2016-01-01

    Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes. PMID:27603016

  4. Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.

    PubMed

    Shindo, Takayuki; Kaschani, Farnusch; Yang, Fan; Kovács, Judit; Tian, Fang; Kourelis, Jiorgos; Hong, Tram Ngoc; Colby, Tom; Shabab, Mohammed; Chawla, Rohini; Kumari, Selva; Ilyas, Muhammad; Hörger, Anja C; Alfano, James R; van der Hoorn, Renier A L

    2016-09-01

    Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes. PMID:27603016

  5. A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer.

    PubMed

    O'Donnell, Kathryn A; Keng, Vincent W; York, Brian; Reineke, Erin L; Seo, Daekwan; Fan, Danhua; Silverstein, Kevin A T; Schrum, Christina T; Xie, Wei Rose; Mularoni, Loris; Wheelan, Sarah J; Torbenson, Michael S; O'Malley, Bert W; Largaespada, David A; Boeke, Jef D

    2012-05-22

    The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy. PMID:22556267

  6. Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Ploier, Birgit; Scharwey, Melanie; Koch, Barbara; Schmidt, Claudia; Schatte, Jessica; Rechberger, Gerald; Kollroser, Manfred; Hermetter, Albin; Daum, Günther

    2013-12-13

    Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.

  7. Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

    PubMed Central

    Choy, John S.; O'Toole, Eileen; Schuster, Breanna M.; Crisp, Matthew J.; Karpova, Tatiana S.; McNally, James G.; Winey, Mark; Gardner, Melissa K.; Basrai, Munira A.

    2013-01-01

    How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles. PMID:23825022

  8. Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis

    PubMed Central

    Stadelmann, Britta; Rufener, Reto; Aeschbacher, Denise; Spiliotis, Markus; Gottstein, Bruno; Hemphill, Andrew

    2016-01-01

    The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load. PMID:26967740

  9. A Functional Screen Reveals an Extensive Layer of Transcriptional and Splicing Control Underlying RAS/MAPK Signaling in Drosophila

    PubMed Central

    Ashton-Beaucage, Dariel; Udell, Christian M.; Gendron, Patrick; Sahmi, Malha; Lefrançois, Martin; Baril, Caroline; Guenier, Anne-Sophie; Duchaine, Jean; Lamarre, Daniel; Lemieux, Sébastien; Therrien, Marc

    2014-01-01

    The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway—including a new protein complex modulating RAF activation—we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing components can also

  10. New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast*

    PubMed Central

    Martin, D. Christian; Kim, Hyemin; Mackin, Nancy A.; Maldonado-Báez, Lymarie; Evangelista, Carlos C.; Beaudry, Veronica G.; Dudgeon, Drew D.; Naiman, Daniel Q.; Erdman, Scott E.; Cunningham, Kyle W.

    2011-01-01

    The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca2+ influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca2+ after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis. PMID:21252230

  11. Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

    PubMed

    Stadelmann, Britta; Rufener, Reto; Aeschbacher, Denise; Spiliotis, Markus; Gottstein, Bruno; Hemphill, Andrew

    2016-03-01

    The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.

  12. Whole genomewide linkage screen for neural tube defects reveals regions of interest on chromosomes 7 and 10

    PubMed Central

    Rampersaud, E; Bassuk, A; Enterline, D; George, T; Siegel, D; Melvin, E; Aben, J; Allen, J; Aylsworth, A; Brei, T; Bodurtha, J; Buran, C; Floyd, L; Hammock, P; Iskandar, B; Ito, J; Kessler, J; Lasarsky, N; Mack, P; Mackey, J; McLone, D; Meeropol, E; Mehltretter, L; Mitchell, L; Oakes, W; Nye, J; Powell, C; Sawin, K; Stevenson, R; Walker, M; West, S; Worley, G; Gilbert, J; Speer, M

    2005-01-01

    Neural tube defects (NTDs) are the second most common birth defects (1 in 1000 live births) in the world. Periconceptional maternal folate supplementation reduces NTD risk by 50–70%; however, studies of folate related and other developmental genes in humans have failed to definitively identify a major causal gene for NTD. The aetiology of NTDs remains unknown and both genetic and environmental factors are implicated. We present findings from a microsatellite based screen of 44 multiplex pedigrees ascertained through the NTD Collaborative Group. For the linkage analysis, we defined our phenotype narrowly by considering individuals with a lumbosacral level myelomeningocele as affected, then we expanded the phenotype to include all types of NTDs. Two point parametric analyses were performed using VITESSE and HOMOG. Multipoint parametric and nonparametric analyses were performed using ALLEGRO. Initial results identified chromosomes 7 and 10, both with maximum parametric multipoint lod scores (Mlod) >2.0. Chromosome 7 produced the highest score in the 24 cM interval between D7S3056 and D7S3051 (parametric Mlod 2.45; nonparametric Mlod 1.89). Further investigation demonstrated that results on chromosome 7 were being primarily driven by a single large pedigree (parametric Mlod 2.40). When this family was removed from analysis, chromosome 10 was the most interesting region, with a peak Mlod of 2.25 at D10S1731. Based on mouse human synteny, two candidate genes (Meox2, Twist1) were identified on chromosome 7. A review of public databases revealed three biologically plausible candidates (FGFR2, GFRA1, Pax2) on chromosome 10. The results from this screen provide valuable positional data for prioritisation of candidate gene assessment in future studies of NTDs. PMID:15831595

  13. Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

    PubMed

    Stadelmann, Britta; Rufener, Reto; Aeschbacher, Denise; Spiliotis, Markus; Gottstein, Bruno; Hemphill, Andrew

    2016-03-01

    The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load. PMID:26967740

  14. A Complex Regulatory Network Coordinating Cell Cycles During C. elegans Development Is Revealed by a Genome-Wide RNAi Screen

    PubMed Central

    Roy, Sarah H.; Tobin, David V.; Memar, Nadin; Beltz, Eleanor; Holmen, Jenna; Clayton, Joseph E.; Chiu, Daniel J.; Young, Laura D.; Green, Travis H.; Lubin, Isabella; Liu, Yuying; Conradt, Barbara; Saito, R. Mako

    2014-01-01

    The development and homeostasis of multicellular animals requires precise coordination of cell division and differentiation. We performed a genome-wide RNA interference screen in Caenorhabditis elegans to reveal the components of a regulatory network that promotes developmentally programmed cell-cycle quiescence. The 107 identified genes are predicted to constitute regulatory networks that are conserved among higher animals because almost half of the genes are represented by clear human orthologs. Using a series of mutant backgrounds to assess their genetic activities, the RNA interference clones displaying similar properties were clustered to establish potential regulatory relationships within the network. This approach uncovered four distinct genetic pathways controlling cell-cycle entry during intestinal organogenesis. The enhanced phenotypes observed for animals carrying compound mutations attest to the collaboration between distinct mechanisms to ensure strict developmental regulation of cell cycles. Moreover, we characterized ubc-25, a gene encoding an E2 ubiquitin-conjugating enzyme whose human ortholog, UBE2Q2, is deregulated in several cancers. Our genetic analyses suggested that ubc-25 acts in a linear pathway with cul-1/Cul1, in parallel to pathways employing cki-1/p27 and lin-35/pRb to promote cell-cycle quiescence. Further investigation of the potential regulatory mechanism demonstrated that ubc-25 activity negatively regulates CYE-1/cyclin E protein abundance in vivo. Together, our results show that the ubc-25-mediated pathway acts within a complex network that integrates the actions of multiple molecular mechanisms to control cell cycles during development. PMID:24584095

  15. Focused screening of mitochondrial metabolism reveals a crucial role for a tumor suppressor Hbp1 in ovarian reserve

    PubMed Central

    Dong, Z; Huang, M; Liu, Z; Xie, P; Dong, Y; Wu, X; Qu, Z; Shen, B; Huang, X; Zhang, T; Li, J; Liu, J; Yanase, T; Zhou, C; Xu, Y

    2016-01-01

    Granulosa cells (GCs) are tightly associated with fertility and the fate of ovarian follicles. Mitochondria are the central executers of apoptosis. However, the genetic basis underlying mitochondrial modulation in GCs during the ovarian development is poorly understood. Here, CRISPR/Cas9-mediated genetic screening was used to identify genes conferring mitochondrial metabolism in human GCs. The results uncovered roles for several tumor suppressors, including HBP1, in the augmentation of mitochondrial function. Focused analysis revealed that high-mobility group (HMG)-box transcription factor 1 (Hbp1) levels regulate mitochondrial biogenesis, which is associated with global changes in transcription including Tfam. The systemic or granulosa-specific but not oocyte-specific ablation of Hbp1 promoted follicle growth and oocyte production, and is associated with the reduced apoptotic signals in mouse GCs. Consistent with increased mitochondrial function and attenuated GC apoptosis, the regulation of Hbp1 conferred substantial protection of ovarian reserve. Thus, the results of the present study provide a critical target to understand the control of the reproductive lifespan. PMID:27206316

  16. Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate.

    PubMed

    Pospisilik, J Andrew; Schramek, Daniel; Schnidar, Harald; Cronin, Shane J F; Nehme, Nadine T; Zhang, Xiaoyun; Knauf, Claude; Cani, Patrice D; Aumayr, Karin; Todoric, Jelena; Bayer, Martina; Haschemi, Arvand; Puviindran, Vijitha; Tar, Krisztina; Orthofer, Michael; Neely, G Gregory; Dietzl, Georg; Manoukian, Armen; Funovics, Martin; Prager, Gerhard; Wagner, Oswald; Ferrandon, Dominique; Aberger, Fritz; Hui, Chi-chung; Esterbauer, Harald; Penninger, Josef M

    2010-01-01

    Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. We performed a systems-level genetic dissection of adiposity regulation using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo and revealed hedgehog signaling as the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, we generated fat-specific hedgehog-activation mutant mice. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals.

  17. A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

    PubMed Central

    Jose, Mini; Tollis, Sylvain; Nair, Deepak; Mitteau, Romain; Velours, Christophe; Massoni-Laporte, Aurelie; Royou, Anne; Sibarita, Jean-Baptiste; McCusker, Derek

    2015-01-01

    The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis. PMID:25947137

  18. Virtual screening reveals a viral-like polymerase inhibitor that complexes with the DNA polymerase of Moniliophthora perniciosa.

    PubMed

    Andrade, B S; Souza, C S; Santos, G; Góes-Neto, A

    2016-01-01

    The filamentous fungus Moniliophthora perniciosa is a basidiomycota that causes the witches' broom disease in cocoa trees (Theobroma cacao L.). The mitochondrial DNA polymerase of M. perniciosa (MpmitDNApol) is classified within the B family of DNA polymerases, which can be found in viruses and cellular organelles. Using virtual screening processes, accessing KEGG, PubChem, and ZINC databases, we selected the 27 best putative nucleoside viral-like polymerase inhibitors to test against MpmitDNApol. We used Autodock Vina to perform docking simulations of the selected molecules and to return energy values in several ligand conformations. Then, we used Pymol v1.7.4.4 to check the stereochemistry of chiral carbons, hydrogen bonding receptors, absence or presence of hydrogen, sub and superstructure, numbers of rings, rotatable bonds, and donor groups. We selected the Entecavir Hydrate, a drug used to control hepatitis B; subsequently AMBER 14 was used to describe the behavior of polymerase-entecavir complex after setting up 3500 ps of simulation in water at a temperature of 300 K. From the simulation, a graph of Potential Energy was generated revealing that the ligand remains in the catalytic site after 3500 ps with a final energy of -612,587.4214 kcal/mol. PMID:27323084

  19. Pharmacologic screens reveal metformin that suppresses GRP78-dependent autophagy to enhance the anti-myeloma effect of bortezomib.

    PubMed

    Jagannathan, S; Abdel-Malek, M A Y; Malek, E; Vad, N; Latif, T; Anderson, K C; Driscoll, J J

    2015-11-01

    Although the therapeutic benefit of proteasome inhibition in multiple myeloma remains unchallenged, drug resistance inevitably emerges through mechanisms that remain elusive. Bortezomib provokes unwanted protein accumulation and the endoplasmic reticulum stress to activate the unfolded protein response (UPR) and autophagy as compensatory mechanisms that restore protein homeostasis. High-throughput screens to detect pharmacologics that modulated autophagy to enhance the anti-myeloma effect of bortezomib revealed metformin, a widely used antidiabetic agent with proven efficacy and limited adverse effects. Metformin co-treatment with bortezomib suppressed induction of the critical UPR effector glucose-regulated protein 78 (GRP78) to impair autophagosome formation and enhance apoptosis. Gene expression profiling of newly diagnosed myeloma patient tumors further correlated the hyperexpression of GRP78-encoding HSPA5 with reduced clinical response to bortezomib. The effect of bortezomib was enhanced with metformin co-treatment using myeloma patient tumor cells and the chemoresistant, stem cell-like side population that may contribute to disease recurrence. The relevance of the findings was confirmed in vivo as shown by metformin co-treatment with bortezomib that delayed the growth of myeloma xenotransplants. Taken together, our results suggest that metformin suppresses GRP78, a key driver of bortezomib-induced autophagy, and support the pharmacologic repositioning of metformin to enhance the anti-myeloma benefit of bortezomib.

  20. SCREENING FOR REFERRAL BY A SPORTS PHYSICAL THERAPIST REVEALS AN EFFORT THROMBOSIS IN A COLLEGIATE PITCHER: A CASE REPORT

    PubMed Central

    Pinerola, Jase; Ogle, Karen Craig; Wallmann, Harvey W.

    2016-01-01

    ABSTRACT Background and Purpose Screening for referral, regardless of setting, is the responsibility of all physical therapists. A serious condition that sports physical therapists may encounter is upper extremity (UE) deep venous thrombosis (DVT), which can result in the important and sometimes fatal complication of pulmonary embolism. Case Description A 22 year-old male right-hand dominant collegiate pitcher was referred for physical therapist evaluation and treatment secondary to acute right UE pain and swelling. The athlete described the onset of these symptoms as insidious, denying any form of trauma. The athlete had undergone testing, which included UE Doppler ultrasound of the bilateral UE veins and a computed tomography (CT) scan of the chest without contrast; both of which were deemed negative. He was subsequently diagnosed with thoracic outlet syndrome and referred to the team physical therapist. After examination, the physical therapist hypothesized the athlete was presenting with a possible vascular compromise. Findings leading to this decision were: 1) insidious onset, 2) inability to account for the athlete's pain with ROM, strength, neurological, or provocation testing, 3) significant swelling of the right UE (arm and forearm), 4) increased discomfort with palpation in the supraclavicular region, and 5) history of strenuous UE use. Outcomes The athlete was referred back to the orthopedist. A venogram CT was ordered, which revealed an axillary and subclavian DVT and the presence of venous collaterals. The athlete was referred to a vascular surgeon who performed a right first rib removal. The athlete was able to complete post-operative rehabilitation and successfully return to competitive throwing the following spring. Discussion The delay in the initial diagnosis may have been due to the vague symptomology associated with venous complications and negative findings upon initial diagnostic testing. Conclusion This case report highlights the importance

  1. Perinatal lethal osteogenesis imperfecta.

    PubMed Central

    Cole, W G; Dalgleish, R

    1995-01-01

    Perinatal lethal osteogenesis imperfecta is the result of heterozygous mutations of the COL1A1 and COL1A2 genes that encode the alpha 1(I) and alpha 2(I) chains of type I collagen, respectively. Point mutations resulting in the substitution of Gly residues in Gly-X-Y amino acid triplets of the triple helical domain of the alpha 1(I) or alpha 2(I) chains are the most frequent mutations. They interrupt the repetitive Gly-X-Y structure that is mandatory for the formation of a stable triple helix. Most babies have their own private de novo mutation. However, the recurrence rate is about 7% owing to germline mosaicism in one parent. The mutations act in a dominant negative manner as the mutant pro alpha chains are incorporated into type I procollagen molecules that also contain normal pro alpha chains. The abnormal molecules are poorly secreted, more susceptible to degradation, and impair the formation of the extracellular matrix. The collagen fibres are abnormally organised and mineralisation is impaired. The severity of the clinical phenotype appears to be related to the type of mutation, its location in the alpha chain, the surrounding amino acid sequences, and the level of expression of the mutant allele. Images PMID:7643358

  2. A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens

    PubMed Central

    Koh, Cho Yeow; Kallur Siddaramaiah, Latha; Ranade, Ranae M.; Nguyen, Jasmine; Jian, Tengyue; Zhang, Zhongsheng; Gillespie, J. Robert; Buckner, Frederick S.; Verlinde, Christophe L. M. J.; Fan, Erkang; Hol, Wim G. J.

    2015-01-01

    American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallo­graphically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS. PMID:26249349

  3. A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.

    PubMed

    Koh, Cho Yeow; Siddaramaiah, Latha Kallur; Ranade, Ranae M; Nguyen, Jasmine; Jian, Tengyue; Zhang, Zhongsheng; Gillespie, J Robert; Buckner, Frederick S; Verlinde, Christophe L M J; Fan, Erkang; Hol, Wim G J

    2015-08-01

    American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

  4. A Network of Conserved Synthetic Lethal Interactions for Exploration of Precision Cancer Therapy.

    PubMed

    Srivas, Rohith; Shen, John Paul; Yang, Chih Cheng; Sun, Su Ming; Li, Jianfeng; Gross, Andrew M; Jensen, James; Licon, Katherine; Bojorquez-Gomez, Ana; Klepper, Kristin; Huang, Justin; Pekin, Daniel; Xu, Jia L; Yeerna, Huwate; Sivaganesh, Vignesh; Kollenstart, Leonie; van Attikum, Haico; Aza-Blanc, Pedro; Sobol, Robert W; Ideker, Trey

    2016-08-01

    An emerging therapeutic strategy for cancer is to induce selective lethality in a tumor by exploiting interactions between its driving mutations and specific drug targets. Here we use a multi-species approach to develop a resource of synthetic lethal interactions relevant to cancer therapy. First, we screen in yeast ∼169,000 potential interactions among orthologs of human tumor suppressor genes (TSG) and genes encoding drug targets across multiple genotoxic environments. Guided by the strongest signal, we evaluate thousands of TSG-drug combinations in HeLa cells, resulting in networks of conserved synthetic lethal interactions. Analysis of these networks reveals that interaction stability across environments and shared gene function increase the likelihood of observing an interaction in human cancer cells. Using these rules, we prioritize ∼10(5) human TSG-drug combinations for future follow-up. We validate interactions based on cell and/or patient survival, including topoisomerases with RAD17 and checkpoint kinases with BLM. PMID:27453043

  5. Antimicrobial, antitumor and brine shrimp lethality assay of Ranunculus arvensis L. extracts.

    PubMed

    Bhatti, Muhammad Zeeshan; Ali, Amjad; Saeed, Asma; Saeed, Ahmad; Malik, Salman Akbar

    2015-05-01

    To investigate the antitumor activity, brine shrimp lethality assay, antibacterial and antifungal activity of Methanol Extract (ME), Water Extract (WE), Acetone Extract (AE), Chloroform Extract (CE), Methanol-Water Extract (MWE), Methanol-Acetone Extract (MAE), Methanol-Chloroform Extract (MCE) of Ranunculus arvensis (L.). Antitumor activity was evaluated with Agrobacterium tumefaciens (At10) induced potato disc assay. Cytotoxicity was evaluated with brine shrimp lethality assay. Antibacterial activity was evaluated with six bacterial strains including Escherichia coli, Enterobacter aerogenes, Bordetella bronchiseptica, Klebsiella pneumoniae, Micrococcus luteus and Streptococcus anginosus and antifungal screening was done against five fungal strains including Aspergillus niger, A. flavus, A. fumigates, Fusarium solani and Mucor species by using disc diffusion method. Best antitumor activity was obtained with ME and WE, having highest IC50 values 20.27 ± 1.62 and 93.01 ± 1.33μg/disc. Brine shrimp lethality assay showed LC50 values of AE, MAE and ME were obtained as 384.66 ± 9.42μg/ml, 724.11 ± 8.01μg/ml and 978.7 ±8.01 μg/ml respectively. WE of R. arvensis revealed weak antimicrobial result against the tested microorganisms. On the other hand, the antifungal activity of the plant extracts was found to be insignificant. These findings demonstrate that extracts of R. arvensis possesses significant antitumor activity. Further extensive study is necessary to assess the therapeutic potential of the plant. PMID:26004705

  6. Lethal outcome in xanthogranulomatous endometritis.

    PubMed

    Noack, Frank; Briese, Juliane; Stellmacher, Florian; Hornung, Daniela; Horny, Hans-Peter

    2006-05-01

    Xanthogranulomatous inflammation is rare, mainly involving the kidneys, while primary xanthogranulomatous endometritis (XE) is a very unusual finding, histologically characterized by partial or complete replacement of the mucosa by granulation tissue with an abundance of foamy histiocytes, siderophages and multinucleated giant cells. We present the case of a 69-year-old woman with a short history of abdominal pain and a palpable mass in the pouch of Douglas. Dilatation of the cervix drained a pyometra. Histological examination of the curettage rendered the diagnosis of XE. Microbiological studies revealed enterococcus spp. and Peptostreptococcus magnus. Despite antibiotic treatment the patient died of heart failure due to systemic inflammation. Autopsy confirmed the diagnosis of XE with transmural extension into the peritoneal cavity. Such a lethal course of XE is extraordinary. Proposed causes of XE include obstruction, infection and hemorrhage. Demonstration of enterococcus spp. and P. magnus supports the probable significance of bacteria in the development of XE. Because this condition may mimic malignant disease macroscopically and histologically, knowledge of XE is of major importance for both pathologists and gynecologists. PMID:16725016

  7. Potential lethal and non-lethal effects of predators on dispersal of spider mites.

    PubMed

    Otsuki, Hatsune; Yano, Shuichi

    2014-11-01

    Predators can affect prey dispersal lethally by direct consumption or non-lethally by making prey hesitate to disperse. These lethal and non-lethal effects are detectable only in systems where prey can disperse between multiple patches. However, most studies have drawn their conclusions concerning the ability of predatory mites to suppress spider mites based on observations of their interactions on a single patch or on heavily infested host plants where spider mites could hardly disperse toward intact patches. In these systems, specialist predatory mites that penetrate protective webs produced by spider mites quickly suppress the spider mites, whereas generalist predators that cannot penetrate the webs were ineffective. By using a connected patch system, we revealed that a generalist ant, Pristomyrmex punctatus Mayr (Hymenoptera: Formicidae), effectively prevented dispersal of spider mites, Tetranychus kanzawai Kishida (Acari: Tetranychidae), by directly consuming dispersing individuals. We also revealed that a generalist predatory mite, Euseius sojaensis Ehara (Acari: Phytoseiidae), prevented between-patch dispersal of T. kanzawai by making them hesitate to disperse. In contrast, a specialist phytoseiid predatory mite, Neoseiulus womersleyi Schicha, allowed spider mites to escape an initial patch, increasing the number of colonized patches within the system. Our results suggest that ants and generalist predatory mites can effectively suppress Tetranychus species under some conditions, and should receive more attention as agents for conservation biological control in agroecosystems.

  8. Approaches to Identifying Synthetic Lethal Interactions in Cancer

    PubMed Central

    Thompson, Jordan M.; Nguyen, Quy H.; Singh, Manpreet; Razorenova, Olga V.

    2015-01-01

    Targeting synthetic lethal interactions is a promising new therapeutic approach to exploit specific changes that occur within cancer cells. Multiple approaches to investigate these interactions have been developed and successfully implemented, including chemical, siRNA, shRNA, and CRISPR library screens. Genome-wide computational approaches, such as DAISY, also have been successful in predicting synthetic lethal interactions from both cancer cell lines and patient samples. Each approach has its advantages and disadvantages that need to be considered depending on the cancer type and its molecular alterations. This review discusses these approaches and examines case studies that highlight their use. PMID:26029013

  9. Virtual screening using MTiOpenScreen and PyRx 0,8 revealed ZINC95486216 as a human acetylcholinesterase inhibitor candidate

    NASA Astrophysics Data System (ADS)

    Sulistyo Dwi K., P.; Arindra Trisna, W.; Vindri Catur P., W.; Wijayanti, Erna; Ichsan, Mochammad

    2016-03-01

    One of the efforts to prevent Alzheimer's disease becomes more severe is by inhibiting the activity of Human acetylcholinesterase enzyme (PDB ID: 4BDT). In this study, virtual screening againts 885 natural compounds from AfroDB has been done using MTIOpenScreen and this step has been successful in identifying ZINC15121024 (-12,9) and ZINC95486216 (-12,7) as the top rank compounds. This data then strengthened by the results of second docking step using Autodock software that has been integrated in PyRx 0.8 software. From this stage, ZINC95486216 (-11,3 kcal/mol) is a compound with the most negative binding affinity compared with four Alzheimer's drugs that have been officially used to date including Rivastigmine (-6,3 Kcal/mol), Donepenzil (-7.9 kcal/mol), Galantamine (-8.4 kcal/mol), and Huprine W (-7.3 kcal/mol). In addition, based on the results of the 2D and 3D visualization using LigPlus and PyMol softwares, respectively, known that the five compounds above are equally capable of binding to several amino acids (Trp 286, Phe295, and Tyr341) located in the active site of Human Acetylcholinesterase enzyme.

  10. Proteomic screening of variola virus reveals a unique NF-kappaB inhibitor that is highly conserved among pathogenic orthopoxviruses.

    PubMed

    Mohamed, Mohamed R; Rahman, Masmudur M; Lanchbury, Jerry S; Shattuck, Donna; Neff, Chris; Dufford, Max; van Buuren, Nick; Fagan, Katharine; Barry, Michele; Smith, Scott; Damon, Inger; McFadden, Grant

    2009-06-01

    Identification of the binary interactions between viral and host proteins has become a valuable tool for investigating viral tropism and pathogenesis. Here, we present the first systematic protein interaction screening of the unique variola virus proteome by using yeast 2-hybrid screening against a variety of human cDNA libraries. Several protein-protein interactions were identified, including an interaction between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1 (NF-kappaB1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-kappaB1/p105. Orthologs of G1R are present in a variety of pathogenic orthopoxviruses, but not in vaccinia virus, and expression of any one of these viral proteins blocks NF-kappaB signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses.

  11. Lethal otogenic Candida meningitis.

    PubMed

    Koch, S; Rudel, B; Tietz, H-J

    2004-10-01

    History revealed a chronic obstructive pulmonary condition which had been treated with prednisolone for a long time. There was a raised temperature with further signs of an acute inflammatory underlying disease and internal hydrocephalus. After performing trepanation, the symptoms of raised intercerebral pressure ceased. Candida albicans could be detected microbiologically in the cerebrospinal fluid. There was no pneumonia at the time of admission. Despite instituting immediate intensive care with administration of antibiotics and antimycotics, the patient died 11 days after inpatient admission. Autopsy revealed a C. albicans mycosis originating from the right middle ear with extensive suppurative meningitis, which was the immediate cause of death. Confluent bronchopneumonia had developed in both lower lung lobes at the time of death, but did not show any signs of mycosis and had contributed indirectly to the death of the patient.

  12. Theories of Lethal Mutagenesis: From Error Catastrophe to Lethal Defection.

    PubMed

    Tejero, Héctor; Montero, Francisco; Nuño, Juan Carlos

    2016-01-01

    RNA viruses get extinct in a process called lethal mutagenesis when subjected to an increase in their mutation rate, for instance, by the action of mutagenic drugs. Several approaches have been proposed to understand this phenomenon. The extinction of RNA viruses by increased mutational pressure was inspired by the concept of the error threshold. The now classic quasispecies model predicts the existence of a limit to the mutation rate beyond which the genetic information of the wild type could not be efficiently transmitted to the next generation. This limit was called the error threshold, and for mutation rates larger than this threshold, the quasispecies was said to enter into error catastrophe. This transition has been assumed to foster the extinction of the whole population. Alternative explanations of lethal mutagenesis have been proposed recently. In the first place, a distinction is made between the error threshold and the extinction threshold, the mutation rate beyond which a population gets extinct. Extinction is explained from the effect the mutation rate has, throughout the mutational load, on the reproductive ability of the whole population. Secondly, lethal defection takes also into account the effect of interactions within mutant spectra, which have been shown to be determinant for the understanding the extinction of RNA virus due to an augmented mutational pressure. Nonetheless, some relevant issues concerning lethal mutagenesis are not completely understood yet, as so survival of the flattest, i.e. the development of resistance to lethal mutagenesis by evolving towards mutationally more robust regions of sequence space, or sublethal mutagenesis, i.e., the increase of the mutation rate below the extinction threshold which may boost the adaptability of RNA virus, increasing their ability to develop resistance to drugs (including mutagens). A better design of antiviral therapies will still require an improvement of our knowledge about lethal

  13. Lethal Skeletal Dysplasia in Mice and Humans Lacking the Golgin GMAP-210

    PubMed Central

    Smits, Patrick; Bolton, Andrew D.; Funari, Vincent; Hong, Minh; Boyden, Eric D.; Lu, Lei; Manning, Danielle K.; Dwyer, Noelle D.; Moran, Jennifer L.; Prysak, Mary; Merriman, Barry; Nelson, Stanley F.; Bonafé, Luisa; Superti-Furga, Andrea; Ikegawa, Shiro; Krakow, Deborah; Cohn, Daniel H.; Kirchhausen, Tom; Warman, Matthew L.; Beier, David R.

    2011-01-01

    BACKGROUND Establishing the genetic basis of phenotypes such as skeletal dysplasia in model organisms can provide insights into biologic processes and their role in human disease. METHODS We screened mutagenized mice and observed a neonatal lethal skeletal dysplasia with an autosomal recessive pattern of inheritance. Through genetic mapping and positional cloning, we identified the causative mutation. RESULTS Affected mice had a nonsense mutation in the thyroid hormone receptor interactor 11 gene (Trip11), which encodes the Golgi microtubule-associated protein 210 (GMAP-210); the affected mice lacked this protein. Golgi architecture was disturbed in multiple tissues, including cartilage. Skeletal development was severely impaired, with chondrocytes showing swelling and stress in the endoplasmic reticulum, abnormal cellular differentiation, and increased cell death. Golgi-mediated glycosylation events were altered in fibroblasts and chondrocytes lacking GMAP-210, and these chondrocytes had intracellular accumulation of perlecan, an extracellular matrix protein, but not of type II collagen or aggrecan, two other extracellular matrix proteins. The similarities between the skeletal and cellular phenotypes in these mice and those in patients with achondrogenesis type 1A, a neonatal lethal form of skeletal dysplasia in humans, suggested that achondrogenesis type 1A may be caused by GMAP-210 deficiency. Sequence analysis revealed loss-of-function mutations in the 10 unrelated patients with achondrogenesis type 1A whom we studied. CONCLUSIONS GMAP-210 is required for the efficient glycosylation and cellular transport of multiple proteins. The identification of a mutation affecting GMAP-210 in mice, and then in humans, as the cause of a lethal skeletal dysplasia underscores the value of screening for abnormal phenotypes in model organisms and identifying the causative mutations. PMID:20089971

  14. Identification of Genes Important for Cutaneous Function Revealed by a Large Scale Reverse Genetic Screen in the Mouse

    PubMed Central

    DiTommaso, Tia; Jones, Lynelle K.; Cottle, Denny L.; Gerdin, Anna-Karin; Vancollie, Valerie E.; Watt, Fiona M.; Ramirez-Solis, Ramiro; Bradley, Allan; Steel, Karen P.; Sundberg, John P.; White, Jacqueline K.; Smyth, Ian M.

    2014-01-01

    The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation. PMID:25340873

  15. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A

    PubMed Central

    Wu, Yuanzhong; Zhou, Liwen; Wang, Xin; Lu, Jinping; Zhang, Ruhua; Liang, Xiaoting; Wang, Li; Deng, Wuguo; Zeng, Yi-Xin; Huang, Haojie; Kang, Tiebang

    2016-01-01

    The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1DCAF8 was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability. PMID:27462461

  16. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.

    PubMed

    Wu, Yuanzhong; Zhou, Liwen; Wang, Xin; Lu, Jinping; Zhang, Ruhua; Liang, Xiaoting; Wang, Li; Deng, Wuguo; Zeng, Yi-Xin; Huang, Haojie; Kang, Tiebang

    2016-01-01

    The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1(DCAF8) was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability. PMID:27462461

  17. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.

    PubMed

    Wu, Yuanzhong; Zhou, Liwen; Wang, Xin; Lu, Jinping; Zhang, Ruhua; Liang, Xiaoting; Wang, Li; Deng, Wuguo; Zeng, Yi-Xin; Huang, Haojie; Kang, Tiebang

    2016-01-01

    The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1(DCAF8) was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability.

  18. Syn-lethality: an integrative knowledge base of synthetic lethality towards discovery of selective anticancer therapies.

    PubMed

    Li, Xue-juan; Mishra, Shital K; Wu, Min; Zhang, Fan; Zheng, Jie

    2014-01-01

    Synthetic lethality (SL) is a novel strategy for anticancer therapies, whereby mutations of two genes will kill a cell but mutation of a single gene will not. Therefore, a cancer-specific mutation combined with a drug-induced mutation, if they have SL interactions, will selectively kill cancer cells. While numerous SL interactions have been identified in yeast, only a few have been known in human. There is a pressing need to systematically discover and understand SL interactions specific to human cancer. In this paper, we present Syn-Lethality, the first integrative knowledge base of SL that is dedicated to human cancer. It integrates experimentally discovered and verified human SL gene pairs into a network, associated with annotations of gene function, pathway, and molecular mechanisms. It also includes yeast SL genes from high-throughput screenings which are mapped to orthologous human genes. Such an integrative knowledge base, organized as a relational database with user interface for searching and network visualization, will greatly expedite the discovery of novel anticancer drug targets based on synthetic lethality interactions. The database can be downloaded as a stand-alone Java application.

  19. Syn-Lethality: An Integrative Knowledge Base of Synthetic Lethality towards Discovery of Selective Anticancer Therapies

    PubMed Central

    Li, Xue-juan; Mishra, Shital K.; Wu, Min; Zhang, Fan

    2014-01-01

    Synthetic lethality (SL) is a novel strategy for anticancer therapies, whereby mutations of two genes will kill a cell but mutation of a single gene will not. Therefore, a cancer-specific mutation combined with a drug-induced mutation, if they have SL interactions, will selectively kill cancer cells. While numerous SL interactions have been identified in yeast, only a few have been known in human. There is a pressing need to systematically discover and understand SL interactions specific to human cancer. In this paper, we present Syn-Lethality, the first integrative knowledge base of SL that is dedicated to human cancer. It integrates experimentally discovered and verified human SL gene pairs into a network, associated with annotations of gene function, pathway, and molecular mechanisms. It also includes yeast SL genes from high-throughput screenings which are mapped to orthologous human genes. Such an integrative knowledge base, organized as a relational database with user interface for searching and network visualization, will greatly expedite the discovery of novel anticancer drug targets based on synthetic lethality interactions. The database can be downloaded as a stand-alone Java application. PMID:24864230

  20. Inhibitors of the Metalloproteinase Anthrax Lethal Factor.

    PubMed

    Goldberg, Allison B; Turk, Benjamin E

    2016-01-01

    Bacillus anthracis, a rod shaped, spore forming, gram positive bacteria, is the etiological agent of anthrax. B. anthracis virulence is partly attributable to two secreted bipartite protein toxins, which act inside host cells to disrupt signaling pathways important for host defense against infection. These toxins may also directly contribute to mortality in late stage infection. The zinc-dependent metalloproteinase anthrax lethal factor (LF) is a critical component of one of these protein toxins and a prime target for inhibitor development to produce anthrax therapeutics. Here, we describe recent efforts to identify specific and potent LF inhibitors. Derivatization of peptide substrate analogs bearing zinc-binding groups has produced potent and specific LF inhibitors, and X-ray crystallography of LFinhibitor complexes has provided insight into features required for high affinity binding. Novel inhibitor scaffolds have been identified through several approaches, including fragment-based drug discovery, virtual screening, and highthroughput screening of diverse compound libraries. Lastly, efforts to discover LF inhibitors have led to the development of new screening strategies, such as the use of full-length proteins as substrates, that may prove useful for other proteases as well. Overall, these efforts have led to a collection of chemically and mechanistically diverse molecules capable of inhibiting LF activity in vitro and in cells, as well as in animal models of anthrax infection. PMID:27072692

  1. In vitro screening of compounds against laboratory and field isolates of human hookworm reveals quantitative differences in anthelmintic susceptibility.

    PubMed

    Treger, Rebecca S; Otchere, Joseph; Keil, Martin F; Quagraine, Josephine E; Rai, Ganesha; Mott, Bryan T; Humphries, Debbie L; Wilson, Michael; Cappello, Michael; Vermeire, Jon J

    2014-01-01

    A panel of 80 compounds was screened for anthelmintic activity against a laboratory strain of Ancylostoma ceylanicum and field isolates of hookworm obtained from school children in the Kintampo North District of the Brong Ahafo Region of Ghana. Although the laboratory strain of A. ceylanicum was more susceptible to the compounds tested than the field isolates of hookworm, a twofold increase in compound concentration resulted in comparable egg hatch percent inhibition for select compounds. These data provide evidence that the efficacy of anthelmintic compounds may be species-dependent and that field and laboratory strains of hookworm differ in their sensitivities to the anthelmintics tested. These data also suggest that both compound concentration and hookworm species must be considered when screening to identify novel anthelmintic compounds.

  2. In vitro Screening of Compounds against Laboratory and Field Isolates of Human Hookworm Reveals Quantitative Differences in Anthelmintic Susceptibility

    PubMed Central

    Treger, Rebecca S.; Otchere, Joseph; Keil, Martin F.; Quagraine, Josephine E.; Rai, Ganesha; Mott, Bryan T.; Humphries, Debbie L.; Wilson, Michael; Cappello, Michael; Vermeire, Jon J.

    2014-01-01

    A panel of 80 compounds was screened for anthelmintic activity against a laboratory strain of Ancylostoma ceylanicum and field isolates of hookworm obtained from school children in the Kintampo North District of the Brong Ahafo Region of Ghana. Although the laboratory strain of A. ceylanicum was more susceptible to the compounds tested than the field isolates of hookworm, a twofold increase in compound concentration resulted in comparable egg hatch percent inhibition for select compounds. These data provide evidence that the efficacy of anthelmintic compounds may be species-dependent and that field and laboratory strains of hookworm differ in their sensitivities to the anthelmintics tested. These data also suggest that both compound concentration and hookworm species must be considered when screening to identify novel anthelmintic compounds. PMID:24297811

  3. Cofactor-independent phosphoglycerate mutase from nematodes has limited druggability, as revealed by two high-throughput screens.

    PubMed

    Crowther, Gregory J; Booker, Michael L; He, Min; Li, Ting; Raverdy, Sylvine; Novelli, Jacopo F; He, Panqing; Dale, Natalie R G; Fife, Amy M; Barker, Robert H; Kramer, Martin L; Van Voorhis, Wesley C; Carlow, Clotilde K S; Wang, Ming-Wei

    2014-01-01

    Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z'-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a "druggability paradox" of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data.

  4. Cofactor-Independent Phosphoglycerate Mutase from Nematodes Has Limited Druggability, as Revealed by Two High-Throughput Screens

    PubMed Central

    Crowther, Gregory J.; Booker, Michael L.; He, Min; Li, Ting; Raverdy, Sylvine; Novelli, Jacopo F.; He, Panqing; Dale, Natalie R. G.; Fife, Amy M.; Barker, Robert H.; Kramer, Martin L.; Van Voorhis, Wesley C.; Carlow, Clotilde K. S.; Wang, Ming-Wei

    2014-01-01

    Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z′-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a “druggability paradox” of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data. PMID:24416464

  5. High throughput kinase inhibitor screens reveal TRB3 and MAPK-ERK/TGFβ pathways as fundamental Notch regulators in breast cancer.

    PubMed

    Izrailit, Julia; Berman, Hal K; Datti, Alessandro; Wrana, Jeffrey L; Reedijk, Michael

    2013-01-29

    Expression of the Notch ligand Jagged 1 (JAG1) and Notch activation promote poor-prognosis in breast cancer. We used high throughput screens to identify elements responsible for Notch activation in this context. Chemical kinase inhibitor and kinase-specific small interfering RNA libraries were screened in a breast cancer cell line engineered to report Notch. Pathway analyses revealed MAPK-ERK signaling to be the predominant JAG1/Notch regulator and this was supported by gene set enrichment analyses in 51 breast cancer cell lines. In accordance with the chemical screen, kinome small interfering RNA high throughput screens identified Tribbles homolog 3 (TRB3), a known regulator of MAPK-ERK, among the most significant hits. We demonstrate that TRB3 is a master regulator of Notch through the MAPK-ERK and TGFβ pathways. Complementary in vitro and in vivo studies underscore the importance of TRB3 for tumor growth. These data demonstrate a dominant role for TRB3 and MAPK-ERK/TGFβ pathways as Notch regulators in breast cancer, establishing TRB3 as a potential therapeutic target.

  6. Induction of Human iPSC-Derived Cardiomyocyte Proliferation Revealed by Combinatorial Screening in High Density Microbioreactor Arrays

    PubMed Central

    Titmarsh, Drew M.; Glass, Nick R.; Mills, Richard J.; Hidalgo, Alejandro; Wolvetang, Ernst J.; Porrello, Enzo R.; Hudson, James E.; Cooper-White, Justin J.

    2016-01-01

    Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways, however, it remains unclear as to what extent these pathways can be exploited, either individually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro, but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, we developed a high-density microbioreactor array (HDMA) – a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt, Hedgehog, IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67, and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration. PMID:27097795

  7. Proteomic screening of variola virus reveals a unique NF-κB inhibitor that is highly conserved among pathogenic orthopoxviruses

    PubMed Central

    Mohamed, Mohamed R.; Rahman, Masmudur M.; Lanchbury, Jerry S.; Shattuck, Donna; Neff, Chris; Dufford, Max; van Buuren, Nick; Fagan, Katharine; Barry, Michele; Smith, Scott; Damon, Inger; McFadden, Grant

    2009-01-01

    Identification of the binary interactions between viral and host proteins has become a valuable tool for investigating viral tropism and pathogenesis. Here, we present the first systematic protein interaction screening of the unique variola virus proteome by using yeast 2-hybrid screening against a variety of human cDNA libraries. Several protein–protein interactions were identified, including an interaction between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1 (NF-κB1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-κB1/p105. Orthologs of G1R are present in a variety of pathogenic orthopoxviruses, but not in vaccinia virus, and expression of any one of these viral proteins blocks NF-κB signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses. PMID:19451633

  8. The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening.

    PubMed

    Chatterjee, Kaushik; Lin-Gibson, Sheng; Wallace, William E; Parekh, Sapun H; Lee, Young Jong; Cicerone, Marcus T; Young, Marian F; Simon, Carl G

    2010-07-01

    Cells are known to sense and respond to the physical properties of their environment and those of tissue scaffolds. Optimizing these cell-material interactions is critical in tissue engineering. In this work, a simple and inexpensive combinatorial platform was developed to rapidly screen three-dimensional (3D) tissue scaffolds and was applied to screen the effect of scaffold properties for tissue engineering of bone. Differentiation of osteoblasts was examined in poly(ethylene glycol) hydrogel gradients spanning a 30-fold range in compressive modulus ( approximately 10 kPa to approximately 300 kPa). Results demonstrate that material properties (gel stiffness) of scaffolds can be leveraged to induce cell differentiation in 3D culture as an alternative to biochemical cues such as soluble supplements, immobilized biomolecules and vectors, which are often expensive, labile and potentially carcinogenic. Gel moduli of approximately 225 kPa and higher enhanced osteogenesis. Furthermore, it is proposed that material-induced cell differentiation can be modulated to engineer seamless tissue interfaces between mineralized bone tissue and softer tissues such as ligaments and tendons. This work presents a combinatorial method to screen biological response to 3D hydrogel scaffolds that more closely mimics the 3D environment experienced by cells in vivo.

  9. A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle

    PubMed Central

    Chockalingam, Karuppiah; Simeon, Rudo L.; Rice, Charles M.; Chen, Zhilei

    2010-01-01

    The hepatitis C virus (HCV) life cycle involves multiple steps, but most current drug candidates target only viral replication. The inability to systematically discover inhibitors targeting multiple steps of the HCV life cycle has hampered antiviral development. We present a simple screen for HCV antivirals based on the alleviation of HCV-mediated cytopathic effect in an engineered cell line—n4mBid. This approach obviates the need for a secondary screen to avoid cytotoxic false-positive hits. Application of our screen to 1280 compounds, many in clinical trials or approved for therapeutic use, yielded >200 hits. Of the 55 leading hits, 47 inhibited one or more aspects of the HCV life cycle by >40%. Six compounds blocked HCV entry to levels similar to an antibody (JS-81) targeting the HCV entry receptor CD81. Seven hits inhibited HCV replication and/or infectious virus production by >100-fold, with one (quinidine) inhibiting infectious virus production by 450-fold relative to HCV replication levels. This approach is simple and inexpensive and should enable the rapid discovery of new classes of HCV life cycle inhibitors. PMID:20142494

  10. A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

    PubMed Central

    Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

    2016-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

  11. A systematic High-Content Screening microscopy approach reveals key roles for Rab33b, OATL1 and Myo6 in nanoparticle trafficking in HeLa cells

    PubMed Central

    Panarella, Angela; Bexiga, Mariana G.; Galea, George; O’ Neill, Elaine D.; Salvati, Anna; Dawson, Kenneth A.; Simpson, Jeremy C.

    2016-01-01

    Synthetic nanoparticles are promising tools for imaging and drug delivery; however the molecular details of cellular internalization and trafficking await full characterization. Current knowledge suggests that following endocytosis most nanoparticles pass from endosomes to lysosomes. In order to design effective drug delivery strategies that can use the endocytic pathway, or by-pass lysosomal accumulation, a comprehensive understanding of nanoparticle uptake and trafficking mechanisms is therefore fundamental. Here we describe and apply an RNA interference-based high-content screening microscopy strategy to assess the intracellular trafficking of fluorescently-labeled polystyrene nanoparticles in HeLa cells. We screened a total of 408 genes involved in cytoskeleton and membrane function, revealing roles for myosin VI, Rab33b and OATL1 in this process. This work provides the first systematic large-scale quantitative assessment of the proteins responsible for nanoparticle trafficking in cells, paving the way for subsequent genome-wide studies. PMID:27374232

  12. [The "lethal white foal" syndrome].

    PubMed

    Blendinger, C; Müller, G; Bostedt, H

    1994-06-01

    The lethal white foal syndrome (congenital intestinal aganglionosis) was diagnosed by history, clinical signs and pathological findings in a female foal, born in March 1992, that was an offspring of two overo-spotted paint horses. The syndrome is a congenital innervation defect of the gastrointestinal tract. A literature review of this condition, relatively unknown in Germany, is given.

  13. Cell-based screen for altered nuclear phenotypes reveals senescence progression in polyploid cells after Aurora kinase B inhibition.

    PubMed

    Sadaie, Mahito; Dillon, Christian; Narita, Masako; Narita, Masashi; Young, Andrew R J; Cairney, Claire J; Godwin, Lauren S; Torrance, Christopher J; Bennett, Dorothy C; Keith, W Nicol; Narita, Masashi

    2015-09-01

    Cellular senescence is a widespread stress response and is widely considered to be an alternative cancer therapeutic goal. Unlike apoptosis, senescence is composed of a diverse set of subphenotypes, depending on which of its associated effector programs are engaged. Here we establish a simple and sensitive cell-based prosenescence screen with detailed validation assays. We characterize the screen using a focused tool compound kinase inhibitor library. We identify a series of compounds that induce different types of senescence, including a unique phenotype associated with irregularly shaped nuclei and the progressive accumulation of G1 tetraploidy in human diploid fibroblasts. Downstream analyses show that all of the compounds that induce tetraploid senescence inhibit Aurora kinase B (AURKB). AURKB is the catalytic component of the chromosome passenger complex, which is involved in correct chromosome alignment and segregation, the spindle assembly checkpoint, and cytokinesis. Although aberrant mitosis and senescence have been linked, a specific characterization of AURKB in the context of senescence is still required. This proof-of-principle study suggests that our protocol is capable of amplifying tetraploid senescence, which can be observed in only a small population of oncogenic RAS-induced senescence, and provides additional justification for AURKB as a cancer therapeutic target.

  14. Cell-based screen for altered nuclear phenotypes reveals senescence progression in polyploid cells after Aurora kinase B inhibition

    PubMed Central

    Sadaie, Mahito; Dillon, Christian; Narita, Masashi; Young, Andrew R. J.; Cairney, Claire J.; Godwin, Lauren S.; Torrance, Christopher J.; Bennett, Dorothy C.; Keith, W. Nicol; Narita, Masashi

    2015-01-01

    Cellular senescence is a widespread stress response and is widely considered to be an alternative cancer therapeutic goal. Unlike apoptosis, senescence is composed of a diverse set of subphenotypes, depending on which of its associated effector programs are engaged. Here we establish a simple and sensitive cell-based prosenescence screen with detailed validation assays. We characterize the screen using a focused tool compound kinase inhibitor library. We identify a series of compounds that induce different types of senescence, including a unique phenotype associated with irregularly shaped nuclei and the progressive accumulation of G1 tetraploidy in human diploid fibroblasts. Downstream analyses show that all of the compounds that induce tetraploid senescence inhibit Aurora kinase B (AURKB). AURKB is the catalytic component of the chromosome passenger complex, which is involved in correct chromosome alignment and segregation, the spindle assembly checkpoint, and cytokinesis. Although aberrant mitosis and senescence have been linked, a specific characterization of AURKB in the context of senescence is still required. This proof-of-principle study suggests that our protocol is capable of amplifying tetraploid senescence, which can be observed in only a small population of oncogenic RAS-induced senescence, and provides additional justification for AURKB as a cancer therapeutic target. PMID:26133385

  15. Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A

    PubMed Central

    Hubert, Christopher G.; Bradley, Robert K.; Ding, Yu; Toledo, Chad M.; Herman, Jacob; Skutt-Kakaria, Kyobi; Girard, Emily J.; Davison, Jerry; Berndt, Jason; Corrin, Philip; Hardcastle, Justin; Basom, Ryan; Delrow, Jeffery J.; Webb, Thomas; Pollard, Steven M.; Lee, Jeongwu; Olson, James M.; Paddison, Patrick J.

    2013-01-01

    To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3′ splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies. PMID:23651857

  16. Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A.

    PubMed

    Hubert, Christopher G; Bradley, Robert K; Ding, Yu; Toledo, Chad M; Herman, Jacob; Skutt-Kakaria, Kyobi; Girard, Emily J; Davison, Jerry; Berndt, Jason; Corrin, Philip; Hardcastle, Justin; Basom, Ryan; Delrow, Jeffery J; Webb, Thomas; Pollard, Steven M; Lee, Jeongwu; Olson, James M; Paddison, Patrick J

    2013-05-01

    To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies. PMID:23651857

  17. A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells

    PubMed Central

    Gowen, Benjamin G; Chim, Bryan; Marceau, Caleb D; Greene, Trever T; Burr, Patrick; Gonzalez, Jeanmarie R; Hesser, Charles R; Dietzen, Peter A; Russell, Teal; Iannello, Alexandre; Coscoy, Laurent; Sentman, Charles L; Carette, Jan E; Muljo, Stefan A; Raulet, David H

    2015-01-01

    Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger. DOI: http://dx.doi.org/10.7554/eLife.08474.001 PMID:26565589

  18. Hidden drivers of low-dose pharmaceutical pollutant mixtures revealed by the novel GSA-QHTS screening method

    PubMed Central

    Rodea-Palomares, Ismael; Gonzalez-Pleiter, Miguel; Gonzalo, Soledad; Rosal, Roberto; Leganes, Francisco; Sabater, Sergi; Casellas, Maria; Muñoz-Carpena, Rafael; Fernández-Piñas, Francisca

    2016-01-01

    The ecological impacts of emerging pollutants such as pharmaceuticals are not well understood. The lack of experimental approaches for the identification of pollutant effects in realistic settings (that is, low doses, complex mixtures, and variable environmental conditions) supports the widespread perception that these effects are often unpredictable. To address this, we developed a novel screening method (GSA-QHTS) that couples the computational power of global sensitivity analysis (GSA) with the experimental efficiency of quantitative high-throughput screening (QHTS). We present a case study where GSA-QHTS allowed for the identification of the main pharmaceutical pollutants (and their interactions), driving biological effects of low-dose complex mixtures at the microbial population level. The QHTS experiments involved the integrated analysis of nearly 2700 observations from an array of 180 unique low-dose mixtures, representing the most complex and data-rich experimental mixture effect assessment of main pharmaceutical pollutants to date. An ecological scaling-up experiment confirmed that this subset of pollutants also affects typical freshwater microbial community assemblages. Contrary to our expectations and challenging established scientific opinion, the bioactivity of the mixtures was not predicted by the null mixture models, and the main drivers that were identified by GSA-QHTS were overlooked by the current effect assessment scheme. Our results suggest that current chemical effect assessment methods overlook a substantial number of ecologically dangerous chemical pollutants and introduce a new operational framework for their systematic identification. PMID:27617294

  19. Hidden drivers of low-dose pharmaceutical pollutant mixtures revealed by the novel GSA-QHTS screening method.

    PubMed

    Rodea-Palomares, Ismael; Gonzalez-Pleiter, Miguel; Gonzalo, Soledad; Rosal, Roberto; Leganes, Francisco; Sabater, Sergi; Casellas, Maria; Muñoz-Carpena, Rafael; Fernández-Piñas, Francisca

    2016-09-01

    The ecological impacts of emerging pollutants such as pharmaceuticals are not well understood. The lack of experimental approaches for the identification of pollutant effects in realistic settings (that is, low doses, complex mixtures, and variable environmental conditions) supports the widespread perception that these effects are often unpredictable. To address this, we developed a novel screening method (GSA-QHTS) that couples the computational power of global sensitivity analysis (GSA) with the experimental efficiency of quantitative high-throughput screening (QHTS). We present a case study where GSA-QHTS allowed for the identification of the main pharmaceutical pollutants (and their interactions), driving biological effects of low-dose complex mixtures at the microbial population level. The QHTS experiments involved the integrated analysis of nearly 2700 observations from an array of 180 unique low-dose mixtures, representing the most complex and data-rich experimental mixture effect assessment of main pharmaceutical pollutants to date. An ecological scaling-up experiment confirmed that this subset of pollutants also affects typical freshwater microbial community assemblages. Contrary to our expectations and challenging established scientific opinion, the bioactivity of the mixtures was not predicted by the null mixture models, and the main drivers that were identified by GSA-QHTS were overlooked by the current effect assessment scheme. Our results suggest that current chemical effect assessment methods overlook a substantial number of ecologically dangerous chemical pollutants and introduce a new operational framework for their systematic identification.

  20. Hidden drivers of low-dose pharmaceutical pollutant mixtures revealed by the novel GSA-QHTS screening method

    PubMed Central

    Rodea-Palomares, Ismael; Gonzalez-Pleiter, Miguel; Gonzalo, Soledad; Rosal, Roberto; Leganes, Francisco; Sabater, Sergi; Casellas, Maria; Muñoz-Carpena, Rafael; Fernández-Piñas, Francisca

    2016-01-01

    The ecological impacts of emerging pollutants such as pharmaceuticals are not well understood. The lack of experimental approaches for the identification of pollutant effects in realistic settings (that is, low doses, complex mixtures, and variable environmental conditions) supports the widespread perception that these effects are often unpredictable. To address this, we developed a novel screening method (GSA-QHTS) that couples the computational power of global sensitivity analysis (GSA) with the experimental efficiency of quantitative high-throughput screening (QHTS). We present a case study where GSA-QHTS allowed for the identification of the main pharmaceutical pollutants (and their interactions), driving biological effects of low-dose complex mixtures at the microbial population level. The QHTS experiments involved the integrated analysis of nearly 2700 observations from an array of 180 unique low-dose mixtures, representing the most complex and data-rich experimental mixture effect assessment of main pharmaceutical pollutants to date. An ecological scaling-up experiment confirmed that this subset of pollutants also affects typical freshwater microbial community assemblages. Contrary to our expectations and challenging established scientific opinion, the bioactivity of the mixtures was not predicted by the null mixture models, and the main drivers that were identified by GSA-QHTS were overlooked by the current effect assessment scheme. Our results suggest that current chemical effect assessment methods overlook a substantial number of ecologically dangerous chemical pollutants and introduce a new operational framework for their systematic identification.

  1. High-throughput functional screening reveals low frequency of antibiotic resistance genes in DNA recovered from the Upper Mississippi River.

    PubMed

    Staley, Christopher; Gould, Trevor J; Wang, Ping; Phillips, Jane; Cotner, James B; Sadowsky, Michael J

    2015-09-01

    In this study, we determined the frequency of antibiotic resistance genes (ARGs) in the Upper Mississippi River using a high-throughput, functional, metagenomic screening procedure. Fosmid libraries containing ∼10,000 clones were screened for resistance to ampicillin, cephalothin, kanamycin, and tetracycline. We hypothesized that nutrient concentrations, land cover type, and taxonomic community composition may select for ARGs. Resistance to ampicillin, cephalothin, and kanamycin was low (<1.00%), and no resistance to tetracycline was detected. Ammonium and total dissolved solids (TDS) concentrations were correlated with kanamycin and cephalothin resistances (r=0.617 and -0.449, P=0.002 and 0.036, respectively). Cephalothin resistance was also positively correlated with the percentage of forested land cover (r=0.444, P=0.039). Only the candidate division OD1, among 35 phyla identified, was correlated with ampicillin resistance (r=0.456, P=0.033), suggesting that minority members of the community may be responsible for dissemination of ARGs in this ecosystem. Results of this study suggest that ammonium and TDS may be involved in a complex selection process for ARGs. Furthermore, we suggest that minority species, potentially contributed in low numbers from sediment and biofilm reservoirs, may be the primary carriers of ARGs in this riverine system. PMID:26322755

  2. A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells.

    PubMed

    Gowen, Benjamin G; Chim, Bryan; Marceau, Caleb D; Greene, Trever T; Burr, Patrick; Gonzalez, Jeanmarie R; Hesser, Charles R; Dietzen, Peter A; Russell, Teal; Iannello, Alexandre; Coscoy, Laurent; Sentman, Charles L; Carette, Jan E; Muljo, Stefan A; Raulet, David H

    2015-01-01

    Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.

  3. Screening for Active Small Molecules in Mitochondrial Complex I Deficient Patient's Fibroblasts, Reveals AICAR as the Most Beneficial Compound

    PubMed Central

    Weissman, Sarah; Link, Gabriela; Wikstrom, Jakob D.; Saada, Ann

    2011-01-01

    Congenital deficiency of the mitochondrial respiratory chain complex I (CI) is a common defect of oxidative phosphorylation (OXPHOS). Despite major advances in the biochemical and molecular diagnostics and the deciphering of CI structure, function assembly and pathomechanism, there is currently no satisfactory cure for patients with mitochondrial complex I defects. Small molecules provide one feasible therapeutic option, however their use has not been systematically evaluated using a standardized experimental system. In order to evaluate potentially therapeutic compounds, we set up a relatively simple system measuring different parameters using only a small amount of patient's fibroblasts, in glucose free medium, where growth is highly OXPOS dependent. Ten different compounds were screened using fibroblasts derived from seven CI patients, harboring different mutations. 5-Aminoimidazole-4-carboxamide ribotide (AICAR) was found to be the most beneficial compound improving growth and ATP content while decreasing ROS production. AICAR also increased mitochondrial biogenesis without altering mitochondrial membrane potential (Δψ). Fluorescence microscopy data supported increased mitochondrial biogenesis and activation of the AMP activated protein kinase (AMPK). Other compounds such as; bezafibrate and oltipraz were rated as favorable while polyphenolic phytochemicals (resverastrol, grape seed extract, genistein and epigallocatechin gallate) were found not significant or detrimental. Although the results have to be verified by more thorough investigation of additional OXPHOS parameters, preliminary rapid screening of potential therapeutic compounds in individual patient's fibroblasts could direct and advance personalized medical treatment. PMID:22046392

  4. High content screening of a kinase-focused library reveals compounds broadly-active against dengue viruses.

    PubMed

    Cruz, Deu John M; Koishi, Andrea Cristine; Taniguchi, Juliana Bosso; Li, Xiaolan; Milan Bonotto, Rafaela; No, Joo Hwan; Kim, Keum Hyun; Baek, Sungmin; Kim, Hee Young; Windisch, Marc Peter; Pamplona Mosimann, Ana Luiza; de Borba, Luana; Liuzzi, Michel; Hansen, Michael Adsetts Edberg; Duarte dos Santos, Claudia Nunes; Freitas-Junior, Lucio Holanda

    2013-01-01

    Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.

  5. Hidden drivers of low-dose pharmaceutical pollutant mixtures revealed by the novel GSA-QHTS screening method.

    PubMed

    Rodea-Palomares, Ismael; Gonzalez-Pleiter, Miguel; Gonzalo, Soledad; Rosal, Roberto; Leganes, Francisco; Sabater, Sergi; Casellas, Maria; Muñoz-Carpena, Rafael; Fernández-Piñas, Francisca

    2016-09-01

    The ecological impacts of emerging pollutants such as pharmaceuticals are not well understood. The lack of experimental approaches for the identification of pollutant effects in realistic settings (that is, low doses, complex mixtures, and variable environmental conditions) supports the widespread perception that these effects are often unpredictable. To address this, we developed a novel screening method (GSA-QHTS) that couples the computational power of global sensitivity analysis (GSA) with the experimental efficiency of quantitative high-throughput screening (QHTS). We present a case study where GSA-QHTS allowed for the identification of the main pharmaceutical pollutants (and their interactions), driving biological effects of low-dose complex mixtures at the microbial population level. The QHTS experiments involved the integrated analysis of nearly 2700 observations from an array of 180 unique low-dose mixtures, representing the most complex and data-rich experimental mixture effect assessment of main pharmaceutical pollutants to date. An ecological scaling-up experiment confirmed that this subset of pollutants also affects typical freshwater microbial community assemblages. Contrary to our expectations and challenging established scientific opinion, the bioactivity of the mixtures was not predicted by the null mixture models, and the main drivers that were identified by GSA-QHTS were overlooked by the current effect assessment scheme. Our results suggest that current chemical effect assessment methods overlook a substantial number of ecologically dangerous chemical pollutants and introduce a new operational framework for their systematic identification. PMID:27617294

  6. A direct pre-screen for marine bacteria producing compounds inhibiting quorum sensing reveals diverse planktonic bacteria that are bioactive.

    PubMed

    Linthorne, Jamie S; Chang, Barbara J; Flematti, Gavin R; Ghisalberti, Emilio L; Sutton, David C

    2015-02-01

    A promising new strategy in antibacterial research is inhibition of the bacterial communication system termed quorum sensing. In this study, a novel and rapid pre-screening method was developed to detect the production of chemical inhibitors of this system (quorum-quenching compounds) by bacteria isolated from marine and estuarine waters. This method involves direct screening of mixed populations on an agar plate, facilitating specific isolation of bioactive colonies. The assay showed that between 4 and 46 % of culturable bacteria from various samples were bioactive, and of the 95 selectively isolated bacteria, 93.7 % inhibited Vibrio harveyi bioluminescence without inhibiting growth, indicating potential production of quorum-quenching compounds. Of the active isolates, 21 % showed further activity against quorum-sensing-regulated pigment production by Serratia marcescens. The majority of bioactive isolates were identified by 16S ribosomal DNA (rDNA) amplification and sequencing as belonging to the genera Vibrio and Pseudoalteromonas. Extracts of two strongly bioactive Pseudoalteromonas isolates (K1 and B2) were quantitatively assessed for inhibition of growth and quorum-sensing-regulated processes in V. harveyi, S. marcescens and Chromobacterium violaceum. Extracts of the isolates reduced V. harveyi bioluminescence by as much as 98 % and C. violaceum pigment production by 36 % at concentrations which had no adverse effect on growth. The activity found in the extracts indicated that the isolates may produce quorum-quenching compounds. This study further supports the suggestion that quorum quenching may be a common attribute among culturable planktonic marine and estuarine bacteria.

  7. High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

    PubMed Central

    Li, Xiaolan; Milan Bonotto, Rafaela; No, Joo Hwan; Kim, Keum Hyun; Baek, Sungmin; Kim, Hee Young; Windisch, Marc Peter; Pamplona Mosimann, Ana Luiza; de Borba, Luana; Liuzzi, Michel; Hansen, Michael Adsetts Edberg; Nunes Duarte dos Santos, Claudia; Freitas-Junior, Lucio Holanda

    2013-01-01

    Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates. PMID:23437413

  8. The Rorschach Suicide Constellation: assessing various degrees of lethality.

    PubMed

    Fowler, J C; Piers, C; Hilsenroth, M J; Holdwick, D J; Padawer, J R

    2001-04-01

    In this article we examine the relation between the Rorschach Comprehensive System's Suicide Constellation (S-CON; Exner, 1993; Exner & Wiley, 1977) and lethality of suicide attempts during the course of patients' hospitalization at the Austen Riggs Center (Stockbridge, MA). Patient records were rated as nonsuicidal (n = 37), parasuicidal (n = 37), or near-lethal (n = 30) based on the presence and lethality of self-destructive acts. Diagnostic efficiency statistics utilizing a cutoff score of 7 or more positive indicators successfully predicted which patients would engage in near-lethal suicidal activity relative to parasuicidal patients (overall correct classification rate [OCC] = .79), nonsuicidal inpatients (OCC = .79), and college students (OCC = .89). Although these predictions were influenced by relatively high base rates in the hospital population (14.5%), base rate estimates were calculated for other hypothetical populations revealing different prediction estimates that should be considered when judging the relative efficacy of the S-CON. Logistic regression analysis revealed that an S-CON score of 7 or more was the sole predictor of near-lethal suicide attempts among 9 psychiatric and demographic variables.

  9. The Rorschach Suicide Constellation: assessing various degrees of lethality.

    PubMed

    Fowler, J C; Piers, C; Hilsenroth, M J; Holdwick, D J; Padawer, J R

    2001-04-01

    In this article we examine the relation between the Rorschach Comprehensive System's Suicide Constellation (S-CON; Exner, 1993; Exner & Wiley, 1977) and lethality of suicide attempts during the course of patients' hospitalization at the Austen Riggs Center (Stockbridge, MA). Patient records were rated as nonsuicidal (n = 37), parasuicidal (n = 37), or near-lethal (n = 30) based on the presence and lethality of self-destructive acts. Diagnostic efficiency statistics utilizing a cutoff score of 7 or more positive indicators successfully predicted which patients would engage in near-lethal suicidal activity relative to parasuicidal patients (overall correct classification rate [OCC] = .79), nonsuicidal inpatients (OCC = .79), and college students (OCC = .89). Although these predictions were influenced by relatively high base rates in the hospital population (14.5%), base rate estimates were calculated for other hypothetical populations revealing different prediction estimates that should be considered when judging the relative efficacy of the S-CON. Logistic regression analysis revealed that an S-CON score of 7 or more was the sole predictor of near-lethal suicide attempts among 9 psychiatric and demographic variables. PMID:11393464

  10. High-Throughput 3D Screening Reveals Differences in Drug Sensitivities between Culture Models of JIMT1 Breast Cancer Cells

    PubMed Central

    Fey, Vidal; Mpindi, John-Patrick; Kleivi Sahlberg, Kristine; Kallioniemi, Olli; Perälä, Merja

    2013-01-01

    The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional monolayers on plastic. However, many cellular features are impaired in these artificial conditions, and large changes in gene expression compared to tumors have been reported. Three-dimensional cell culture models have become increasingly popular and are suggested to be better models than two-dimensional monolayers due to improved cell-to-cell contact and structures that resemble in vivo architecture. The aim of this study was to develop a simple high-throughput three-dimensional drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in two dimensions, in poly(2-hydroxyethyl methacrylate) induced anchorage-independent three-dimensional models, and in Matrigel three-dimensional cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating, or they were allowed to grow in three-dimensional cultures for 4 days before the drug treatment. Large variations in drug responses were observed between the models indicating that comparisons of culture model-influenced drug sensitivities cannot be made based on the effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in two-dimensional cultures, while the responses of cells grown in poly(2-hydroxyethyl methacrylate) resembled those of the two-dimensional cultures. Furthermore, comparing the gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study, we also suggest that three-dimensional cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode

  11. Functional genomic screening reveals splicing of the EWS-FLI1 fusion transcript as a vulnerability in Ewing sarcoma

    PubMed Central

    Grohar, Patrick J.; Kim, Suntae; Rangel Rivera, Guillermo O.; Sen, Nirmalya; Haddock, Sara; Harlow, Matt L.; Maloney, Nichole K.; Zhu, Jack; O’Neill, Maura; Jones, Tamara L.; Huppi, Konrad; Grandin, Magdalena; Gehlhaus, Kristen; Klumpp-Thomas, Carleen A.; Buehler, Eugen; Helman, Lee J.; Martin, Scott E.; Caplen, Natasha J.

    2016-01-01

    Summary Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss-of-function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1 driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells. PMID:26776507

  12. Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma.

    PubMed

    Grohar, Patrick J; Kim, Suntae; Rangel Rivera, Guillermo O; Sen, Nirmalya; Haddock, Sara; Harlow, Matt L; Maloney, Nichole K; Zhu, Jack; O'Neill, Maura; Jones, Tamara L; Huppi, Konrad; Grandin, Magdalena; Gehlhaus, Kristen; Klumpp-Thomas, Carleen A; Buehler, Eugen; Helman, Lee J; Martin, Scott E; Caplen, Natasha J

    2016-01-26

    Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.

  13. Screening of spider mites (Acari: Tetranychidae) for reproductive endosymbionts reveals links between co-infection and evolutionary history.

    PubMed

    Zhang, Yan-Kai; Chen, Ya-Ting; Yang, Kun; Qiao, Ge-Xia; Hong, Xiao-Yue

    2016-01-01

    Reproductive endosymbionts have been shown to have wide-ranging effects on many aspects of their hosts' biology. A first step to understanding how these endosymbionts interact with their hosts is to determine their incidences. Here, we screened for four reproductive endosymbionts (Wolbachia, Cardinium, Spiroplasma and Rickettsia) in 28 populations of spider mites (Acari: Tetranychidae) representing 12 species. Each of the four endosymbionts were identified in at least some of the tested specimens, and their infection patterns showed variations at the species-level and population-level, suggesting their distributions can be correlated with both the phylogeny and ecology of the hosts. Co-infections of unrelated bacteria, especially double infections of Wolbachia and Cardinium within the same individuals were common. Spiroplasma and Rickettsia infections were specific to particular host species, respectively. Further, the evolutionary histories of these endosymbionts were inferred by comparing the phylogenies of them and their hosts. These findings can help to clarify the interactions between endosymbionts and arthropods.

  14. Systematic E2 screening reveals a UBE2D-RNF138-CtIP axis promoting DNA repair

    PubMed Central

    Sczaniecka-Clift, Matylda; Coates, Julia; Jhujh, Satpal; Demir, Mukerrem; Cornwell, Matthew; Beli, Petra; Jackson, Stephen P

    2016-01-01

    Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumourigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2-enzymes for impacts on cellular DSB responses. Applying a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3-ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies. PMID:26502057

  15. Pharmacophore modeling, virtual screening, and in vitro testing reveal haloperidol, eprazinone, and fenbutrazate as neurokinin receptors ligands.

    PubMed

    Krautscheid, Yvonne; Senning, Carl Johann Åke; Sartori, Simone B; Singewald, Nicolas; Schuster, Daniela; Stuppner, Hermann

    2014-06-23

    Neurokinin receptors (NKRs) have been shown to be involved in many physiological processes, rendering them promising novel drug targets, but also making them the possible cause for side effects of several drugs. Aiming to answer the question whether the binding to NKRs could have a share in the side effects or even the desired effects of already licensed drugs, we generated a set of ligand-based common feature pharmacophore models based on the structural information about subtype-selective and nonselective NKR antagonists and screened an in-house database mainly composed of licensed drugs. The prospective pharmacological investigations of the virtual hits haloperidol, eprazinone, and fenbutrazate confirmed them to be NKR ligands in vitro. By the identification of licensed drugs as so far unknown NKR ligands, this study contributes to establishing an activity profile of the investigated compounds and confirms the presented pharmacophore models as useful tools for this purpose. PMID:24849814

  16. Assembly of anthrax toxin pore: Lethal-factor complexes into lipid nanodiscs

    PubMed Central

    Akkaladevi, N; Hinton-Chollet, L; Katayama, H; Mitchell, J; Szerszen, L; Mukherjee, S; Gogol, E P; Pentelute, B L; Collier, R J; Fisher, M T

    2013-01-01

    We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LFN) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (∼43%). Three-dimensional analysis of negatively stained single particles revealed the LFN-PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LFN molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LFN-PA nanodisc structures and use of advanced automated particle selection methods. PMID:23389868

  17. A direct screen for c-di-GMP modulators reveals a Salmonella Typhimurium periplasmic ʟ-arginine-sensing pathway.

    PubMed

    Mills, Erez; Petersen, Erik; Kulasekara, Bridget R; Miller, Samuel I

    2015-06-01

    Cyclic-di-GMP (c-di-GMP) is a bacterial second messenger that transduces internal and external signals and regulates bacterial motility and biofilm formation. Some organisms encode more than 100 c-di-GMP-modulating enzymes, but only for a few has a signal been defined that modulates their activity. We developed and applied a high-throughput, real-time flow cytometry method that uses a fluorescence resonance energy transfer (FRET)-based biosensor of free c-di-GMP to screen for signals that modulate its concentration within Salmonella Typhimurium. We identified multiple compounds, including glucose, N-acetyl-d-glucosamine, salicylic acid, and ʟ-arginine, that modulated the FRET signal and therefore the free c-di-GMP concentration. By screening a library of mutants, we identified proteins required for the c-di-GMP response to each compound. Furthermore, low micromolar concentrations of ʟ-arginine induced a rapid translation-independent increase in c-di-GMP concentrations and c-di-GMP-dependent cellulose synthesis, responses that required the regulatory periplasmic domain of the diguanylate cyclase STM1987. ʟ-Arginine signaling also required the periplasmic putative ʟ-arginine-binding protein ArtI, implying that ʟ-arginine sensing occurred in the periplasm. Among the 20 commonly used amino acids, S. Typhimurium specifically responded to ʟ-arginine with an increase in c-di-GMP, suggesting that ʟ-arginine may serve as a signal during S. Typhimurium infection. Our results demonstrate that a second-messenger biosensor can be used to identify environmental signals and define pathways that alter microbial behavior.

  18. A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide.

    PubMed

    Wittmann, Christine; Reischl, Markus; Shah, Asmi H; Kronfuss, Eva; Mikut, Ralf; Liebel, Urban; Grabher, Clemens

    2015-01-01

    Tissue injury and infection trigger innate immune responses. However, dysregulation may result in chronic inflammation and is commonly treated with corticosteroids and non-steroidal anti-inflammatory drugs. Unfortunately, long-term administration of both therapeutic classes can cause unwanted side effects. To identify alternative immune-modulatory compounds we have previously established a novel screening method using zebrafish larvae. Using this method we here present results of an in vivo high-content drug-repurposing screen, identifying 63 potent anti-inflammatory drugs that are in clinical use for other indications. Our approach reveals a novel pro-inflammatory role of nitric oxide. Nitric oxide affects leukocyte recruitment upon peripheral sensory nervous system or epithelial injury in zebrafish larvae both via soluble guanylate cyclase and in a soluble guanylate cyclase -independent manner through protein S-nitrosylation. Together, we show that our screening method can help to identify novel immune-modulatory activities and provide new mechanistic insights into the regulation of inflammatory processes.

  19. Zygotic Lethals with Specific Maternal Effect Phenotypes in Drosophila Melanogaster. I. Loci on the X Chromosome

    PubMed Central

    Perrimon, N.; Engstrom, L.; Mahowald, A. P.

    1989-01-01

    In order to identify all X-linked zygotic lethal loci that exhibit a specific maternal effect on embryonic development, germline clonal analyses of X-linked zygotic lethal mutations have been performed. Two strategies were employed. In Screen A germline clonal analysis of 441 mutations at 211 previously mapped X-linked loci within defined regions was performed. In Screen B germline clonal analysis of 581 larval and pupal mutations distributed throughout the entire length of the X chromosome was performed. These approaches provide an 86% level of saturation for X-linked late zygotic lethals (larval and pupal) with specific maternal effect embryonic lethal phenotypes. The maternal effect phenotypes of these mutations are described. PMID:2499512

  20. RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27.

    PubMed

    Puri, V; Virbasius, J V; Guilherme, A; Czech, M P

    2008-01-01

    Adipose tissue modulates whole body metabolism and insulin sensitivity by controlling circulating lipid levels and producing molecules that can regulate fatty acid metabolism in such tissues as muscle and liver. We have developed RNA interference (RNAi) screens to identify genes in cultured adipocytes that regulate insulin signalling and key metabolic pathways. These short interfering RNA (siRNA)-based screens identified the transcriptional corepressor receptor interacting protein 140 (RIP140) (J Clin Invest 116: 125, 2006) and the mitogen-activated protein kinase (MAP4k4) (Proc Natl Acad Sci USA 103: 2087, 2006) as negative regulators of insulin-responsive hexose uptake and oxidative metabolism. Gene expression profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis and oxidative phosphorylation. RIP140-null mice resist weight gain on a high-fat diet and display enhanced glucose tolerance. MAP4k4 depletion in adipocytes increases many of the RIP140-sensitive genes, increases adipogenesis and mediates some actions of tumour necrosis factor-alpha (TNF-alpha). Remarkably, another hit in our RNAi screens was fat specific protein 27 (FSP27), a highly expressed isoform of Cidea. We discovered that FSP27 unexpectedly associates specifically with lipid droplets and regulates fat storage. We conclude that RIP140, MAP4k4 and the novel lipid droplet protein FSP27 are powerful regulators of adipose tissue metabolism and are potential therapeutic targets for controlling metabolic disease. The discovery of these novel proteins validates the power of RNAi screening for discovery of new therapeutic approaches to type 2 diabetes and obesity. PMID:18171433

  1. A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase

    PubMed Central

    Sabogal, Alex; Rio, Donald C

    2010-01-01

    Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions. PMID:20842711

  2. Lethal mutagenesis and evolutionary epidemiology.

    PubMed

    Martin, Guillaume; Gandon, Sylvain

    2010-06-27

    The lethal mutagenesis hypothesis states that within-host populations of pathogens can be driven to extinction when the load of deleterious mutations is artificially increased with a mutagen, and becomes too high for the population to be maintained. Although chemical mutagens have been shown to lead to important reductions in viral titres for a wide variety of RNA viruses, the theoretical underpinnings of this process are still not clearly established. A few recent models sought to describe lethal mutagenesis but they often relied on restrictive assumptions. We extend this earlier work in two novel directions. First, we derive the dynamics of the genetic load in a multivariate Gaussian fitness landscape akin to classical quantitative genetics models. This fitness landscape yields a continuous distribution of mutation effects on fitness, ranging from deleterious to beneficial (i.e. compensatory) mutations. We also include an additional class of lethal mutations. Second, we couple this evolutionary model with an epidemiological model accounting for the within-host dynamics of the pathogen. We derive the epidemiological and evolutionary equilibrium of the system. At this equilibrium, the density of the pathogen is expected to decrease linearly with the genomic mutation rate U. We also provide a simple expression for the critical mutation rate leading to extinction. Stochastic simulations show that these predictions are accurate for a broad range of parameter values. As they depend on a small set of measurable epidemiological and evolutionary parameters, we used available information on several viruses to make quantitative and testable predictions on critical mutation rates. In the light of this model, we discuss the feasibility of lethal mutagenesis as an efficient therapeutic strategy.

  3. An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication.

    PubMed

    Mauthe, Mario; Langereis, Martijn; Jung, Jennifer; Zhou, Xingdong; Jones, Alex; Omta, Wienand; Tooze, Sharon A; Stork, Björn; Paludan, Søren Riis; Ahola, Tero; Egan, Dave; Behrends, Christian; Mokry, Michal; de Haan, Cornelis; van Kuppeveld, Frank; Reggiori, Fulvio

    2016-08-29

    Autophagy is a catabolic process regulated by the orchestrated action of the autophagy-related (ATG) proteins. Recent work indicates that some of the ATG proteins also have autophagy-independent roles. Using an unbiased siRNA screen approach, we explored the extent of these unconventional functions of ATG proteins. We determined the effects of the depletion of each ATG proteome component on the replication of six different viruses. Our screen reveals that up to 36% of the ATG proteins significantly alter the replication of at least one virus in an unconventional fashion. Detailed analysis of two candidates revealed an undocumented role for ATG13 and FIP200 in picornavirus replication that is independent of their function in autophagy as part of the ULK complex. The high numbers of unveiled ATG gene-specific and pathogen-specific functions of the ATG proteins calls for caution in the interpretation of data, which rely solely on the depletion of a single ATG protein to specifically ablate autophagy. PMID:27573464

  4. Biochemical evaluation of virtual screening methods reveals a cell-active inhibitor of the cancer-promoting phosphatases of regenerating liver.

    PubMed

    Hoeger, Birgit; Diether, Maren; Ballester, Pedro J; Köhn, Maja

    2014-12-17

    Computationally supported development of small molecule inhibitors has successfully been applied to protein tyrosine phosphatases in the past, revealing a number of cell-active compounds. Similar approaches have also been used to screen for small molecule inhibitors for the cancer-related phosphatases of regenerating liver (PRL) family. Still, selective and cell-active compounds are of limited availability. Since especially PRL-3 remains an attractive drug target due to its clear role in cancer metastasis, such compounds are highly demanded. In this study, we investigated various virtual screening approaches for their applicability to identify novel small molecule entities for PRL-3 as target. Biochemical evaluation of purchasable compounds revealed ligand-based approaches as well suited for this target, compared to docking-based techniques that did not perform well in this context. The best hit of this study, a 2-cyano-2-ene-ester and hence a novel chemotype targeting the PRLs, was further optimized by a structure-activity-relationship (SAR) study, leading to a low micromolar PRL inhibitor with acceptable selectivity over other protein tyrosine phosphatases. The compound is active in cells, as shown by its ability to specifically revert PRL-3 induced cell migration, and exhibits similar effects on PRL-1 and PRL-2. It is furthermore suitable for fluorescence microscopy applications, and it is commercially available. These features make it the only purchasable, cell-active and acceptably selective PRL inhibitor to date that can be used in various cellular applications.

  5. A Genomic Screen Revealing the Importance of Vesicular Trafficking Pathways in Genome Maintenance and Protection against Genotoxic Stress in Diploid Saccharomyces cerevisiae Cells

    PubMed Central

    Krol, Kamil; Brozda, Izabela; Skoneczny, Marek; Bretne, Maria; Skoneczna, Adrianna

    2015-01-01

    The ability to survive stressful conditions is important for every living cell. Certain stresses not only affect the current well-being of cells but may also have far-reaching consequences. Uncurbed oxidative stress can cause DNA damage and decrease cell survival and/or increase mutation rates, and certain substances that generate oxidative damage in the cell mainly act on DNA. Radiomimetic zeocin causes oxidative damage in DNA, predominantly by inducing single- or double-strand breaks. Such lesions can lead to chromosomal rearrangements, especially in diploid cells, in which the two sets of chromosomes facilitate excessive and deleterious recombination. In a global screen for zeocin-oversensitive mutants, we selected 133 genes whose deletion reduces the survival of zeocin-treated diploid Saccharomyces cerevisiae cells. The screen revealed numerous genes associated with stress responses, DNA repair genes, cell cycle progression genes, and chromatin remodeling genes. Notably, the screen also demonstrated the involvement of the vesicular trafficking system in cellular protection against DNA damage. The analyses indicated the importance of vesicular system integrity in various pathways of cellular protection from zeocin-dependent damage, including detoxification and a direct or transitional role in genome maintenance processes that remains unclear. The data showed that deleting genes involved in vesicular trafficking may lead to Rad52 focus accumulation and changes in total DNA content or even cell ploidy alterations, and such deletions may preclude proper DNA repair after zeocin treatment. We postulate that functional vesicular transport is crucial for sustaining an integral genome. We believe that the identification of numerous new genes implicated in genome restoration after genotoxic oxidative stress combined with the detected link between vesicular trafficking and genome integrity will reveal novel molecular processes involved in genome stability in diploid cells

  6. A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

    PubMed Central

    Finlayson, Alexander E.; Freeman, Kevin W.

    2009-01-01

    Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis. PMID:19924305

  7. Histochemical screening, metabolite profiling and expression analysis reveal Rosaceae roots as the site of flavan-3-ol biosynthesis.

    PubMed

    Hoffmann, T; Friedlhuber, R; Steinhauser, C; Tittel, I; Skowranek, K; Schwab, W; Fischer, T C

    2012-01-01

    Histochemical screening of 30 Rosaceae genera representing all classic subfamilies demonstrated flavan-3-ols (catechins) as general secondary metabolites in roots of Rosaceae. Semi-quantitative LC-MS analyses confirmed the presence of catechin, epicatechin and various dimeric flavan-3-ols (also representing higher polymeric proanthocyanidins) as prominent polyphenols in root tips of Fragaria (strawberry), Malus (apple), Rosa (rose), Pyrus (pear) and Prunus (plum). Distinct patterns of flavan-3-ol distribution at the cellular level were found in strawberry (Fragaria × ananassa) and apple (Malus × domestica) root tips. The calyptras (root caps) showed the most prominent flavan-3-ol staining for these two genera. Border cells of Fragaria and Malus, as first demonstrated here for Rosaceae, were also found to contain flavan-3-ols. Transcript analyses with cDNA demonstrated root expression of known flavonoid genes expressed in the respective fruits and leaves. Primarily, this proves in situ biosynthesis of flavan-3-ols in these roots. Knowledge of the distinct cellular distribution patterns and their in situ biosynthesis in roots provides a basis for analysis of the functional roles of Rosaceae root flavan-3-ols.

  8. Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection

    PubMed Central

    Foo, Chwan Hong; Rootes, Christina L.; Gould, Cathryn M.; Grusovin, Julian; Monaghan, Paul; Lo, Michael K.; Tompkins, S. Mark; Adams, Timothy E.; Lowenthal, John W.; Simpson, Kaylene J.; Stewart, Cameron R.; Bean, Andrew G. D.; Wang, Lin-Fa

    2016-01-01

    Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections. PMID:27010548

  9. An extracellular biochemical screen reveals that FLRTs and Unc5s mediate neuronal subtype recognition in the retina

    PubMed Central

    Visser, Jasper J; Cheng, Yolanda; Perry, Steven C; Chastain, Andrew Benjamin; Parsa, Bayan; Masri, Shatha S; Ray, Thomas A; Kay, Jeremy N; Wojtowicz, Woj M

    2015-01-01

    In the inner plexiform layer (IPL) of the mouse retina, ~70 neuronal subtypes organize their neurites into an intricate laminar structure that underlies visual processing. To find recognition proteins involved in lamination, we utilized microarray data from 13 subtypes to identify differentially-expressed extracellular proteins and performed a high-throughput biochemical screen. We identified ~50 previously-unknown receptor-ligand pairs, including new interactions among members of the FLRT and Unc5 families. These proteins show laminar-restricted IPL localization and induce attraction and/or repulsion of retinal neurites in culture, placing them in an ideal position to mediate laminar targeting. Consistent with a repulsive role in arbor lamination, we observed complementary expression patterns for one interaction pair, FLRT2-Unc5C, in vivo. Starburst amacrine cells and their synaptic partners, ON-OFF direction-selective ganglion cells, express FLRT2 and are repelled by Unc5C. These data suggest a single molecular mechanism may have been co-opted by synaptic partners to ensure joint laminar restriction. DOI: http://dx.doi.org/10.7554/eLife.08149.001 PMID:26633812

  10. Functional mutagenesis screens reveal the ‘cap structure’ formation in disulfide-bridge free TASK channels

    PubMed Central

    Goldstein, Matthias; Rinné, Susanne; Kiper, Aytug K.; Ramírez, David; Netter, Michael F.; Bustos, Daniel; Ortiz-Bonnin, Beatriz; González, Wendy; Decher, Niels

    2016-01-01

    Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels. PMID:26794006

  11. Screening of spider mites (Acari: Tetranychidae) for reproductive endosymbionts reveals links between co-infection and evolutionary history.

    PubMed

    Zhang, Yan-Kai; Chen, Ya-Ting; Yang, Kun; Qiao, Ge-Xia; Hong, Xiao-Yue

    2016-01-01

    Reproductive endosymbionts have been shown to have wide-ranging effects on many aspects of their hosts' biology. A first step to understanding how these endosymbionts interact with their hosts is to determine their incidences. Here, we screened for four reproductive endosymbionts (Wolbachia, Cardinium, Spiroplasma and Rickettsia) in 28 populations of spider mites (Acari: Tetranychidae) representing 12 species. Each of the four endosymbionts were identified in at least some of the tested specimens, and their infection patterns showed variations at the species-level and population-level, suggesting their distributions can be correlated with both the phylogeny and ecology of the hosts. Co-infections of unrelated bacteria, especially double infections of Wolbachia and Cardinium within the same individuals were common. Spiroplasma and Rickettsia infections were specific to particular host species, respectively. Further, the evolutionary histories of these endosymbionts were inferred by comparing the phylogenies of them and their hosts. These findings can help to clarify the interactions between endosymbionts and arthropods. PMID:27291078

  12. A screen for constituents of motor control and decision making in Drosophila reveals visual distance-estimation neurons

    PubMed Central

    Triphan, Tilman; Nern, Aljoscha; Roberts, Sonia F.; Korff, Wyatt; Naiman, Daniel Q.; Strauss, Roland

    2016-01-01

    Climbing over chasms larger than step size is vital to fruit flies, since foraging and mating are achieved while walking. Flies avoid futile climbing attempts by processing parallax-motion vision to estimate gap width. To identify neuronal substrates of climbing control, we screened a large collection of fly lines with temporarily inactivated neuronal populations in a novel high-throughput assay described here. The observed climbing phenotypes were classified; lines in each group are reported. Selected lines were further analysed by high-resolution video cinematography. One striking class of flies attempts to climb chasms of unsurmountable width; expression analysis guided us to C2 optic-lobe interneurons. Inactivation of C2 or the closely related C3 neurons with highly specific intersectional driver lines consistently reproduced hyperactive climbing whereas strong or weak artificial depolarization of C2/C3 neurons strongly or mildly decreased climbing frequency. Contrast-manipulation experiments support our conclusion that C2/C3 neurons are part of the distance-evaluation system. PMID:27255169

  13. Screening of spider mites (Acari: Tetranychidae) for reproductive endosymbionts reveals links between co-infection and evolutionary history

    PubMed Central

    Zhang, Yan-Kai; Chen, Ya-Ting; Yang, Kun; Qiao, Ge-Xia; Hong, Xiao-Yue

    2016-01-01

    Reproductive endosymbionts have been shown to have wide-ranging effects on many aspects of their hosts’ biology. A first step to understanding how these endosymbionts interact with their hosts is to determine their incidences. Here, we screened for four reproductive endosymbionts (Wolbachia, Cardinium, Spiroplasma and Rickettsia) in 28 populations of spider mites (Acari: Tetranychidae) representing 12 species. Each of the four endosymbionts were identified in at least some of the tested specimens, and their infection patterns showed variations at the species-level and population-level, suggesting their distributions can be correlated with both the phylogeny and ecology of the hosts. Co-infections of unrelated bacteria, especially double infections of Wolbachia and Cardinium within the same individuals were common. Spiroplasma and Rickettsia infections were specific to particular host species, respectively. Further, the evolutionary histories of these endosymbionts were inferred by comparing the phylogenies of them and their hosts. These findings can help to clarify the interactions between endosymbionts and arthropods. PMID:27291078

  14. A screen for constituents of motor control and decision making in Drosophila reveals visual distance-estimation neurons.

    PubMed

    Triphan, Tilman; Nern, Aljoscha; Roberts, Sonia F; Korff, Wyatt; Naiman, Daniel Q; Strauss, Roland

    2016-01-01

    Climbing over chasms larger than step size is vital to fruit flies, since foraging and mating are achieved while walking. Flies avoid futile climbing attempts by processing parallax-motion vision to estimate gap width. To identify neuronal substrates of climbing control, we screened a large collection of fly lines with temporarily inactivated neuronal populations in a novel high-throughput assay described here. The observed climbing phenotypes were classified; lines in each group are reported. Selected lines were further analysed by high-resolution video cinematography. One striking class of flies attempts to climb chasms of unsurmountable width; expression analysis guided us to C2 optic-lobe interneurons. Inactivation of C2 or the closely related C3 neurons with highly specific intersectional driver lines consistently reproduced hyperactive climbing whereas strong or weak artificial depolarization of C2/C3 neurons strongly or mildly decreased climbing frequency. Contrast-manipulation experiments support our conclusion that C2/C3 neurons are part of the distance-evaluation system. PMID:27255169

  15. Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity

    PubMed Central

    Virreira Winter, Sebastian; Zychlinsky, Arturo; Bardoel, Bart W.

    2016-01-01

    Staphylococcus aureus causes a wide variety of infections and antibiotic resistant strains are a major problem in hospitals. One of the best studied virulence factors of S. aureus is the pore-forming toxin alpha hemolysin (αHL) whose mechanism of action is incompletely understood. We performed a genome-wide loss-of-function screen using CRISPR/Cas9 technology to identify host targets required for αHL susceptibility in human myeloid cells. We found gRNAs for ten genes enriched after intoxication with αHL and focused on the top five hits. Besides a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), the host receptor for αHL, we identified three proteins, Sys1 golgi trafficking protein (SYS1), ADP-ribosylation factor 1 (ARFRP1), and tetraspanin-14 (TSPAN14) which regulate the presentation of ADAM10 on the plasma membrane post-translationally. Interestingly, we also showed that cells lacking sphingomyelin synthase 1 (SGMS1) resist αHL intoxication, but have only a slightly reduced ADAM10 surface expression. SGMS1 regulates lipid raft formation, suggesting that αHL requires these membrane microdomains for attachment and cytotoxicity. PMID:27066838

  16. Proteomic Screening and Lasso Regression Reveal Differential Signaling in Insulin and Insulin-like Growth Factor I (IGF1) Pathways.

    PubMed

    Erdem, Cemal; Nagle, Alison M; Casa, Angelo J; Litzenburger, Beate C; Wang, Yu-Fen; Taylor, D Lansing; Lee, Adrian V; Lezon, Timothy R

    2016-09-01

    Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. First, acetyl-CoA carboxylase (ACC) knock-down was shown to increase the level of mitogen-activated protein kinase (MAPK) phosphorylation. Second, stable knock-down of E-Cadherin increased the phospho-Akt protein levels. Both of the knock-down perturbations incurred phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro.

  17. Proteomic Screening and Lasso Regression Reveal Differential Signaling in Insulin and Insulin-like Growth Factor I (IGF1) Pathways.

    PubMed

    Erdem, Cemal; Nagle, Alison M; Casa, Angelo J; Litzenburger, Beate C; Wang, Yu-Fen; Taylor, D Lansing; Lee, Adrian V; Lezon, Timothy R

    2016-09-01

    Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. First, acetyl-CoA carboxylase (ACC) knock-down was shown to increase the level of mitogen-activated protein kinase (MAPK) phosphorylation. Second, stable knock-down of E-Cadherin increased the phospho-Akt protein levels. Both of the knock-down perturbations incurred phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro. PMID:27364358

  18. Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

    PubMed Central

    van der Ploeg, René; Goudelis, Spyridon Theodoros; den Blaauwen, Tanneke

    2015-01-01

    The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors. PMID:26263980

  19. A mutant screen reveals RNase E as a silencer of group II intron retromobility in Escherichia coli

    PubMed Central

    Coros, Colin J.; Piazza, Carol Lyn; Chalamcharla, Venkata R.; Belfort, Marlene

    2008-01-01

    Group II introns are mobile retroelements that invade their hosts. The Lactococcus lactis group II intron recruits cellular polymerases, nucleases, and DNA ligase to complete the retromobility process in Escherichia coli. Here we describe a genetic screen with a Tn5 transposon library to identify other E. coli functions involved in retromobility of the L. lactis LtrB intron. Thirteen disruptions that reproducibly resulted in increased or decreased retrohoming levels into the E. coli chromosome were isolated. These functions were classified as factors involved in RNA processing, DNA replication, energy metabolism, and global regulation. Here we characterize a novel mutant in the rne promoter region, which regulates RNase E expression. Retrohoming and retrotransposition levels are elevated in the rne∷Tn5 mutant. The stimulatory effect of the mutation on retromobility results from intron RNA accumulation in the RNase E mutant. These results suggest that RNase E, which is the central component of the RNA degradosome, could regulate retrohoming levels in response to cellular physiology. PMID:18945808

  20. Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function.

    PubMed

    de Lange, Job; Faramarz, Atiq; Oostra, Anneke B; de Menezes, Renee X; van der Meulen, Ida H; Rooimans, Martin A; Rockx, Davy A; Brakenhoff, Ruud H; van Beusechem, Victor W; King, Randall W; de Winter, Johan P; Wolthuis, Rob M F

    2015-01-01

    Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31(comet). A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers. PMID:26423134

  1. Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function

    PubMed Central

    de Lange, Job; Faramarz, Atiq; Oostra, Anneke B.; de Menezes, Renee X.; van der Meulen, Ida H.; Rooimans, Martin A.; Rockx, Davy A.; Brakenhoff, Ruud H.; van Beusechem, Victor W.; King, Randall W.; de Winter, Johan P.; Wolthuis, Rob M. F.

    2015-01-01

    Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31comet. A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers. PMID:26423134

  2. Synthetic antibodies with a human framework that protect mice from lethal Sudan ebolavirus challenge.

    PubMed

    Chen, Gang; Koellhoffer, Jayne F; Zak, Samantha E; Frei, Julia C; Liu, Nina; Long, Hua; Ye, Wei; Nagar, Kaajal; Pan, Guohua; Chandran, Kartik; Dye, John M; Sidhu, Sachdev S; Lai, Jonathan R

    2014-10-17

    The ebolaviruses cause severe and rapidly progressing hemorrhagic fever. There are five ebolavirus species; although much is known about Zaire ebolavirus (EBOV) and its neutralization by antibodies, little is known about Sudan ebolavirus (SUDV), which is emerging with increasing frequency. Here we describe monoclonal antibodies containing a human framework that potently inhibit infection by SUDV and protect mice from lethal challenge. The murine antibody 16F6, which binds the SUDV envelope glycoprotein (GP), served as the starting point for design. Sequence and structural alignment revealed similarities between 16F6 and YADS1, a synthetic antibody with a humanized scaffold. A focused phage library was constructed and screened to impart 16F6-like recognition properties onto the YADS1 scaffold. A panel of 17 antibodies were characterized and found to have a range of neutralization potentials against a pseudotype virus infection model. Neutralization correlated with GP binding as determined by ELISA. Two of these clones, E10 and F4, potently inhibited authentic SUDV and conferred protection and memory immunity in mice from lethal SUDV challenge. E10 and F4 were further shown to bind to the same epitope on GP as 16F6 with comparable affinities. These antibodies represent strong immunotherapeutic candidates for treatment of SUDV infection. PMID:25140871

  3. Empirical complexities in the genetic foundations of lethal mutagenesis.

    PubMed

    Bull, James J; Joyce, Paul; Gladstone, Eric; Molineux, Ian J

    2013-10-01

    From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias-mutagenesis of virions-but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

  4. A Screen of Coxiella burnetii Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis

    PubMed Central

    Newton, Hayley J.; Kohler, Lara J.; McDonough, Justin A.; Temoche-Diaz, Morayma; Crabill, Emerson; Hartland, Elizabeth L.; Roy, Craig R.

    2014-01-01

    Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle. PMID:25080348

  5. Pharmacophore-based screening targeted at upregulated FN1, MMP-9, APP reveals therapeutic compounds for nasopharyngeal carcinoma.

    PubMed

    Lai, Catherine Jessica; Tay, Boon Hunt

    2016-02-01

    Nasopharyngeal carcinoma (NpC) is rare in the west but common in Southeast Asia and only a few other locations. With the limited geographic incidence, it is relatively under-studied. It also has as co-determinant the Epstein-Barr virus (EBV), which may adapt to NpC therapies, so not only must a therapeutic compound be found, the discovery process must be rapid, to cope with the changing basis of the EBV. An R-based computer workbench, Mendel, was developed so biologists could quickly upload genomic data, pre-process them, and identify upregulated and downregulated genes. Mendel was used on 10 control and 31 diseased cell lines to discover 3 differentially expressed genes (DEGs) that meet thresholds on fold-changes, 3-clique membership, pathway constraints, and druggability. From the DEGs, we conducted a pharmacophore-based screening of 22,723,923 compounds using protein-protein interaction anchor-residue clusters as binding sites. Of the 4 hits, 3 passed all the ADME-Tox tests. These 3 hit compounds, 6-(4-iminiocyclohexa-2,5-dien-1-ylidene)-4-(thiazol-2-ylcarbamoyl)-1H-pyrimidine-2-thiolate, 1-[4-[2-[(3R)-3-hydroxy-2-oxo-indolin-3-yl]acetyl]phenyl]-3-phenyl-urea, and (2R)-N4-[4-(1-piperidyl)cyclohexyl]morpholine-2,4-dicarboxamide have predicted pIC50 values superior to the current drugs fluorouracil (5-FU) and taxotere, which have side effects and face EBV drug resistance. PMID:26773938

  6. An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription.

    PubMed

    Giraud, Matthieu; Jmari, Nada; Du, Lina; Carallis, Floriane; Nieland, Thomas J F; Perez-Campo, Flor M; Bensaude, Olivier; Root, David E; Hacohen, Nir; Mathis, Diane; Benoist, Christophe

    2014-01-28

    Aire induces the expression of a large set of autoantigen genes in the thymus, driving immunological tolerance in maturing T cells. To determine the full spectrum of molecular mechanisms underlying the Aire transactivation function, we screened an AIRE-dependent gene-expression system with a genome-scale lentiviral shRNA library, targeting factors associated with chromatin architecture/function, transcription, and mRNA processing. Fifty-one functional allies were identified, with a preponderance of factors that impact transcriptional elongation compared with initiation, in particular members of the positive transcription elongation factor b (P-TEFb) involved in the release of "paused" RNA polymerases (CCNT2 and HEXIM1); mRNA processing and polyadenylation factors were also highlighted (HNRNPL/F, SFRS1, SFRS3, and CLP1). Aire's functional allies were validated on transfected and endogenous target genes, including the generation of lentigenic knockdown (KD) mice. We uncovered the effect of the splicing factor Hnrnpl on Aire-induced transcription. Transcripts sensitive to the P-TEFb inhibitor flavopiridol were reduced by Hnrnpl knockdown in thymic epithelial cells, independently of their dependence on Aire, therefore indicating a general effect of Hnrnpl on RNA elongation. This conclusion was substantiated by demonstration of HNRNPL interactions with P-TEFb components (CDK9, CCNT2, HEXIM1, and the small 7SK RNA). Aire-containing complexes include 7SK RNA, the latter interaction disrupted by HNRNPL knockdown, suggesting that HNRNPL may partake in delivering inactive P-TEFb to Aire. Thus, these results indicate that mRNA processing factors cooperate with Aire to release stalled polymerases and to activate ectopic expression of autoantigen genes in the thymus.

  7. A screen of Coxiella burnetii mutants reveals important roles for Dot/Icm effectors and host autophagy in vacuole biogenesis.

    PubMed

    Newton, Hayley J; Kohler, Lara J; McDonough, Justin A; Temoche-Diaz, Morayma; Crabill, Emerson; Hartland, Elizabeth L; Roy, Craig R

    2014-07-01

    Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.

  8. Screening a yeast library of temperature-sensitive mutants reveals a role for actin in tombusvirus RNA recombination.

    PubMed

    Prasanth, K Reddisiva; Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Baker, Jannine; Nagy, Peter D

    2016-02-01

    Genetic recombination in RNA viruses drives the evolutionary arms race with host's antiviral strategies and recombination also facilitates adaptation of viruses to new hosts. In this paper, the authors used tombusvirus and a temperature-sensitive (ts) mutant library of yeast to identify 40 host proteins affecting viral recombination in yeast model host. Subsequent detailed analysis with two identified actin-related proteins, Act1p and Arp3p, has revealed that the wt actin network helps TBSV to maintain low level viral recombination. Pharmacological inhibition of actin in plant protoplasts confirmed the role of the actin network in tombusvirus recombination. An in vitro approach revealed the altered activity of the tombusvirus replicase in the presence of mutated Act1p. The authors show more efficient recruitment of a cellular DEAD-box helicase, which enhances tombusvirus recombination, into the membrane-bound replicase in Act1p mutant yeast. Overall, this work shows that the actin network affects tombusvirus recombination in yeast and plant cells.

  9. Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis gene cheB2 in Pseudomonas aeruginosa virulence.

    PubMed

    Garvis, Steven; Munder, Antje; Ball, Geneviève; de Bentzmann, Sophie; Wiehlmann, Lutz; Ewbank, Jonathan J; Tümmler, Burkhard; Filloux, Alain

    2009-08-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.

  10. An EST screen from the annelid Pomatoceros lamarckii reveals patterns of gene loss and gain in animals

    PubMed Central

    2009-01-01

    Background Since the drastic reorganisation of the phylogeny of the animal kingdom into three major clades of bilaterians; Ecdysozoa, Lophotrochozoa and Deuterostomia, it became glaringly obvious that the selection of model systems with extensive molecular resources was heavily biased towards only two of these three clades, namely the Ecdysozoa and Deuterostomia. Increasing efforts have been put towards redressing this imbalance in recent years, and one of the principal phyla in the vanguard of this endeavour is the Annelida. Results In the context of this effort we here report our characterisation of an Expressed Sequence Tag (EST) screen in the serpulid annelid, Pomatoceros lamarckii. We have sequenced over 5,000 ESTs which consolidate into over 2,000 sequences (clusters and singletons). These sequences are used to build phylogenetic trees to estimate relative branch lengths amongst different taxa and, by comparison to genomic data from other animals, patterns of gene retention and loss are deduced. Conclusion The molecular phylogenetic trees including the P. lamarckii sequences extend early observations that polychaetes tend to have relatively short branches in such trees, and hence are useful taxa with which to reconstruct gene family evolution. Also, with the availability of lophotrochozoan data such as that of P. lamarckii, it is now possible to make much more accurate reconstructions of the gene complement of the ancestor of the bilaterians than was previously possible from comparisons of ecdysozoan and deuterostome genomes to non-bilaterian outgroups. It is clear that the traditional molecular model systems for protostomes (e.g. Drosophila melanogaster and Caenorhabditis elegans), which are restricted to the Ecdysozoa, have undergone extensive gene loss during evolution. These ecdysozoan systems, in terms of gene content, are thus more derived from the bilaterian ancestral condition than lophotrochozoan systems like the polychaetes, and thus cannot be used

  11. iTRAQ-based chromatin proteomic screen reveals CHD4-dependent recruitment of MBD2 to sites of DNA damage.

    PubMed

    Sun, Yazhou; Yang, Yeran; Shen, Hongyan; Huang, Min; Wang, Zhifeng; Liu, Yang; Zhang, Hui; Tang, Tie-Shan; Guo, Caixia

    2016-02-26

    Many DNA repair proteins can be recruited to DNA damage sites upon genotoxic stress. In order to search potential DNA repair proteins involved in cellular response to mitomycin C treatment, we utilized a quantitative proteome to uncover proteins that manifest differentially enrichment in the chromatin fraction after DNA damage. 397 proteins were identified, among which many factors were shown to be involved in chromatin modification and DNA repair by GO analysis. Specifically, methyl-CpG-binding domain protein 2 (MBD2) is revealed to be recruited to DNA damage sites after laser microirradiation, which was mediated through MBD domain and MBD2 C-terminus. Additionally, the recruitment of MBD2 is dependent on poly (ADP-ribose) and chromodomain helicase DNA-binding protein 4 (CHD4). Moreover, knockdown of MBD2 by CRISPR-Cas9 technique results in MMC sensitivity in mammalian cells. PMID:26827827

  12. A novel mutation in the mitochondrial tRNA{sup Asn} gene associated with a lethal disease

    SciTech Connect

    Coulbault, Laurent; Herlicoviez, Danielle; Chapon, Francoise; Read, Marie-Helene; Penniello, Marie-Jose; Reynier, Pascal; Fayet, Guillemette; Lombes, Anne; Jauzac, Philippe; Allouche, Stephane . E-mail: allouche-s@chu-caen.fr

    2005-04-15

    We describe a lethal mitochondrial disease in a 10-month-old child who presented with encephalomyopathy. Histochemical and electron microscopy examinations of skeletal muscle biopsy revealed abnormal mitochondria associated with a combined deficiency of complexes I and IV. After excluding mitochondrial DNA deletions and depletion, direct sequencing was used to screen for mutation in all transfer RNA (tRNA) genes. A T-to-C substitution at position 5693 in the tRNA{sup Asn} gene was found in blood and muscle. Microdissection of muscle biopsy and its analysis revealed the highest level of this mutation in cytochrome c oxidase (COX)-negative fibres. We suggest that this novel mutation would affect the anticodon loop structure of the tRNA{sup Asn} and cause a fatal mitochondrial disease.

  13. Virtual and In Vitro Screens Reveal a Potential Pharmacophore that Avoids the Fibrillization of Aβ1-42.

    PubMed

    Hernández-Rodríguez, Maricarmen; Correa-Basurto, José; Nicolás-Vázquez, María Inés; Miranda-Ruvalcaba, René; Benítez-Cardoza, Claudia Guadalupe; Reséndiz-Albor, Aldo Arturo; Méndez-Méndez, Juan Vicente; Rosales-Hernández, Martha C

    2015-01-01

    Among the multiple factors that induce Alzheimer's disease, aggregation of the amyloid β peptide (Aβ) is considered the most important due to the ability of the 42-amino acid Aβ peptides (Aβ1-42) to form oligomers and fibrils, which constitute Aβ pathological aggregates. For this reason, the development of inhibitors of Aβ1-42 pathological aggregation represents a field of research interest. Several Aβ1-42 fibrillization inhibitors possess tertiary amine and aromatic moieties. In the present study, we selected 26 compounds containing tertiary amine and aromatic moieties with or without substituents and performed theoretical studies that allowed us to select four compounds according to their free energy values for Aβ1-42 in α-helix (Aβ-α), random coil (Aβ-RC) and β-sheet (Aβ-β) conformations. Docking studies revealed that compound 5 had a higher affinity for Aβ-α and Aβ-RC than the other compounds. In vitro, this compound was able to abolish Thioflavin T fluorescence and favored an RC conformation of Aβ1-42 in circular dichroism studies, resulting in the formation of amorphous aggregates as shown by atomic force microscopy. The results obtained from quantum studies allowed us to identify a possible pharmacophore that can be used to design Aβ1-42 aggregation inhibitors. In conclusion, compounds with higher affinity for Aβ-α and Aβ-RC prevented the formation of oligomeric species.

  14. Virtual and In Vitro Screens Reveal a Potential Pharmacophore that Avoids the Fibrillization of Aβ1–42

    PubMed Central

    Hernández-Rodríguez, Maricarmen; Correa-Basurto, José; Nicolás-Vázquez, María Inés; Miranda-Ruvalcaba, René; Benítez-Cardoza, Claudia Guadalupe; Reséndiz-Albor, Aldo Arturo; Méndez-Méndez, Juan Vicente; Rosales-Hernández, Martha C.

    2015-01-01

    Among the multiple factors that induce Alzheimer’s disease, aggregation of the amyloid β peptide (Aβ) is considered the most important due to the ability of the 42-amino acid Aβ peptides (Aβ1–42) to form oligomers and fibrils, which constitute Aβ pathological aggregates. For this reason, the development of inhibitors of Aβ1–42 pathological aggregation represents a field of research interest. Several Aβ1–42 fibrillization inhibitors possess tertiary amine and aromatic moieties. In the present study, we selected 26 compounds containing tertiary amine and aromatic moieties with or without substituents and performed theoretical studies that allowed us to select four compounds according to their free energy values for Aβ1–42 in α-helix (Aβ-α), random coil (Aβ-RC) and β-sheet (Aβ-β) conformations. Docking studies revealed that compound 5 had a higher affinity for Aβ-α and Aβ-RC than the other compounds. In vitro, this compound was able to abolish Thioflavin T fluorescence and favored an RC conformation of Aβ1–42 in circular dichroism studies, resulting in the formation of amorphous aggregates as shown by atomic force microscopy. The results obtained from quantum studies allowed us to identify a possible pharmacophore that can be used to design Aβ1–42 aggregation inhibitors. In conclusion, compounds with higher affinity for Aβ-α and Aβ-RC prevented the formation of oligomeric species. PMID:26172152

  15. In silico screening reveals structurally diverse, nanomolar inhibitors of NQO2 that are functionally active in cells and can modulate NFκB signalling

    PubMed Central

    Nolan, Karen A.; Dunstan, Mark S.; Caraher, Mary C.; Scott, Katherine A.; Leys, David; Stratford, Ian J.

    2011-01-01

    The NCI chemical database has been screened using in silico docking to identify novel nanomolar inhibitors of NRH:quinone oxidoreductase 2 (NQO2). The inhibitors identified from the screen exhibit a diverse range of scaffolds and the structure of one of the inhibitors, NSC13000 co-crystalized with NQO2, has been solved. This has been used to aid the generation of a structure/activity relationship between the computationally derived binding affinity and experimentally measured enzyme inhibitory potency. Many of the compounds are functionally active as inhibitors of NQO2 in cells at non toxic concentrations. To demonstrate this, advantage was taken of the NQO2-mediated toxicity of the chemotherapeutic drug CB1954. The toxicity of this drug is substantially reduced when the function of NQO2 is inhibited and many of the compounds achieve this in cells at nanomolar concentrations. The NQO2 inhibitors also attenuated TNFα-mediated, NFκB-driven transcriptional activity. The link between NQO2 and the regulation of NFκB was confirmed by using siRNA to NQO2 and by the observation that NRH, the cofactor for NQO2 enzyme activity, could regulate NFκB activity in an NQO2 dependent manner. NFκB is a potential therapeutic target and this study reveals an underlying mechanism that may exploitable for developing new anti-cancer drugs. PMID:22090421

  16. A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair

    PubMed Central

    Shahar, Or David; Kalousi, Alkmini; Eini, Lital; Fisher, Benoit; Weiss, Amelie; Darr, Jonatan; Mazina, Olga; Bramson, Shay; Kupiec, Martin; Eden, Amir; Meshorer, Eran; Mazin, Alexander V.; Brino, Laurent; Goldberg, Michal; Soutoglou, Evi

    2014-01-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy. PMID:24682826

  17. A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair.

    PubMed

    Shahar, Or David; Kalousi, Alkmini; Eini, Lital; Fisher, Benoit; Weiss, Amelie; Darr, Jonatan; Mazina, Olga; Bramson, Shay; Kupiec, Martin; Eden, Amir; Meshorer, Eran; Mazin, Alexander V; Brino, Laurent; Goldberg, Michal; Soutoglou, Evi

    2014-05-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy.

  18. Electroshock weapons can be lethal!

    NASA Astrophysics Data System (ADS)

    Lundquist, Marjorie

    2008-03-01

    Electroshock weapons (EWs)-stun guns, tasers, riot shields-are electroconductive devices designed to safely incapacitate healthy men neuromuscularly, so they are called nonlethal or less-lethal. EW firms seeking large nonmilitary markets targeted law enforcement and corrections personnel, who began using EWs in prisons/jails and on public patrol in 1980 in the USA. This shifted the EW-shocked population from healthy soldiers to a heterogeneous mix of both sexes, ages 6-92, in a wide variety of health conditions! An EW operates by disrupting normal physiological processes, producing transient effects in healthy people. But if a person's health is sufficiently compromised, the margin of safety can be lost, resulting in death or permanent health problems. 325 people have died after EW shock since 1980. Did the EW cause these deaths? Evidence indicates that EWs do play a causal role in most such deaths. EWs can be lethal for people in diabetic shock^1 (hypoglycemia), which may be why Robert Dziekanski-a Polish immigrant to Canada-died so quickly after he was tasered at Vancouver Airport: not having eaten for over 10 hours, he likely was severely hypoglycemic. The EW death rate in North America is 30 times higher than need be, because EW users have not been properly trained to use EWs on a heterogeneous population safely! ^1J. Clinical Engineering 30(3):111(2005).

  19. Arenavirus extinction through lethal mutagenesis.

    PubMed

    de la Torre, Juan Carlos

    2005-02-01

    Viral hemorrhagic fevers represent serious human public health problems causing devastating and often lethal disease. Several hemorrhagic fevers are caused by arenaviruses including Lassa fever virus (LFV) and the South American viral hemorrhagic fevers (SAHF). In recent years, increased air travel between Africa and other areas has led to the importation of LFV into the US, Europe, Japan, and Canada. This has raised awareness about arenaviruses as potential emerging viruses. Moreover, because of its severe morbidity and high mortality, and transmissibility from human to human, weaponized forms of LFV poses a real threat as agent of bioterrorism. No licensed vaccine is available in the US, and currently there is not efficacious therapy to treat these infections. Therefore, the importance of developing novel effective antiviral drugs to combat HF arenaviruses, for which the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) provides us with an excellent model system. Recent findings have shown that LCMV multiplication both in cultured cells and in vivo is highly susceptible to the mutagenic agent 5-fluorouracil (FU). FU-mediated extinction of LCMV was associated with only modest increases in virus mutation frequencies, but did not significantly affect virus replication and transcription, or virus particle formation. These findings indicate that, as with other riboviruses, lethal mutagenesis is effective also against LCMV raising the possibility of using this novel antiviral strategy to combat pathogenic arenaviruses. PMID:15649566

  20. Misonidazole and potentially lethal damage

    SciTech Connect

    Korbelik, M.; Palcic, B.; Skov, K.; Skarsgard, L.

    1982-03-01

    The existence of potentially lethal damage (PLD) is demonstrated in exponentially growing CHO cells exposed to misonidazole in hypoxia. The method of hypertonic post-treatment of cells was used in these studies. Misonidazole-induced PLD differs in many characteristics from radiation-induced PLD.The repair kinetics of misonidazole-induced PLD are much slower than for the repair of radiation-induced PLD (hours vs. minutes). No significant repair of misonidazole-induced PLD took place at 25/sup 0/C. Other differences are discussed. Hypertonic post-treatment of irradiated cells which had been pre-incubated with misonidazole to non-toxic levels, gave survival data consistent with the interpretation that no radiation PLD can be induced in such cells.

  1. Ethical language and decision-making for prenatally diagnosed lethal malformations.

    PubMed

    Wilkinson, Dominic; de Crespigny, Lachlan; Xafis, Vicki

    2014-10-01

    In clinical practice, and in the medical literature, severe congenital malformations such as trisomy 18, anencephaly, and renal agenesis are frequently referred to as 'lethal' or as 'incompatible with life'. However, there is no agreement about a definition of lethal malformations, nor which conditions should be included in this category. Review of outcomes for malformations commonly designated 'lethal' reveals that prolonged survival is possible, even if rare. This article analyses the concept of lethal malformations and compares it to the problematic concept of 'futility'. We recommend avoiding the term 'lethal' and suggest that counseling should focus on salient prognostic features instead. For conditions with a high chance of early death or profound impairment in survivors despite treatment, perinatal and neonatal palliative care would be ethical. However, active obstetric and neonatal management, if desired, may also sometimes be appropriate. PMID:25200733

  2. Gene essentiality and synthetic lethality in haploid human cells.

    PubMed

    Blomen, Vincent A; Májek, Peter; Jae, Lucas T; Bigenzahn, Johannes W; Nieuwenhuis, Joppe; Staring, Jacqueline; Sacco, Roberto; van Diemen, Ferdy R; Olk, Nadine; Stukalov, Alexey; Marceau, Caleb; Janssen, Hans; Carette, Jan E; Bennett, Keiryn L; Colinge, Jacques; Superti-Furga, Giulio; Brummelkamp, Thijn R

    2015-11-27

    Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology. PMID:26472760

  3. Gene essentiality and synthetic lethality in haploid human cells.

    PubMed

    Blomen, Vincent A; Májek, Peter; Jae, Lucas T; Bigenzahn, Johannes W; Nieuwenhuis, Joppe; Staring, Jacqueline; Sacco, Roberto; van Diemen, Ferdy R; Olk, Nadine; Stukalov, Alexey; Marceau, Caleb; Janssen, Hans; Carette, Jan E; Bennett, Keiryn L; Colinge, Jacques; Superti-Furga, Giulio; Brummelkamp, Thijn R

    2015-11-27

    Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology.

  4. Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M.

    PubMed

    Pradeepkiran, Jangampalli Adi; Sainath, Sri Bhashyam; Kumar, Konidala Kranthi; Bhaskar, Matcha

    2015-01-01

    Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes) to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50%) to Silicibacter pomeroyi DUF1285 family protein (2RE3). A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the glycerol structural analogs from the PubChem database. We identified five best inhibitors with strong affinities, stable interactions, and also with reliable drug-like properties. Hence, these leads might be used as the most effective inhibitors of modeled protein. The outcome of the present work of virtual screening of putative gene targets might facilitate design of potential drugs for better treatment against brucellosis.

  5. Histopathological effects of anthrax lethal factor on rat liver.

    PubMed

    Altunkaynak, Berrin Zuhal; Ozbek, Elvan

    2015-01-01

    Bacillus anthracis, the causative agent of anthrax, has become an increasingly important scientific topic due to its potential role in bioterrorism. The lethal toxin (LT) of B. anthracis consists of lethal factor (LF) and a protective antigen (PA). This study investigated whether only lethal factor was efficient as a hepatotoxin in the absence of the PA. To achieve this aim, LF (100 µg/kg body weight, dissolved in sterile distilled water) or distilled water vehicle were intraperitoneally injected once into adult rats. At 24 h post-injection, the hosts were euthanized and their livers removed and tissue samples examined under light and electron microscopes. As a result of LF application, hepatic injury - including cytoplasmic and nuclear damage in hepatocytes, sinusoidal dilatation, and hepatocellular lysis - became apparent. Further, light microscopic analyses of liver sections from the LF-injected rats revealed ballooning degeneration and cytoplasmic loss within hepatocytes, as well as peri-sinusoidal inflammation. Additionally, an increase in the numbers of Kupffer cells was evident. Common vascular injuries were also found in the liver samples; these injuries caused hypoxia and pathological changes. In addition, some cytoplasmic and nuclear changes were detected within the liver ultrastructure. The results of these studies allow one to suggest that LF could be an effective toxicant alone and that PA might act in situ to modify the effect of this agent (or the reverse situation wherein LF modifies effects of PA) such that lethality results.

  6. Uncovering buffered pleiotropy: a genome-scale screen for mel-28 genetic interactors in Caenorhabditis elegans.

    PubMed

    Fernandez, Anita G; Mis, Emily K; Lai, Allison; Mauro, Michael; Quental, Angela; Bock, Carly; Piano, Fabio

    2014-01-10

    mel-28 (maternal-effect-lethal-28) encodes a conserved protein required for nuclear envelope function and chromosome segregation in Caenorhabditis elegans. Because mel-28 is a strict maternal-effect lethal gene, its function is required in the early embryo but appears to be dispensable for larval development. We wanted to test the idea that mel-28 has postembryonic roles that are buffered by the contributions of other genes. To find genes that act coordinately with mel-28, we did an RNA interference-based genetic interaction screen using mel-28 and wild-type larvae. We screened 18,364 clones and identified 65 genes that cause sterility in mel-28 but not wild-type worms. Some of these genes encode components of the nuclear pore. In addition we identified genes involved in dynein and dynactin function, vesicle transport, and cell-matrix attachments. By screening mel-28 larvae we have bypassed the requirement for mel-28 in the embryo, uncovering pleiotropic functions for mel-28 later in development that are normally provided by other genes. This work contributes toward revealing the gene networks that underlie cellular processes and reveals roles for a maternal-effect lethal gene later in development.

  7. Acute and sub-lethal response to mercury in Arctic and boreal calanoid copepods.

    PubMed

    Overjordet, Ida Beathe; Altin, Dag; Berg, Torunn; Jenssen, Bjørn Munro; Gabrielsen, Geir Wing; Hansen, Bjørn Henrik

    2014-10-01

    Acute lethal toxicity, expressed as LC50 values, is a widely used parameter in risk assessment of chemicals, and has been proposed as a tool to assess differences in species sensitivities to chemicals between climatic regions. Arctic Calanus glacialis and boreal Calanus finmarchicus were exposed to mercury (Hg(2+)) under natural environmental conditions including sea temperatures of 2° and 10°C, respectively. Acute lethal toxicity (96 h LC50) and sub-lethal molecular response (GST expression; in this article gene expression is used as a synonym of gene transcription, although it is acknowledged that gene expression is also regulated, e.g., at translation and protein stability level) were studied. The acute lethal toxicity was monitored for 96 h using seven different Hg concentrations. The sub-lethal experiment was set up on the basis of nominal LC50 values for each species using concentrations equivalent to 50, 5 and 0.5% of their 96 h LC50 value. No significant differences were found in acute lethal toxicity between the two species. The sub-lethal molecular response revealed large differences both in response time and the fold induction of GST, where the Arctic species responded both faster and with higher mRNA levels of GST after 48 h exposure. Under the natural exposure conditions applied in the present study, the Arctic species C. glacialis may potentially be more susceptible to mercury exposure on the sub-lethal level.

  8. Alcohol Consumption and Nearly Lethal Suicide Attempts.

    ERIC Educational Resources Information Center

    Powell, Kenneth E.; Kresnow, Marcie-jo; Mercy, James A.; Potter, Lloyd B.; Swann, Alan C.; Frankowski, Ralph F.; Lee, Roberta K.; Bayer, Timothy L.

    2002-01-01

    Presents a case-control study of the association between nearly lethal suicide attempts and facets of alcohol consumption; namely, drinking frequency, drinking quantity, binge drinking, alcoholism, drinking within 3 hours of suicide attempt, and age began drinking. In bivariate analyses, all measures were associated with nearly lethal suicide…

  9. The repair of sub-lethal damage and the stimulated repair of potentially lethal damage in Saintpaulia.

    PubMed

    Leenhouts, H P; Sijsma, M J; Litwiniszyn, M; Chadwick, K H

    1981-10-01

    The repair of sublethal and potentially lethal damage in stationary resting epidermal cells of Saintpaulia has been investigated. Fractionation experiments reveal an efficient repair of sublethal damage with a half-life of 1.9 hours. No repair of potentially lethal damage was noted when cultivation of the leaves was delayed for 24 hours after irradiation. At delay times of 2, 3 and 4 days some repair of potentially lethal damage has been found. A small pre-dose given 24 hours before a challenging dose improved the cells' chance to regenerate and the improvement has been shown to be compatible with an improved repair of potentially lethal damage induced by X-rays and fast neutrons. It hs been shown that the stimulated repair process takes 12 to 24 hours to develop, is dependent on the size of the pre-dose, has single-hit dose kinetics, and an r.b.e. of 1 for neutrons. With delayed cultivation of 2 days the stimulated repair process leads to an alteration in the shape of the regeneration (survival)-dose relationship which increases the low dose r.b.e. for neutrons from 10 to 35. PMID:6975252

  10. A trans-Amazonian screening of mtDNA reveals deep intraspecific divergence in forest birds and suggests a vast underestimation of species diversity.

    PubMed

    Milá, Borja; Tavares, Erika S; Muñoz Saldaña, Alberto; Karubian, Jordan; Smith, Thomas B; Baker, Allan J

    2012-01-01

    The Amazonian avifauna remains severely understudied relative to that of the temperate zone, and its species richness is thought to be underestimated by current taxonomy. Recent molecular systematic studies using mtDNA sequence reveal that traditionally accepted species-level taxa often conceal genetically divergent subspecific lineages found to represent new species upon close taxonomic scrutiny, suggesting that intraspecific mtDNA variation could be useful in species discovery. Surveys of mtDNA variation in Holarctic species have revealed patterns of variation that are largely congruent with species boundaries. However, little information exists on intraspecific divergence in most Amazonian species. Here we screen intraspecific mtDNA genetic variation in 41 Amazonian forest understory species belonging to 36 genera and 17 families in 6 orders, using 758 individual samples from Ecuador and French Guiana. For 13 of these species, we also analyzed trans-Andean populations from the Ecuadorian Chocó. A consistent pattern of deep intraspecific divergence among trans-Amazonian haplogroups was found for 33 of the 41 taxa, and genetic differentiation and genetic diversity among them was highly variable, suggesting a complex range of evolutionary histories. Mean sequence divergence within families was the same as that found in North American birds (13%), yet mean intraspecific divergence in Neotropical species was an order of magnitude larger (2.13% vs. 0.23%), with mean distance between intraspecific lineages reaching 3.56%. We found no clear relationship between genetic distances and differentiation in plumage color. Our results identify numerous genetically and phenotypically divergent lineages which may result in new species-level designations upon closer taxonomic scrutiny and thorough sampling, although lineages in the tropical region could be older than those in the temperate zone without necessarily representing separate species. In-depth phylogeographic surveys

  11. High throughput screening for inhibitors of REST in neural derivatives of human embryonic stem cells reveals a chemical compound that promotes expression of neuronal genes.

    PubMed

    Charbord, Jérémie; Poydenot, Pauline; Bonnefond, Caroline; Feyeux, Maxime; Casagrande, Fabrice; Brinon, Benjamin; Francelle, Laetitia; Aurégan, Gwenaelle; Guillermier, Martine; Cailleret, Michel; Viegas, Pedro; Nicoleau, Camille; Martinat, Cécile; Brouillet, Emmanuel; Cattaneo, Elena; Peschanski, Marc; Lechuga, Marc; Perrier, Anselme L

    2013-09-01

    Decreased expression of neuronal genes such as brain-derived neurotrophic factor (BDNF) is associated with several neurological disorders. One molecular mechanism associated with Huntington disease (HD) is a discrete increase in the nuclear activity of the transcriptional repressor REST/NRSF binding to repressor element-1 (RE1) sequences. High-throughput screening of a library of 6,984 compounds with luciferase-assay measuring REST activity in neural derivatives of human embryonic stem cells led to identify two benzoimidazole-5-carboxamide derivatives that inhibited REST silencing in a RE1-dependent manner. The most potent compound, X5050, targeted REST degradation, but neither REST expression, RNA splicing nor binding to RE1 sequence. Differential transcriptomic analysis revealed the upregulation of neuronal genes targeted by REST in wild-type neural cells treated with X5050. This activity was confirmed in neural cells produced from human induced pluripotent stem cells derived from a HD patient. Acute intraventricular delivery of X5050 increased the expressions of BDNF and several other REST-regulated genes in the prefrontal cortex of mice with quinolinate-induced striatal lesions. This study demonstrates that the use of pluripotent stem cell derivatives can represent a crucial step toward the identification of pharmacological compounds with therapeutic potential in neurological affections involving decreased expression of neuronal genes associated to increased REST activity, such as Huntington disease. PMID:23712629

  12. Virtual Screening of Plant Volatile Compounds Reveals a High Affinity of Hylamorpha elegans (Coleoptera: Scarabaeidae) Odorant-Binding Proteins for Sesquiterpenes From Its Native Host.

    PubMed

    González-González, Angélica; Palma-Millanao, Rubén; Yáñez, Osvaldo; Rojas, Maximiliano; Mutis, Ana; Venthur, Herbert; Quiroz, Andrés; Ramírez, Claudio C

    2016-01-01

    Hylamorpha elegans(Burmeister) is a native Chilean scarab beetle considered to be a relevant agricultural pest to pasture and cereal and small fruit crops. Because of their cryptic habits, control with conventional methods is difficult; therefore, alternative and environmentally friendly control strategies are highly desirable. The study of proteins that participate in the recognition of odorants, such as odorant-binding proteins (OBPs), offers interesting opportunities to identify new compounds with the potential to modify pest behavior and computational screening of compounds, which is commonly used in drug discovery, may help to accelerate the discovery of new semiochemicals. Here, we report the discovery of four OBPs inH. elegans as well as six new volatiles released by its native host Nothofagus obliqua(Mirbel). Molecular docking performed between OBPs and new and previously reported volatiles from N. oblique revealed the best binding energy values for sesquiterpenic compounds. Despite remarkable divergence at the amino acid level, three of the four OBPs evaluated exhibited the best interaction energy for the same ligands. Molecular dynamics investigation reinforced the importance of sesquiterpenes, showing that hydrophobic residues of the OBPs interacted most frequently with the tested ligands, and binding free energy calculations demonstrated van der Waals and hydrophobic interactions to be the most important. Altogether, the results suggest that sesquiterpenes are interesting candidates for in vitro and in vivo assays to assess their potential application in pest management strategies.

  13. Formation of hydrogen sulfide from cysteine in Saccharomyces cerevisiae BY4742: genome wide screen reveals a central role of the vacuole.

    PubMed

    Winter, Gal; Cordente, Antonio G; Curtin, Chris

    2014-01-01

    Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes. PMID:25517415

  14. Formation of Hydrogen Sulfide from Cysteine in Saccharomyces cerevisiae BY4742: Genome Wide Screen Reveals a Central Role of the Vacuole

    PubMed Central

    Winter, Gal; Cordente, Antonio G.; Curtin, Chris

    2014-01-01

    Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes. PMID:25517415

  15. Deep recombination centers in C u2ZnSnS e4 revealed by screened-exchange hybrid density functional theory

    NASA Astrophysics Data System (ADS)

    Yee, Ye Sheng; Magyari-Köpe, Blanka; Nishi, Yoshio; Bent, Stacey F.; Clemens, Bruce M.

    2015-11-01

    We present a comprehensive study of the thermodynamic and electronic properties of intrinsic point defects in the solar energy conversion materials C u2ZnSnS e4 and CuInS e2 based on the screened-exchange hybrid density functional theory. A comparison between the defect transition levels for C u2ZnSnS e4 and CuInS e2 reveals that in C u2ZnSnS e4 , the S nCu and S nZn antisite defects can be recombination centers with defect states close to midgap, while the I nCu antisite defect has a shallow defect level in CuInS e2 . The resultant higher Shockley-Read-Hall recombination rate in C u2ZnSnS e4 reduces the steady-state concentration of minority carriers and quasi-Fermi level separation under illumination. This may explain the origin of the low open-circuit voltage values for C u2ZnSnS e4 solar cells compared to CuInS e2 solar cells.

  16. Virtual Screening of Plant Volatile Compounds Reveals a High Affinity of Hylamorpha elegans (Coleoptera: Scarabaeidae) Odorant-Binding Proteins for Sesquiterpenes From Its Native Host

    PubMed Central

    Palma-Millanao, Rubén; Yáñez, Osvaldo; Rojas, Maximiliano; Mutis, Ana; Venthur, Herbert; Quiroz, Andrés; Ramírez, Claudio C.

    2016-01-01

    Hylamorpha elegans (Burmeister) is a native Chilean scarab beetle considered to be a relevant agricultural pest to pasture and cereal and small fruit crops. Because of their cryptic habits, control with conventional methods is difficult; therefore, alternative and environmentally friendly control strategies are highly desirable. The study of proteins that participate in the recognition of odorants, such as odorant-binding proteins (OBPs), offers interesting opportunities to identify new compounds with the potential to modify pest behavior and computational screening of compounds, which is commonly used in drug discovery, may help to accelerate the discovery of new semiochemicals. Here, we report the discovery of four OBPs in H. elegans as well as six new volatiles released by its native host Nothofagus obliqua (Mirbel). Molecular docking performed between OBPs and new and previously reported volatiles from N. obliqua revealed the best binding energy values for sesquiterpenic compounds. Despite remarkable divergence at the amino acid level, three of the four OBPs evaluated exhibited the best interaction energy for the same ligands. Molecular dynamics investigation reinforced the importance of sesquiterpenes, showing that hydrophobic residues of the OBPs interacted most frequently with the tested ligands, and binding free energy calculations demonstrated van der Waals and hydrophobic interactions to be the most important. Altogether, the results suggest that sesquiterpenes are interesting candidates for in vitro and in vivo assays to assess their potential application in pest management strategies. PMID:27012867

  17. Cultivation-Independent Screening Revealed Hot Spots of IncP-1, IncP-7 and IncP-9 Plasmid Occurrence in Different Environmental Habitats

    PubMed Central

    Dealtry, Simone; Ding, Guo-Chun; Weichelt, Viola; Dunon, Vincent; Schlüter, Andreas; Martini, María Carla; Papa, María Florencia Del; Lagares, Antonio; Amos, Gregory Charles Auton; Wellington, Elizabeth Margaret Helen; Gaze, William Hugo; Sipkema, Detmer; Sjöling, Sara; Springael, Dirk; Heuer, Holger; van Elsas, Jan Dirk; Thomas, Christopher; Smalla, Kornelia

    2014-01-01

    IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are “hot spots” of plasmids potentially carrying catabolic genes. PMID:24587126

  18. Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

    PubMed Central

    Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A.

    2016-01-01

    Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3ʹ end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. PMID:27172183

  19. Formation of hydrogen sulfide from cysteine in Saccharomyces cerevisiae BY4742: genome wide screen reveals a central role of the vacuole.

    PubMed

    Winter, Gal; Cordente, Antonio G; Curtin, Chris

    2014-01-01

    Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes.

  20. Lethal entanglement in baleen whales.

    PubMed

    Cassoff, Rachel M; Moore, Kathleen M; McLellan, William A; Barco, Susan G; Rotsteins, David S; Moore, Michael J

    2011-10-01

    Understanding the scenarios whereby fishing gear entanglement of large whales induces mortality is important for the development of mitigation strategies. Here we present a series of 21 cases involving 4 species of baleen whales in the NW Atlantic, describing the available sighting history, necropsy observations, and subsequent data analyses that enabled the compilation of the manners in which entanglement can be lethal. The single acute cause of entanglement mortality identified was drowning from entanglement involving multiple body parts, with the animal's inability to surface. More protracted causes of death included impaired foraging during entanglement, resulting in starvation after many months; systemic infection arising from open, unresolved entanglement wounds; and hemorrhage or debilitation due to severe gear-related damage to tissues. Serious gear-induced injury can include laceration of large vessels, occlusion of the nares, embedding of line in growing bone, and massive periosteal proliferation of new bone in an attempt to wall off constricting, encircling lines. These data show that baleen whale entanglement is not only a major issue for the conservation of some baleen whale populations, but is also a major concern for the welfare of each affected individual.

  1. Lethal photosensitization of Helicobacter species

    NASA Astrophysics Data System (ADS)

    Millson, Charles E.; Wilson, Michael; MacRobert, Alexander J.; Thurrell, Wendy; Mlkvy, Peter; Davies, Claire; Bown, Stephen G.

    1995-01-01

    Helicobacter pylori (H. pylori) is associated with a large number of gastroduodenal disorders. Clearance of the bacteria has been shown to benefit patients with duodenal ulcers, gastric ulcers, and certain rare types of gastric tumors. Broad-spectrum antibiotics are the mainstay of current treatment strategies but side-effects, poor compliance, and drug resistance limit their usefulness. We sensitized H. pylori with toluidine blue, haematoporphyrin derivative, aluminum disulphonated phthalocyanine, methylene blue or protoporphyrin IX prior to exposure to low-power laser light from either a gallium aluminum arsenide laser or a helium neon gas laser. All 5 sensitizers caused reductions of greater than 1000-fold in the number of viable bacteria. Light alone had no effect and only HpD caused a significant decrease in bacterial numbers without laser light. Next, we sensitized H. mustelae on explanted ferret gastric mucosa (ex vivo) with the same sensitizers and exposed them to light from a copper vapor pumped dye laser tuned appropriately. MB caused significant reductions in bacterial counts. Successful lethal photosensitization of Helicobacter pylori both in vitro and ex vivo raises the possibility of a local method for eradicating the bacteria, especially as the bacteria are only found in those parts of the upper gastrointestinal tract that are accessible to the endoscope.

  2. The flap by flap dissection in terminal ballistic applied to less lethal weapons.

    PubMed

    de Freminville, Humbert; Rongieras, Fréderic; Prat, Nicolas; Voiglio, Eric J

    2011-06-01

    Medical examiners often have to solve questions such as firing distance and bullet trajectory for lethal weapons. Knowledge in the field of terminal ballistics has increased during the last 30 years and layer by layer dissection reveals superficial wounds that can be linked with the permanent cavity. At the end of the 1990s, terminal ballistics also focused on less lethal weapons and their wounds. Here, 2 different less lethal weapons with single bullets were tested on nonembalmed and undressed cadavers (N = 26) at different ranges and speeds. We have developed a technique for dissection which we call flap by flap dissection that reveals the advantage of the bullet-skin-bone entity, the absence of wounds linking its components and range of less lethal weapons.

  3. High-throughput Screening of ToxCast" Phase I Chemicals in an Embryonic Stem Cell Assay Reveals Potential Disruption of a Critical Developmental Signaling Pathway

    EPA Science Inventory

    Little is known about the developmental toxicity of the expansive chemical landscape in existence today. Significant efforts are being made to apply novel methods to predict developmental activity of chemicals utilizing high-throughput screening (HTS) and high-content screening (...

  4. Sequence-Based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families

    PubMed Central

    Maimanakos, Janine; Chow, Jennifer; Gaßmeyer, Sarah K.; Güllert, Simon; Busch, Florian; Kourist, Robert; Streit, Wolfgang R.

    2016-01-01

    Arylmalonate Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta-, and Gamma-proteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the tripartite tricarboxylate transporters family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes. PMID:27610105

  5. Large Scale Screening of Digeneans for Neorickettsia Endosymbionts Using Real-Time PCR Reveals New Neorickettsia Genotypes, Host Associations and Geographic Records

    PubMed Central

    Greiman, Stephen E.; Tkach, Vasyl V.; Pulis, Eric; Fayton, Thomas J.; Curran, Stephen S.

    2014-01-01

    Digeneans are endoparasitic flatworms with complex life cycles including one or two intermediate hosts (first of which is always a mollusk) and a vertebrate definitive host. Digeneans may harbor intracellular endosymbiotic bacteria belonging to the genus Neorickettsia (order Rickettsiales, family Anaplasmataceae). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of wildlife and humans. In this study we report the results of screening 771 digenean samples for Neorickettsia collected from various vertebrates in terrestrial, freshwater, brackish, and marine habitats in the United States, China and Australia. Neorickettsia were detected using a newly designed real-time PCR protocol targeting a 152 bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1371 bp long region of 16S rRNA. Eight isolates of Neorickettsia have been obtained. Sequence comparison and phylogenetic analysis demonstrated that 7 of these isolates, provisionally named Neorickettsia sp. 1–7 (obtained from allocreadiid Crepidostomum affine, haploporids Saccocoelioides beauforti and Saccocoelioides lizae, faustulid Bacciger sprenti, deropegid Deropegus aspina, a lecithodendriid, and a pleurogenid) represent new genotypes and one (obtained from Metagonimoides oregonensis) was identical to a published sequence of Neorickettsia known as SF agent. All digenean species reported in this study represent new host records. Three of the 6 digenean families (Haploporidae, Pleurogenidae, and Faustulidae) are also reported for the first time as hosts of Neorickettsia. We have detected Neorickettsia in digeneans from China and Australia for the first time based on PCR and sequencing evidence. Our findings suggest that further surveys from broader geographic regions and wider selection of digenean taxa are likely to reveal new Neorickettsia lineages as well as new digenean host associations. PMID

  6. miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma.

    PubMed

    Khalil, Susanna; Fabbri, Enrica; Santangelo, Alessandra; Bezzerri, Valentino; Cantù, Cinzia; Di Gennaro, Gianfranco; Finotti, Alessia; Ghimenton, Claudio; Eccher, Albino; Dechecchi, Maria; Scarpa, Aldo; Hirshman, Brian; Chen, Clark; Ferracin, Manuela; Negrini, Massimo; Gambari, Roberto; Cabrini, Giulio

    2016-05-10

    The levels of expression of O6-methylguanine-DNA methyltransferase (MGMT) are relevant in predicting the response to the alkylating chemotherapy in patients affected by glioblastoma. MGMT promoter methylation and the published MGMT regulating microRNAs (miRNAs) do not completely explain the expression pattern of MGMT in clinical glioblastoma specimens. Here we used a genome-wide microarray-based approach to identify MGMT regulating miRNAs. Our screen unveiled three novel MGMT regulating miRNAs, miR-127-3p, miR-409-3p, and miR-124-3p, in addition to the previously identified miR-181d-5p. Transfection of these three novel miRNAs into the T98G glioblastoma cell line suppressed MGMT mRNA and protein expression. However, their MGMT- suppressive effects are 30-50% relative that seen with miR-181d-5p transfection. In silico analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) revealed that miR-181d-5p is the only miRNA that consistently exhibited inverse correlation with MGMT mRNA expression. However, statistical models incorporating both miR-181d-5p and miR-409-3p expression better predict MGMT expression relative to models involving either miRNA alone. Our results confirmed miR-181d-5p as the key MGMT-regulating miRNA. Other MGMT regulating miRNAs, including the miR-409-3p identified in this report, modify the effect of miR-181d-5p on MGMT expression. MGMT expression is, thus, regulated by cooperative interaction between key MGMT-regulating miRNAs. PMID:27057640

  7. miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

    PubMed Central

    Khalil, Susanna; Fabbri, Enrica; Santangelo, Alessandra; Bezzerri, Valentino; Cantù, Cinzia; Gennaro, Gianfranco Di; Finotti, Alessia; Ghimenton, Claudio; Eccher, Albino; Dechecchi, Maria; Scarpa, Aldo; Hirshman, Brian; Chen, Clark; Ferracin, Manuela; Negrini, Massimo; Gambari, Roberto; Cabrini, Giulio

    2016-01-01

    The levels of expression of O6-methylguanine-DNA methyltransferase (MGMT) are relevant in predicting the response to the alkylating chemotherapy in patients affected by glioblastoma. MGMT promoter methylation and the published MGMT regulating microRNAs (miRNAs) do not completely explain the expression pattern of MGMT in clinical glioblastoma specimens. Here we used a genome-wide microarray-based approach to identify MGMT regulating miRNAs. Our screen unveiled three novel MGMT regulating miRNAs, miR-127-3p, miR-409-3p, and miR-124-3p, in addition to the previously identified miR-181d-5p. Transfection of these three novel miRNAs into the T98G glioblastoma cell line suppressed MGMT mRNA and protein expression. However, their MGMT- suppressive effects are 30–50% relative that seen with miR-181d-5p transfection. In silico analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) revealed that miR-181d-5p is the only miRNA that consistently exhibited inverse correlation with MGMT mRNA expression. However, statistical models incorporating both miR-181d-5p and miR-409-3p expression better predict MGMT expression relative to models involving either miRNA alone. Our results confirmed miR-181d-5p as the key MGMT-regulating miRNA. Other MGMT regulating miRNAs, including the miR-409-3p identified in this report, modify the effect of miR-181d-5p on MGMT expression. MGMT expression is, thus, regulated by cooperative interaction between key MGMT-regulating miRNAs. PMID:27057640

  8. Sequence-Based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.

    PubMed

    Maimanakos, Janine; Chow, Jennifer; Gaßmeyer, Sarah K; Güllert, Simon; Busch, Florian; Kourist, Robert; Streit, Wolfgang R

    2016-01-01

    Arylmalonate Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica's prototype appeared to be limited to the classes of Alpha-, Beta-, and Gamma-proteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the tripartite tricarboxylate transporters family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes. PMID:27610105

  9. Sequence-Based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families

    PubMed Central

    Maimanakos, Janine; Chow, Jennifer; Gaßmeyer, Sarah K.; Güllert, Simon; Busch, Florian; Kourist, Robert; Streit, Wolfgang R.

    2016-01-01

    Arylmalonate Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta-, and Gamma-proteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the tripartite tricarboxylate transporters family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes.

  10. [Synthetic lethal genes to mutant p53].

    PubMed

    Tongyang, Liu; Haiqiang, Guo; Meiyan, Zhu; Yingze, Huang; Shuting, Jia; Ying, Luo; Jihong, Zhang

    2015-04-01

    Targeted therapy has become a powerful approach for cancer treatment. Better understanding of oncogenes as well as synthetic lethal interactions with oncogenes will lead to new strategies for tumor-specific treatment. It is well known that mutant p53 plays an important role in tumorigenesis and tumor development. Thus, understanding the synthetic lethal relationship between p53 mutations and interacting genes in tumor is critical for the personalized treatments of p53 mutant tumors. Synthetic lethal genes to mutant p53 can be divided into cell cycle regulators and non-cell cycle regulators. This paper review show these two types of target genes contribute to synthetic lethal interactions with p53 mutations and potential applications of these interactions in anticancer therapy.

  11. On lethal injections and the death penalty.

    PubMed

    1982-10-01

    A brief account is given of the recent adoption by several states of capital punishment by lethal injection and the objections that this move has aroused. Critics argue that, because of difficulties in administering the drugs and in determining the proper dosage, this method may actually be less humane than other means of execution. Articles in medical journals have forcefully expressed the profession's opposition to lethal injections and to physician involvement in their administration.

  12. Theory of lethal mutagenesis for viruses.

    PubMed

    Bull, J J; Sanjuán, R; Wilke, C O

    2007-03-01

    Mutation is the basis of adaptation. Yet, most mutations are detrimental, and elevating mutation rates will impair a population's fitness in the short term. The latter realization has led to the concept of lethal mutagenesis for curing viral infections, and work with drugs such as ribavirin has supported this perspective. As yet, there is no formal theory of lethal mutagenesis, although reference is commonly made to Eigen's error catastrophe theory. Here, we propose a theory of lethal mutagenesis. With an obvious parallel to the epidemiological threshold for eradication of a disease, a sufficient condition for lethal mutagenesis is that each viral genotype produces, on average, less than one progeny virus that goes on to infect a new cell. The extinction threshold involves an evolutionary component based on the mutation rate, but it also includes an ecological component, so the threshold cannot be calculated from the mutation rate alone. The genetic evolution of a large population undergoing mutagenesis is independent of whether the population is declining or stable, so there is no runaway accumulation of mutations or genetic signature for lethal mutagenesis that distinguishes it from a level of mutagenesis under which the population is maintained. To detect lethal mutagenesis, accurate measurements of the genome-wide mutation rate and the number of progeny per infected cell that go on to infect new cells are needed. We discuss three methods for estimating the former. Estimating the latter is more challenging, but broad limits to this estimate may be feasible.

  13. Structural basis for a lethal mutation in U6 RNA.

    PubMed

    Sashital, Dipali G; Allmann, Anne M; Van Doren, Steven R; Butcher, Samuel E

    2003-02-18

    U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal. The U80G mutation at the essential magnesium binding site of the U6 3' intramolecular stem-loop region (ISL) is lethal in yeast. To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy. The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson-Crick base pair (C67 x G80), and disrupts a protonated C67 x A79 wobble pair that forms in the wild-type structure. Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs. The thermodynamic stability of the U80G mutant is significantly increased (Delta Delta G(fold) = -3.6 +/- 1.9 kcal/mol), consistent with formation of the Watson-Crick pair. Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem-loop hyperstabilization. This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4. PMID:12578359

  14. Crystallographic studies of the Anthrax lethal toxin. Annual report

    SciTech Connect

    Frederick, C.A.

    1996-07-01

    The lethal form of Anthrax results from the inhalation of anthrax spores. Death is primarily due to the effects of the lethal toxin (Protective Antigen (PA) + Lethal Factor) from the causative agent, Bacillus anthracis. All the Anthrax vaccines currently in use or under development contain or produce PA, the major antigenic component of anthrax toxin, and there is a clear need for an improved vaccine for human use. In the previous report we described the first atomic resolution structure of PA, revealing that the molecule is composed largely of beta-sheets organized into four domains. This information can be used in the design. of recombinant PA vaccines. In this report we describe additional features of the full-length PA molecule derived from further crystallographic refinement and careful examination of the structure. We compare two crystal forms of PA grown at different pH values and discuss the functional implications. A complete definition of the function of each domain must await the crystal structure of the PA63 heptamer. We have grown crystals of the heptamer under both detergent and detergent-free conditions, and made substantial progress towards the crystal structure. The mechanism of anthrax intoxication in the light of our results is reviewed.

  15. Lethal Effects of Helianthemum lippii (L.) on Acanthamoeba castellanii Cysts in Vitro

    PubMed Central

    Badria, F.A.; Hetta, M.H.; Sarhan, Rania M.; Ezz El-Din, M.H.

    2014-01-01

    Acanthamoeba spp. commonly cause Acanthamoeba keratitis which is typically associated with the wear of contact lenses. Therefore, finding an economic, efficient, and safe therapy of natural origin is of outmost importance. This study examined the in vitro lethal potential of ethyl acetate and methanol extracts of Helianthemum lippii (L.) (sun roses) against Acanthamoeba castellanii cysts isolated from patients with amoebic keratitis. Both extracts proved to be potent as regard to their lethal effects on A. castellanii cysts with comparable results to chlorhexidine. The ethyl acetate was more promising with cumulative lethality. It showed a highly significant lethal percentage along the duration of treatment. The analysis of the more potent ethyl acetate extract revealed the presence of 2.96 mg/100 g of total phenolics, 0.289 mg/100 ml of total flavonoids and 37 mg/100 mg of total tannins which highlighted their phytomedicinal role. PMID:25031463

  16. Enhancing the stability and ecological safety of mass-reared transgenic strains for field release by redundant conditional lethality systems.

    PubMed

    Handler, Alfred M

    2016-04-01

    The genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically-based population control programs, most notably the Sterile Insect Technique (SIT), for both plant and animal insect pests. Tetracycline-suppressible (Tet-off) conditional lethal systems may function together so that transgenic strains will be viable and fertile on a tetracycline-containing diet, but female-lethal and male sterile in tetracycline-free conditions. This would allow their most efficacious use in a unified system for sterile male-only production for SIT. A critical consideration for the field release of such transgenic insect strains, however, is a determination of the frequency and genetic basis of lethality revertant survival. This will provide knowledge essential to evaluating the genetic stability of the lethality system, its environmental safety, and provide the basis for modifications ensuring optimal efficacy. For Tet-off lethal survival determinations, development of large-scale screening protocols should also allow the testing of these modifications, and test the ability of other conditional lethal systems to fully suppress propagation of rare Tet-off survivors. If a dominant temperature sensitive (DTS) pupal lethality system proves efficient for secondary lethality in Drosophila, it may provide the safeguard needed to support the release of sexing/sterility strains, and potentially, the release of unisex lethality strains as a form of genetic male sterility. Should the DTS Prosβ2(1) mutation prove effective for redundant lethality, its high level of structural and functional conservation should allow host-specific cognates to be created for a wide range of insect species.

  17. New form of platyspondylic lethal chondrodysplasia.

    PubMed

    Akaba, K; Nishimura, G; Hashimoto, M; Wakabayashi, T; Kanasugi, H; Hayasaka, K

    1996-12-30

    We report on a sporadic case of hitherto unknown lethal skeletal dysplasia. The cardinal clinical manifestations consisted of frontal bossing, cloudy corneae, low nasal ridge, and micrognathia, hypoplastic thorax, and rhizomelic micromelia. Laryngoscopy and neck CT disclosed laryngeal stenosis, and brain CT demonstrated hypoplasia of the corpus callosum. Skeletal survey demonstrated hypoplasia of facial bones and short skull base, extremely severe platyspondyly, hypoplastic ilia, and delayed epiphyseal ossification and rhizomelic shortness of tubular bones. The long bones appeared overtubulated with exaggerated metaphyseal flaring. The humeri were particularly short and bowed. Bowing of the radii and ulnae with subluxation of radial heads presented as a Madelung-like deformity. Unlike the long bones, the short tubular bones were not short and normally modeled. The skeletal changes were superficially similar to those in a group of lethal platyspondylic chondrodysplasias, but were inconsistent with any known subtypes of this group or other lethal skeletal dysplasias. PMID:8989469

  18. Inadequate anaesthesia in lethal injection for execution.

    PubMed

    Koniaris, Leonidas G; Zimmers, Teresa A; Lubarsky, David A; Sheldon, Jonathan P

    Anaesthesia during lethal injection is essential to minimise suffering and to maintain public acceptance of the practice. Lethal injection is usually done by sequential administration of thiopental, pancuronium, and potassium chloride. Protocol information from Texas and Virginia showed that executioners had no anaesthesia training, drugs were administered remotely with no monitoring for anaesthesia, data were not recorded and no peer-review was done. Toxicology reports from Arizona, Georgia, North Carolina, and South Carolina showed that post-mortem concentrations of thiopental in the blood were lower than that required for surgery in 43 of 49 executed inmates (88%); 21 (43%) inmates had concentrations consistent with awareness. Methods of lethal injection anaesthesia are flawed and some inmates might experience awareness and suffering during execution.

  19. Live deaths online: internet suicide and lethality.

    PubMed

    Klein, Carolina A

    2012-01-01

    The Internet provides an infinite platform for the portrayal of lethal events. Beyond mere display, however, it dispenses information, allows for participation and sharing of content, and constitutes a virtual interactive forum. The Internet may ultimately shape society's approach to perceiving and dealing with death. Thus, psychiatrists may wish to be aware of these matters so that they may be considered in assessments and clinical care. In this article, the author attempts to identify key online locations where lethality is portrayed and how it may affect the individual patient and practitioner and the population at large.

  20. Therapeutically targeting RNA viruses via lethal mutagenesis.

    PubMed

    Graci, Jason D; Cameron, Craig E

    2008-11-01

    RNA viruses exhibit increased mutation frequencies relative to other organisms. Recent work has attempted to exploit this unique feature by increasing the viral mutation frequency beyond an extinction threshold, an antiviral strategy known as lethal mutagenesis. A number of novel nucleoside analogs have been designed around this premise. Herein, we review the quasispecies nature of RNA viruses and survey the antiviral, biological and biochemical characteristics of mutagenic nucleoside analogs, including clinically-used ribavirin. Biological implications of modulating viral replication fidelity are discussed in the context of translating lethal mutagenesis into a clinically-useful antiviral strategy.

  1. High-throughput Screening of ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell (mESC) Assay Reveals Disruption of Potential Toxicity Pathways

    EPA Science Inventory

    Little information is available regarding the potential for many commercial chemicals to induce developmental toxicity. The mESC Adherent Cell Differentiation and Cytoxicity (ACDC) assay is a high-throughput screen used to close this data gap. Thus, ToxCast™ Phase I chemicals wer...

  2. Preparation and characterization of cobalt-substituted anthrax lethal factor

    SciTech Connect

    Saebel, Crystal E.; Carbone, Ryan; Dabous, John R.; Lo, Suet Y.; Siemann, Stefan

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Cobalt-substituted anthrax lethal factor (CoLF) is highly active. Black-Right-Pointing-Pointer CoLF can be prepared by bio-assimilation and direct exchange. Black-Right-Pointing-Pointer Lethal factor binds cobalt tightly. Black-Right-Pointing-Pointer The electronic spectrum of CoLF reveals penta-coordination. Black-Right-Pointing-Pointer Interaction of CoLF with thioglycolic acid follows a 2-step mechanism. -- Abstract: Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of mitogen-activated protein kinase kinases near their N-termini. The current report concerns the preparation of cobalt-substituted LF (CoLF) and its characterization by electronic spectroscopy. Two strategies to produce CoLF were explored, including (i) a bio-assimilation approach involving the cultivation of LF-expressing Bacillus megaterium cells in the presence of CoCl{sub 2}, and (ii) direct exchange by treatment of zinc-LF with CoCl{sub 2}. Independent of the method employed, the protein was found to contain one Co{sup 2+} per LF molecule, and was shown to be twice as active as its native zinc counterpart. The electronic spectrum of CoLF suggests the Co{sup 2+} ion to be five-coordinate, an observation similar to that reported for other Co{sup 2+}-substituted gluzincins, but distinct from that documented for the crystal structure of native LF. Furthermore, spectroscopic studies following the exposure of CoLF to thioglycolic acid (TGA) revealed a sequential mechanism of metal removal from LF, which likely involves the formation of an enzyme: Co{sup 2+}:TGA ternary complex prior to demetallation of the active site. CoLF reported herein constitutes the first spectroscopic probe of LF's active site, which may be utilized in future studies to gain further insight into the enzyme's mechanism and inhibitor interactions.

  3. Identification of a De Novo Heterozygous Missense FLNB Mutation in Lethal Atelosteogenesis Type I by Exome Sequencing

    PubMed Central

    Jeon, Ga Won; Lee, Mi-Na; Jung, Ji Mi; Hong, Seong Yeon; Kim, Young Nam; Sin, Jong Beom

    2014-01-01

    Background Atelosteogenesis type I (AO-I) is a rare lethal skeletal dysplastic disorder characterized by severe short-limbed dwarfism and dislocated hips, knees, and elbows. AO-I is caused by mutations in the filamin B (FLNB) gene; however, several other genes can cause AO-like lethal skeletal dysplasias. Methods In order to screen all possible genes associated with AO-like lethal skeletal dysplasias simultaneously, we performed whole-exome sequencing in a female newborn having clinical features of AO-I. Results Exome sequencing identified a novel missense variant (c.517G>A; p.Ala173Thr) in exon 2 of the FLNB gene in the patient. Sanger sequencing validated this variant, and genetic analysis of the patient's parents suggested a de novo occurrence of the variant. Conclusions This study shows that exome sequencing can be a useful tool for the identification of causative mutations in lethal skeletal dysplasia patients. PMID:24624349

  4. Comparative analysis of RNAi screening technologies at genome-scale reveals an inherent processing inefficiency of the plasmid-based shRNA hairpin

    PubMed Central

    Bhinder, Bhavneet; Shum, David; Djaballah, Hakim

    2014-01-01

    RNAi screening in combination with the genome-sequencing projects would constitute the Holy Grail of modern genetics; enabling discovery and validation towards a better understanding of fundamental biology leading to novel targets to combat disease. Hit discordance at inter-screen level together with the lack of reproducibility is emerging as the technology's main pitfalls. To examine some of the underlining factors leading to such discrepancies, we reasoned that perhaps there is an inherent difference in knockdown efficiency of the various RNAi technologies. For this purpose, we utilized the two most popular ones, chemically synthesized siRNA duplex and plasmid-based shRNA hairpin, in order to perform a head to head comparison. Using a previously developed gain-of-function assay probing modulators of the miRNA biogenesis pathway, we first executed on a siRNA screen against the Silencer Select V4.0 library (AMB) nominating 1,273, followed by an shRNA screen against the TRC1 library (TRC1) nominating 497 gene candidates. We observed a poor overlap of only 29 hits given that there are 15,068 overlapping genes between the two libraries; with DROSHA as the only common hit out of the seven known core miRNA biogenesis genes. Distinct genes interacting with the same biogenesis regulators were observed in both screens, with a dismal cross-network overlap of only 3 genes (DROSHA, TGFBR1, and DIS3). Taken together, our study demonstrates differential knockdown activities between the two technologies, possibly due to the inefficient intracellular processing and potential cell-type specificity determinants in generating intended targeting sequences for the plasmid-based shRNA hairpins; and suggests this observed inefficiency as potential culprit in addressing the lack of reproducibility. PMID:24433414

  5. Development of ELISA based detection system for lethal toxin of Clostridium sordellii

    PubMed Central

    Arya, Preetika; Ponmariappan, S.; Singh, Lokendra; Kumar, Om

    2013-01-01

    Background & objectives: Clostridium sordellii and its toxins are associated with diseases in animals as well as human. C. sordellii produces two protein toxins (lethal toxin and haemorrhagic toxin). Lethal toxin has gained more importance due its high toxicity. The present study was carried out to develop a sandwich ELISA for detection of lethal toxin of C. sordellii. Methods: The catalytic domain (1.6kb) of lethal toxin of C. sordellii was PCR amplified, cloned into pQE30 UA vector and transformed into Escherichia coli SG 13009. Expression conditions were optimized and the recombinant protein was purified under native condition using Ni-NTA affinity chromatography, confirmed by SDS-PAGE and Western blot. Antibody was generated against the purified recombinant protein using Freund's complete and incomplete adjuvants (FCA and FIA) in BALB/c mice and rabbit. A sandwich ELISA was optimized for the detection of lethal toxin. Results: The maximum recombinant protein expression was achieved at 0.5 mM IPTG (isopropylthiogalactoside) induction 4.0 h of post-induction. The polyclonal antibody raised in mice and rabbit showed a titre up to 1:512000. The produced antibody was highly sensitive with the detection limit of 0.3 ng/ml of lethal toxin at 1:4000 dilutions of mice (capturing) and rabbit (revealing) antibody. Interpretation & conclusions: An ELISA based detection system was developed for the detection of lethal toxin of C. sordellii. The developed detection system was found to be specific as there was no cross-reactivity with any other clostridial toxins. It will be useful for the detection of lethal toxin of C. sordellii in clinical and environmental samples. PMID:23852299

  6. Macrophage cytotoxicity in lethal and non-lethal murine malaria and the effect of vaccination.

    PubMed Central

    Taverne, J; Treagust, J D; Playfair, J H

    1986-01-01

    We investigated the development of cell-mediated immunity in lethal and non-lethal malarial infections by assaying the cytotoxic activity of spleen cells for L929 tumour cells at different times after infection of mice with the lethal P. berghei, a lethal variant of Plasmodium yoelii and the non-lethal P. yoelii and P. chabaudi. In all cases the cytotoxicity increased to a peak during the first week and then diminished but the time of the peak varied with the infection; its activity was lowest with P. berghei. A second peak occurred in the non-lethal infections at the time of recovery. A protective vaccine accelerated and enhanced the early peak of cytotoxicity. The activity was mediated by adherent phagocytic cells, probably through the release of tumour necrosis factor (TNF) by macrophages since it was inhibited by antiserum against recombinant mouse TNF and did not destroy TNF-resistant L929 cells. Its induction was not dependent on T cells since it occurred in T cell-deficient mice infected with non-lethal P. yoelii. However, the accelerated increase associated with vaccination could be adoptively transferred by spleen lymphocytes from vaccinated mice. PMID:3542317

  7. Suicide intent and accurate expectations of lethality: predictors of medical lethality of suicide attempts.

    PubMed

    Brown, Gregory K; Henriques, Gregg R; Sosdjan, Daniella; Beck, Aaron T

    2004-12-01

    The degree of intent to commit suicide and the severity of self-injury were examined in individuals (N = 180) who had recently attempted suicide. Although a minimal association was found between the degree of suicide intent and the degree of lethality of the attempt, the accuracy of expectations about the likelihood of dying was found to moderate the relationship between suicide intent and lethality. Specifically, higher levels of suicide intent were associated with more lethal attempts but only for those individuals who had more accurate expectations about the likelihood of dying from their attempts.

  8. Deadly Lessons: Understanding Lethal School Violence.

    ERIC Educational Resources Information Center

    Moore, Mark H., Ed.; Petrie, Carol V., Ed.; Braga, Anthony A., Ed.; McLaughlin, Brenda L., Ed.

    This collection of papers is the outcome of the National Academies' effort to glean information from six different case studies of student-perpetrated school shootings. Part 1, "Case Studies of Lethal School Violence," includes: "The Copycat Factor: Mental Illness, Guns, and the Shooting Incident at Heritage High School, Rockdale County, Georgia"…

  9. The evolution of lethal intergroup violence

    PubMed Central

    Kelly, Raymond C.

    2005-01-01

    Recent findings and analyses in evolutionary biology, archaeology, and ethnology provide a favorable conjuncture for examining the evolution of lethal intergroup violence among hominids during the 2.9-million-year Paleolithic time span. Here, I seek to identify and investigate the main turning points in this evolutionary trajectory and to delineate the periodization that follows from this inquiry. PMID:16129826

  10. Analysis of mutants from a genetic screening reveals the control of intestine and liver development by many common genes in zebrafish.

    PubMed

    Jiang, Faming; Chen, Jiehui; Ma, Xirui; Huang, Chao; Zhu, Shicheng; Wang, Fei; Li, Li; Luo, Lingfei; Ruan, Hua; Huang, Honghui

    2015-05-01

    Both the intestine and liver develop from the endoderm, yet little is known how these two digestive organs share and differ in their developmental programs, at the molecular level. A classical forward genetic screen, with no gene bias, is an effective way to address this question by examining the defects of the intestine and liver in obtained mutants to assess mutated genes responsible for the development of either organ or both. We report here such a screen in zebrafish. ENU was used as the mutagen because of its high mutagenic efficiency and no site preference. Embryos were collected at 3.5 dpf for RNA whole mount in situ hybridization with a cocktail probe of the intestine marker ifabp and the liver marker lfabp to check phenotypes and determine their parental heterozygosis. A total of 52 F2 putative mutants were identified, and those with general developmental defects were aborted. To rule out non-inheritable phenotypes caused by high mutation background, F2 putative mutants were outcrossed with wild type fish and a re-screen in F3 generations was performed. After complementation tests between F3 mutants with similar phenotypes originating from the same F2 families, a total of 37 F3 mutant lines originated from 22 F2 families were identified after screening 78 mutagenized genomes. Classification of mutant phenotypes indicated that 31 out of the 37 mutants showed defects in both the intestine and liver. In addition, four "intestine specific mutants" and two "liver specific mutants" showed selectively more severe phenotype in the intestine and liver respectively. These results suggested that the intestine and liver share a substantial number of essential genes during both organs development in zebrafish. Further studies of the mutants are likely to shed more insights into the molecular basis of the digestive system development in the zebrafish and vertebrate.

  11. Genetic Panel Screening of Nearly 100 Mutations Reveals New Insights into the Breed Distribution of Risk Variants for Canine Hereditary Disorders

    PubMed Central

    Donner, Jonas; Möller, Fredrik; Kyöstilä, Kaisa; Sankari, Satu; Hytönen, Marjo; Giger, Urs; Lohi, Hannes

    2016-01-01

    Background The growing number of identified genetic disease risk variants across dog breeds challenges the current state-of-the-art of population screening, veterinary molecular diagnostics, and genetic counseling. Multiplex screening of such variants is now technologically feasible, but its practical potential as a supportive tool for canine breeding, disease diagnostics, pet care, and genetics research is still unexplored. Results To demonstrate the utility of comprehensive genetic panel screening, we tested nearly 7000 dogs representing around 230 breeds for 93 disease-associated variants using a custom-designed genotyping microarray (the MyDogDNA® panel test). In addition to known breed disease-associated mutations, we discovered 15 risk variants in a total of 34 breeds in which their presence was previously undocumented. We followed up on seven of these genetic findings to demonstrate their clinical relevance. We report additional breeds harboring variants causing factor VII deficiency, hyperuricosuria, lens luxation, von Willebrand’s disease, multifocal retinopathy, multidrug resistance, and rod-cone dysplasia. Moreover, we provide plausible molecular explanations for chondrodysplasia in the Chinook, cerebellar ataxia in the Norrbottenspitz, and familiar nephropathy in the Welsh Springer Spaniel. Conclusions These practical examples illustrate how genetic panel screening represents a comprehensive, efficient and powerful diagnostic and research discovery tool with a range of applications in veterinary care, disease research, and breeding. We conclude that several known disease alleles are more widespread across different breeds than previously recognized. However, careful follow up studies of any unexpected discoveries are essential to establish genotype-phenotype correlations, as is readiness to provide genetic counseling on their implications for the dog and its breed. PMID:27525650

  12. Cholesterol Metabolism and Prostate Cancer Lethality.

    PubMed

    Stopsack, Konrad H; Gerke, Travis A; Sinnott, Jennifer A; Penney, Kathryn L; Tyekucheva, Svitlana; Sesso, Howard D; Andersson, Swen-Olof; Andrén, Ove; Cerhan, James R; Giovannucci, Edward L; Mucci, Lorelei A; Rider, Jennifer R

    2016-08-15

    Cholesterol metabolism has been implicated in prostate cancer pathogenesis. Here, we assessed the association of intratumoral mRNA expression of cholesterol synthesis enzymes, transporters, and regulators in tumor specimen at diagnosis and lethal prostate cancer, defined as mortality or metastases from prostate cancer in contrast to nonlethal disease without evidence of metastases after at least 8 years of follow-up. We analyzed the prospective prostate cancer cohorts within the Health Professionals Follow-up Study (n = 249) and the Physicians' Health Study (n = 153) as well as expectantly managed patients in the Swedish Watchful Waiting Study (n = 338). The expression of squalene monooxygenase (SQLE) was associated with lethal cancer in all three cohorts. Men with high SQLE expression (>1 standard deviation above the mean) were 8.3 times (95% confidence interval, 3.5 to 19.7) more likely to have lethal cancer despite therapy compared with men with the mean level of SQLE expression. Absolute SQLE expression was associated with lethal cancer independently from Gleason grade and stage, as was a SQLE expression ratio in tumor versus surrounding benign prostate tissue. Higher SQLE expression was tightly associated with increased histologic markers of angiogenesis. Collectively, this study establishes the prognostic value of intratumoral cholesterol synthesis as measured via SQLE, its second rate-limiting enzyme. SQLE expression at cancer diagnosis is prognostic for lethal prostate cancer both after curative-intent prostatectomy and in a watchful waiting setting, possibly by facilitating micrometastatic disease. Cancer Res; 76(16); 4785-90. ©2016 AACR.

  13. 5-Lipoxygenase Deficiency Reduces Acetaminophen-Induced Hepatotoxicity and Lethality

    PubMed Central

    Hohmann, Miriam S. N.; Cardoso, Renato D. R.; Pinho-Ribeiro, Felipe A.; Crespigio, Jefferson; Cunha, Thiago M.; Alves-Filho, José C.; da Silva, Rosiane V.; Pinge-Filho, Phileno; Ferreira, Sergio H.; Cunha, Fernando Q.; Casagrande, Rubia; Verri, Waldiceu A.

    2013-01-01

    5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO−/−) mice and background wild type mice were challenged with APAP (0.3–6 g/kg) or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB4, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3 g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO−/− mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1β, TNF-α, IFN-γ, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-β-D-glucosaminidase activity, Nrf2 and gp91phox mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate) assay were prevented in 5-LO−/− mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage. PMID:24288682

  14. 3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems.

    PubMed

    Howes, Amy L; Richardson, Robyn D; Finlay, Darren; Vuori, Kristiina

    2014-01-01

    3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery. PMID:25247711

  15. Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato.

    PubMed

    Burra, D D; Mühlenbock, P; Andreasson, E

    2015-09-01

    Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro-based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone- related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis-related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya.

  16. Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato.

    PubMed

    Burra, D D; Mühlenbock, P; Andreasson, E

    2015-09-01

    Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro-based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone- related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis-related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya. PMID:25903921

  17. The Spatial Concentration of Southern Whites and Argument-Based Lethal Violence

    ERIC Educational Resources Information Center

    Lee, Matthew R.; Shihadeh, Edward S.

    2009-01-01

    This analysis examines how the spatial concentration of Southern whites is associated with white argument-based lethal violence. Using a well-known measure of spatial segregation (V, the adjusted P* index) among Southern-born whites in U.S. counties in 2000, the results reveal that the spatial concentration of Southern-born whites is only…

  18. Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

    PubMed

    Ando, Akira; Nakamura, Toshihide; Murata, Yoshinori; Takagi, Hiroshi; Shima, Jun

    2007-03-01

    Yeasts used in bread making are exposed to freeze-thaw stress during frozen-dough baking. To clarify the genes required for freeze-thaw tolerance, genome-wide screening was performed using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 58 gene deletions that conferred freeze-thaw sensitivity. These genes were then classified based on their cellular function and on the localization of their products. The results showed that the genes required for freeze-thaw tolerance were frequently involved in vacuole functions and cell wall biogenesis. The highest numbers of gene products were components of vacuolar H(+)-ATPase. Next, the cross-sensitivity of the freeze-thaw-sensitive mutants to oxidative stress and to cell wall stress was studied; both of these are environmental stresses closely related to freeze-thaw stress. The results showed that defects in the functions of vacuolar H(+)-ATPase conferred sensitivity to oxidative stress and to cell wall stress. In contrast, defects in gene products involved in cell wall assembly conferred sensitivity to cell wall stress but not to oxidative stress. Our results suggest the presence of at least two different mechanisms of freeze-thaw injury: oxidative stress generated during the freeze-thaw process, and defects in cell wall assembly. PMID:16989656

  19. Prevalence of lethal osteochondrodysplasias in Denmark.

    PubMed

    Andersen, P E

    1989-04-01

    The point prevalence at birth of lethal osteochondrodysplasias in a subregion of Denmark was estimated by a study of all children born January 1970 through December 1983. Two cases of thanatophoric dysplasia, one case of thanatophoric dysplasia with cloverleaf skull, two cases of micromelic bone dysplasia with cloverleaf skull, two cases of achondrogenesis type III, and three cases of achondrogenesis type IV were found. Two cases were unclassifiable due to lack of radiographs. In total, the point prevalence at birth was 15.4 per 100,000. Thus lethal osteochondrodysplasias seem to be more common than is generally assumed. The clinical and radiographic findings in micromelic bone dysplasia with cloverleaf skull are discussed in relation to thanatophoric dysplasia and achondrogenesis type IV. PMID:2789000

  20. Brine shrimp lethality assay of Bacopa monnieri.

    PubMed

    D'Souza, Prashanth; Deepak, Mundkinajeddu; Rani, Padmaja; Kadamboor, Sandhya; Mathew, Anjana; Chandrashekar, Arun P; Agarwal, Amit

    2002-03-01

    Successive petroleum ether, chloroform, ethanol and water extracts, a saponin rich fraction (SRF) and bacoside A isolated from Bacopa monnieri were tested for brine shrimp lethality. Successive ethanol extracts and SRF showed potent activity. Bacoside A showed the maximum activity with a LC(50) of 38.3 microg/mL. The results confirmed the previous reports of an anticancer effect of Bacopa monnieri and suggest bacoside A as the active constituent. PMID:11933129

  1. Brine shrimp lethality assay of Bacopa monnieri.

    PubMed

    D'Souza, Prashanth; Deepak, Mundkinajeddu; Rani, Padmaja; Kadamboor, Sandhya; Mathew, Anjana; Chandrashekar, Arun P; Agarwal, Amit

    2002-03-01

    Successive petroleum ether, chloroform, ethanol and water extracts, a saponin rich fraction (SRF) and bacoside A isolated from Bacopa monnieri were tested for brine shrimp lethality. Successive ethanol extracts and SRF showed potent activity. Bacoside A showed the maximum activity with a LC(50) of 38.3 microg/mL. The results confirmed the previous reports of an anticancer effect of Bacopa monnieri and suggest bacoside A as the active constituent.

  2. Specific ultrasonographic features of perinatal lethal hypophosphatasia.

    PubMed

    Zankl, Andreas; Mornet, Etienne; Wong, Shell

    2008-05-01

    Prenatal diagnosis of perinatal lethal hypophosphatasia (PL-HPH) by ultrasonography is difficult as PL-HPH must be differentiated from other skeletal dysplasias with short long bones and poor mineralization of the skeleton, such as osteogenesis imperfecta type II and achondrogenesis/hypochondrogenesis. Here we present a case of molecularly confirmed PL-HPH and illustrate specific ultrasonographic findings that help to distinguish PL-HPH from similar conditions. PMID:18386808

  3. Cartilage matrix deficiency (cmd): a new autosomal recessive lethal mutation in the mouse.

    PubMed

    Rittenhouse, E; Dunn, L C; Cookingham, J; Calo, C; Spiegelman, M; Dooher, G B; Bennett, D

    1978-02-01

    A new autosomal recessive lethal mutation in the mouse designated cartilage matrix deficiency (cmd) is described. Homozygotes are dwarfed, and have abnormally short trunk, limbs, tail and snout, as well as a protruding tongue and cleft palate. The abdomen is distended because the foreshortened rib cage and spinal column forces the liver ventrad from its normal location. Histological and electron microscopic study reveals a deficiency of cartilage matrix in tracheal cartilage and in all cartilagenous bones examined. The syndrome closely resembles the rare lethal condition achondrogenesis, found in human infants, which is also believed to be due to an autosomal recessive gene. PMID:632744

  4. Structure-Based Systematic Isolation of Conditional-Lethal Mutations in the Single Yeast Calmodulin Gene

    PubMed Central

    Ohya, Y.; Botstein, D.

    1994-01-01

    Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe -> Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium, 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl(2) in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks. PMID:7896089

  5. Lethal interpersonal violence in the Middle Pleistocene.

    PubMed

    Sala, Nohemi; Arsuaga, Juan Luis; Pantoja-Pérez, Ana; Pablos, Adrián; Martínez, Ignacio; Quam, Rolf M; Gómez-Olivencia, Asier; Bermúdez de Castro, José María; Carbonell, Eudald

    2015-01-01

    Evidence of interpersonal violence has been documented previously in Pleistocene members of the genus Homo, but only very rarely has this been posited as the possible manner of death. Here we report the earliest evidence of lethal interpersonal violence in the hominin fossil record. Cranium 17 recovered from the Sima de los Huesos Middle Pleistocene site shows two clear perimortem depression fractures on the frontal bone, interpreted as being produced by two episodes of localized blunt force trauma. The type of injuries, their location, the strong similarity of the fractures in shape and size, and the different orientations and implied trajectories of the two fractures suggest they were produced with the same object in face-to-face interpersonal conflict. Given that either of the two traumatic events was likely lethal, the presence of multiple blows implies an intention to kill. This finding shows that the lethal interpersonal violence is an ancient human behavior and has important implications for the accumulation of bodies at the site, supporting an anthropic origin. PMID:26018668

  6. Lethal Interpersonal Violence in the Middle Pleistocene

    PubMed Central

    Sala, Nohemi; Arsuaga, Juan Luis; Pantoja-Pérez, Ana; Pablos, Adrián; Martínez, Ignacio; Quam, Rolf M.; Gómez-Olivencia, Asier; Bermúdez de Castro, José María; Carbonell, Eudald

    2015-01-01

    Evidence of interpersonal violence has been documented previously in Pleistocene members of the genus Homo, but only very rarely has this been posited as the possible manner of death. Here we report the earliest evidence of lethal interpersonal violence in the hominin fossil record. Cranium 17 recovered from the Sima de los Huesos Middle Pleistocene site shows two clear perimortem depression fractures on the frontal bone, interpreted as being produced by two episodes of localized blunt force trauma. The type of injuries, their location, the strong similarity of the fractures in shape and size, and the different orientations and implied trajectories of the two fractures suggest they were produced with the same object in face-to-face interpersonal conflict. Given that either of the two traumatic events was likely lethal, the presence of multiple blows implies an intention to kill. This finding shows that the lethal interpersonal violence is an ancient human behavior and has important implications for the accumulation of bodies at the site, supporting an anthropic origin. PMID:26018668

  7. Lethal interpersonal violence in the Middle Pleistocene.

    PubMed

    Sala, Nohemi; Arsuaga, Juan Luis; Pantoja-Pérez, Ana; Pablos, Adrián; Martínez, Ignacio; Quam, Rolf M; Gómez-Olivencia, Asier; Bermúdez de Castro, José María; Carbonell, Eudald

    2015-01-01

    Evidence of interpersonal violence has been documented previously in Pleistocene members of the genus Homo, but only very rarely has this been posited as the possible manner of death. Here we report the earliest evidence of lethal interpersonal violence in the hominin fossil record. Cranium 17 recovered from the Sima de los Huesos Middle Pleistocene site shows two clear perimortem depression fractures on the frontal bone, interpreted as being produced by two episodes of localized blunt force trauma. The type of injuries, their location, the strong similarity of the fractures in shape and size, and the different orientations and implied trajectories of the two fractures suggest they were produced with the same object in face-to-face interpersonal conflict. Given that either of the two traumatic events was likely lethal, the presence of multiple blows implies an intention to kill. This finding shows that the lethal interpersonal violence is an ancient human behavior and has important implications for the accumulation of bodies at the site, supporting an anthropic origin.

  8. High content image-based screening of a protease inhibitor library reveals compounds broadly active against Rift Valley fever virus and other highly pathogenic RNA viruses.

    PubMed

    Mudhasani, Rajini; Kota, Krishna P; Retterer, Cary; Tran, Julie P; Whitehouse, Chris A; Bavari, Sina

    2014-08-01

    High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥ 50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and

  9. Inferred metagenomic comparison of mucosal and fecal microbiota from individuals undergoing routine screening colonoscopy reveals similar differences observed during active inflammation

    PubMed Central

    Tang, Mei San; Poles, Jordan; Leung, Jacqueline M; Wolff, Martin J; Davenport, Michael; Lee, Soo Ching; Lim, Yvonne Al; Chua, Kek Heng; Loke, P'ng; Cho, Ilseung

    2015-01-01

    The mucosal microbiota lives in close proximity with the intestinal epithelium and may interact more directly with the host immune system than the luminal/fecal bacteria. The availability of nutrients in the mucus layer of the epithelium is also very different from the gut lumen environment. Inferred metagenomic analysis for microbial function of the mucosal microbiota is possible by PICRUSt. We recently found that by using this approach, actively inflamed tissue of ulcerative colitis (UC) patients have mucosal communities enriched for genes involved in lipid and amino acid metabolism, and reduced for carbohydrate and nucleotide metabolism. Here, we find that the same bacterial taxa (e.g. Acinetobacter) and predicted microbial pathways enriched in actively inflamed colitis tissue are also enriched in the mucosa of subjects undergoing routine screening colonoscopies, when compared with paired samples of luminal/fecal bacteria. These results suggest that the mucosa of healthy individuals may be a reservoir of aerotolerant microbial communities expanded during colitis. PMID:25559083

  10. A Functional Screen for Myc-Responsive Genes Reveals Serine Hydroxymethyltransferase, a Major Source of the One-Carbon Unit for Cell Metabolism

    PubMed Central

    Nikiforov, Mikhail A.; Chandriani, Sanjay; O'Connell, Brenda; Petrenko, Oleksi; Kotenko, Iulia; Beavis, Andrew; Sedivy, John M.; Cole, Michael D.

    2002-01-01

    A cDNA library enriched with Myc-responsive cDNAs but depleted of myc cDNAs was used in a functional screen for growth enhancement in c-myc-null cells. A cDNA clone for mitochondrial serine hydroxymethyltransferase (mSHMT) that was capable of partial complementation of the growth defects of c-myc-null cells was identified. Expression analysis and chromatin immunoprecipitation demonstrated that mSHMT is a direct Myc target gene. Furthermore, a separate gene encoding the cytoplasmic isoform of the same enzyme is also a direct target of Myc regulation. SHMT enzymes are the major source of the one-carbon unit required for folate metabolism and for the biosynthesis of nucleotides and amino acids. Our data establish a novel functional link between Myc and the regulation of cellular metabolism. PMID:12138190

  11. Functional Screening of the Cronobacter sakazakii BAA-894 Genome reveals a role for ProP (ESA_02131) in carnitine uptake

    PubMed Central

    Feeney, Audrey; Sleator, Roy D

    2015-01-01

    Cronobacter sakazakii is a neonatal pathogen responsible for up to 80% of fatalities in infected infants. Low birth weight infants and neonates infected with C. sakazakii suffer necrotizing enterocolitis, bacteraemia and meningitis. The mode of transmission most often associated with infection is powdered infant formula (PIF) which, with an aw of ∼0.2, is too low to allow most microorganisms to persist. Survival of C. sakazakii in environments subject to extreme hyperosmotic stress has previously been attributed to the uptake of compatible solutes including proline and betaine. Herein, we report the construction and screening of a C. sakazakii genome bank and the identification of ProP (ESA_02131) as a carnitine uptake system. PMID:25915804

  12. Drug repurposing screen reveals FDA-approved inhibitors of human HMG-CoA reductase and isoprenoid synthesis that block Cryptosporidium parvum growth.

    PubMed

    Bessoff, Kovi; Sateriale, Adam; Lee, K Kyungae; Huston, Christopher D

    2013-04-01

    Cryptosporidiosis, a diarrheal disease usually caused by Cryptosporidium parvum or Cryptosporidium hominis in humans, can result in fulminant diarrhea and death in AIDS patients and chronic infection and stunting in children. Nitazoxanide, the current standard of care, has limited efficacy in children and is no more effective than placebo in patients with advanced AIDS. Unfortunately, the lack of financial incentives and the technical difficulties associated with working with Cryptosporidium parasites have crippled efforts to develop effective treatments. In order to address these obstacles, we developed and validated (Z' score = 0.21 to 0.47) a cell-based high-throughput assay and screened a library of drug repurposing candidates (the NIH Clinical Collections), with the hopes of identifying safe, FDA-approved drugs to treat cryptosporidiosis. Our screen yielded 21 compounds with confirmed activity against C. parvum growth at concentrations of <10 μM, many of which had well-defined mechanisms of action, making them useful tools to study basic biology in addition to being potential therapeutics. Additional work, including structure-activity relationship studies, identified the human 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor itavastatin as a potent inhibitor of C. parvum growth (50% inhibitory concentration [IC(50)] = 0.62 μM). Bioinformatic analysis of the Cryptosporidium genomes indicated that the parasites lack all known enzymes required for the synthesis of isoprenoid precursors. Additionally, itavastatin-induced growth inhibition of C. parvum was partially reversed by the addition of exogenous isopentenyl pyrophosphate, suggesting that itavastatin reduces Cryptosporidium growth via on-target inhibition of host HMG-CoA reductase and that the parasite is dependent on the host cell for synthesis of isoprenoid precursors.

  13. Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3 mutation-positive leukemia

    PubMed Central

    Agarwal, Anupriya; MacKenzie, Ryan J.; Eide, Christopher A.; Davare, Monika A.; Watanabe-Smith, Kevin; Tognon, Cristina E.; Mongoue-Tchokote, Solange; Park, Byung; Braziel, Rita M.; Tyner, Jeffrey W.; Druker, Brian J.

    2014-01-01

    To understand the role for cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis we designed a functional RNAi screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for the JAK3A572V mutation-positive AML cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3A572V as well as the transforming potential of additional JAK3 activating mutations such as JAK3M511I. In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant but not wild type JAK3 increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation positive leukemia. Additionally, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth. PMID:25109334

  14. A High-Throughput Fatty Acid Profiling Screen Reveals Novel Variations in Fatty Acid Biosynthesis in Chlamydomonas reinhardtii and Related Algae

    PubMed Central

    Pflaster, Erin L.; Schwabe, Michael J.; Becker, Joyanne; Wilkinson, Melissa S.; Parmer, Ashley; Clemente, Thomas E.; Cahoon, Edgar B.

    2014-01-01

    Analysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green alga Chlamydomonas for variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain of C. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains of Chlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus. PMID:25239975

  15. A Genome-wide In Vitro Bacterial-Infection Screen Reveals Human Variation in the Host Response Associated with Inflammatory Disease

    PubMed Central

    Ko, Dennis C.; Shukla, Kajal P.; Fong, Christine; Wasnick, Michael; Brittnacher, Mitchell J.; Wurfel, Mark M.; Holden, Tarah D.; O'Keefe, Grant E.; Van Yserloo, Brian; Akey, Joshua M.; Miller, Samuel I.

    2009-01-01

    Recent progress in cataloguing common genetic variation has made possible genome-wide studies that are beginning to elucidate the causes and consequences of our genetic differences. Approaches that provide a mechanistic understanding of how genetic variants function to alter disease susceptibility and why they were substrates of natural selection would complement other approaches to human-genome analysis. Here we use a novel cell-based screen of bacterial infection to identify human variation in Salmonella-induced cell death. A loss-of-function allele of CARD8, a reported inhibitor of the proinflammatory protease caspase-1, was associated with increased cell death in vitro (p = 0.013). The validity of this association was demonstrated through overexpression of alternative alleles and RNA interference in cells of varying genotype. Comparison of mammalian CARD8 orthologs and examination of variation among different human populations suggest that the increase in infectious-disease burden associated with larger animal groups (i.e., herds and colonies), and possibly human population expansion, may have naturally selected for loss of CARD8. We also find that the loss-of-function CARD8 allele shows a modest association with an increased risk of systemic inflammatory response syndrome in a small study (p = 0.05). Therefore, a by-product of the selected benefit of loss of CARD8 could be increased inflammatory diseases. These results demonstrate the utility of genome-wide cell-based association screens with microbes in the identification of naturally selected variants that can impact human health. PMID:19664744

  16. A Screen for Dominant Negative Mutants of SEC18 Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis

    PubMed Central

    Steel, Gregor J.; Harley, Carol; Boyd, Alan; Morgan, Alan

    2000-01-01

    An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum–Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure–function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr394→Pro). Thr394 is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast α-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts. PMID:10749934

  17. Dual microRNA Screens Reveal That the Immune-Responsive miR-181 Promotes Henipavirus Entry and Cell-Cell Fusion

    PubMed Central

    Foo, Chwan Hong; Rootes, Christina L.; Marsh, Glenn A.; Gould, Cathryn M.; Klein, Reuben; Riddell, Sarah J.; Middleton, Deborah; Simpson, Kaylene J.; Bean, Andrew G. D.; Stewart, Cameron R.

    2016-01-01

    Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development. PMID:27783670

  18. Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1.

    PubMed

    Trabold, Peter A; Weinberger, Martin; Feng, Li; Burhans, William C

    2005-04-01

    Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA. PMID:15647270

  19. Lethal and sub-lethal effects of UVB on juvenile Biomphalaria glabrata (Mollusca: Pulmonata).

    PubMed

    Ruelas, Debbie S; Karentz, Deneb; Sullivan, John T

    2006-11-01

    Although Schistosoma mansoni occurs mainly in the tropics, where intense levels of solar radiation are present, the impact of ultraviolet (UV) light on schistosome transmission is not known. The purpose of this study was to investigate potential effects of UVB (290-320nm) on juvenile Biomphalaria glabrata, the snail intermediate host of S. mansoni. Albino and wild-type snails were exposed to doses of UVB from UV-fluorescent lamps, and the following were measured: survival, photoreactivation (light-mediated DNA repair), effects on feeding behavior, and morphological tissue abnormalities. Irradiation with UVB is lethal to B. glabrata in a dose-dependent manner. Exposure to white light subsequent to UVB irradiation enhances survival, probably by photoreactivation. The shell offers some, but not complete, protection. Experiments in which UVB transmittance through the shell was blocked with black nail polish suggest that injury to both exposed (headfoot) and shell-enclosed (mantle and visceral mass) tissues contributes to mortality in lethally irradiated snails. Wild-type (pigmented) snails are less susceptible to lethal effects of UVB than albino snails, and they may be more capable of photoreactivation. UVB exposure inhibits snail feeding behavior, and causes tentacle forks and growths on the headfoot. Thus, UVB may influence the life cycle of S. mansoni by both lethal and sub-lethal damage to the snail intermediate host. However, the ability of snails to photoreactivate may mitigate these effects. PMID:16996081

  20. Lethal and sub-lethal effects of UVB on juvenile Biomphalaria glabrata (Mollusca: Pulmonata).

    PubMed

    Ruelas, Debbie S; Karentz, Deneb; Sullivan, John T

    2006-11-01

    Although Schistosoma mansoni occurs mainly in the tropics, where intense levels of solar radiation are present, the impact of ultraviolet (UV) light on schistosome transmission is not known. The purpose of this study was to investigate potential effects of UVB (290-320nm) on juvenile Biomphalaria glabrata, the snail intermediate host of S. mansoni. Albino and wild-type snails were exposed to doses of UVB from UV-fluorescent lamps, and the following were measured: survival, photoreactivation (light-mediated DNA repair), effects on feeding behavior, and morphological tissue abnormalities. Irradiation with UVB is lethal to B. glabrata in a dose-dependent manner. Exposure to white light subsequent to UVB irradiation enhances survival, probably by photoreactivation. The shell offers some, but not complete, protection. Experiments in which UVB transmittance through the shell was blocked with black nail polish suggest that injury to both exposed (headfoot) and shell-enclosed (mantle and visceral mass) tissues contributes to mortality in lethally irradiated snails. Wild-type (pigmented) snails are less susceptible to lethal effects of UVB than albino snails, and they may be more capable of photoreactivation. UVB exposure inhibits snail feeding behavior, and causes tentacle forks and growths on the headfoot. Thus, UVB may influence the life cycle of S. mansoni by both lethal and sub-lethal damage to the snail intermediate host. However, the ability of snails to photoreactivate may mitigate these effects.

  1. Early Cytokine Dysregulation and Viral Replication Are Associated with Mortality During Lethal Influenza Infection

    PubMed Central

    Vogel, Alexander J.; Harris, Seth; Marsteller, Nathan; Condon, Shirley A.

    2014-01-01

    Abstract Infection with influenza A virus (IAV) leads to acute lung injury and possibly fatal complications, especially in immunocompromised, elderly, or chronically infected individuals. Therefore, it is important to study the factors that lead to pathology and mortality in infected hosts. In this report, we analyze immune responses to infection at a sublethal (0.1 LD50) and lethal (1 LD50) dose of the highly pathogenic IAV A/Puerto Rico/8/34 (PR8). Our experiments revealed that infection with a 1 LD50 dose induced peak viral titers at day 2 compared to day 4 in the 0.1 LD50 dose. Moreover, early cytokine dysregulation was observed in the lethal dose with significantly elevated levels of IFN-α, TNF-α, CXCL9, IL-6, and MCP-1 produced at day 2. Early inflammatory responses following infection with 1 LD50 correlated with a greater influx of neutrophils into the lung. However, depletion of neutrophils enhanced morbidity following IAV infection. Though no differences in CD8+ cell function were observed, CD4+ effector responses were impaired in the lungs 8 days after infection with 1 LD50. Histological analysis revealed significant pathology in lethally infected mice at day 2 and day 6 postinfection, when viral titers remained high. Treating lethally infected mice with oseltamivir inhibited viral titers to sublethal levels, and abrogated the pathology associated with the lethal dose. Together, these results suggest that early cytokine dysregulation and viral replication play a role in pulmonary damage and high mortality in lethally infected mice. PMID:24787235

  2. Evaluating the lethal and pre-lethal effects of a range of fungi against adult Anopheles stephensi mosquitoes

    PubMed Central

    2012-01-01

    Background Insecticide resistance is seriously undermining efforts to eliminate malaria. In response, research on alternatives to the use of chemical insecticides against adult mosquito vectors has been increasing. Fungal entomopathogens formulated as biopesticides have received much attention and have shown considerable potential. This research has necessarily focused on relatively few fungal isolates in order to ‘prove concept’. Further, most attention has been paid to examining fungal virulence (lethality) and not the other properties of fungal infection that might also contribute to reducing transmission potential. Here, a range of fungal isolates were screened to examine variation in virulence and how this relates to additional pre-lethal reductions in feeding propensity. Methods The Asian malaria vector, Anopheles stephensi was exposed to 17 different isolates of entomopathogenic fungi belonging to species of Beauveria bassiana, Metarhizium anisopliae, Metarhizium acridum and Isaria farinosus. Each isolate was applied to a test substrate at a standard dose rate of 1×109 spores ml-1 and the mosquitoes exposed for six hours. Subsequently the insects were removed to mesh cages where survival was monitored over the next 14 days. During this incubation period the mosquitoes’ propensity to feed was assayed for each isolate by offering a feeding stimulant at the side of the cage and recording the number probing. Results and conclusions Fungal isolates showed a range of virulence to A. stephensi with some causing >80% mortality within 7 days, while others caused little increase in mortality relative to controls over the study period. Similarly, some isolates had a large impact on feeding propensity, causing >50% pre-lethal reductions in feeding rate, whereas other isolates had very little impact. There was clear correlation between fungal virulence and feeding reduction with virulence explaining nearly 70% of the variation in feeding reduction. However, there

  3. Screening of different metal oxide nanoparticles reveals selective toxicity and inflammatory potential of silica nanoparticles in lung epithelial cells and macrophages.

    PubMed

    Panas, A; Marquardt, C; Nalcaci, O; Bockhorn, H; Baumann, W; Paur, H-R; Mülhopt, S; Diabaté, S; Weiss, C

    2013-05-01

    In cell culture studies, foetal calf serum (FCS) comprising numerous different proteins is added, which might coat the surface of engineered nanomaterials (ENMs) and thus could profoundly alter their biological activities. In this study, a panel of industrially most relevant metal oxide nanoparticles (NPs) was screened for toxic effects in A549 lung epithelial cells and RAW264.7 macrophages in the presence and absence of FCS. In medium without FCS amorphous SiO2-NPs were the most cytotoxic NPs and induced a significant pro-inflammatory response in both cell types. An increased anti-oxidative response after exposure to SiO2-NPs was, however, only observed in RAW264.7 macrophages. Furthermore, pre-coating of SiO2-NPs with FCS proteins or simply bovine serum albumin abrogated responses in A549 lung epithelial cells. Thus, the protein corona bound to the surface of SiO2-NPs suppresses their biological effects, an issue which needs to be more carefully considered for in vitro-in vivo extrapolations.

  4. A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage.

    PubMed Central

    Schulz, Cordula; Kiger, Amy A; Tazuke, Salli I; Yamashita, Yukiko M; Pantalena-Filho, Luiz C; Jones, D Leanne; Wood, Cricket G; Fuller, Margaret T

    2004-01-01

    Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. Here we utilize the Drosophila system to identify genes that cause defects in the male GSC lineage when forcibly expressed. We conducted a gain-of-function screen using a collection of 2050 EP lines and found 55 EP lines that caused defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, our analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGF beta signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGF beta signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation. PMID:15238523

  5. A cell-based screen for inhibitors of flagella-driven motility in Chlamydomonas reveals a novel modulator of ciliary length and retrograde actin flow.

    PubMed

    Engel, Benjamin D; Ishikawa, Hiroaki; Feldman, Jessica L; Wilson, Christopher W; Chuang, Pao-Tien; Snedecor, June; Williams, Janice; Sun, Zhaoxia; Marshall, Wallace F

    2011-03-01

    Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound "ciliabrevin" due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug.

  6. In Vitro Screening of the Open-Source Medicines for Malaria Venture Malaria Box Reveals Novel Compounds with Profound Activities against Theileria annulata Schizonts.

    PubMed

    Hostettler, Isabel; Müller, Joachim; Hemphill, Andrew

    2016-06-01

    Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leukocytes and thereby cause fatal diseases. The hydroxynaphthoquinone buparvaquone is currently the only option for the treatment of theileriosis, and resistance development has been reported. It is therefore tempting to investigate the repurposing of compounds effective against related apicomplexan parasites, such as Plasmodium Here, we present the results of a screen of 400 compounds included in the open-access Medicines for Malaria Venture (MMV) malaria box on TaC12 cells, a macrophage-derived cell line immortalized by T. annulata schizonts. Using a combination of the classical alamarBlue vitality assay and a recently developed quantitative reverse transcriptase real-time PCR method based on the Theileria TaSP gene, we have identified 5 compounds, characterized their effects on the ultrastructure of TaC12 cells, and investigated whether they easily induce resistance formation. Two compounds, the quinolinols MMV666022 and MMV666054, have 50% inhibitory concentrations (IC50s) of 0.5 and 0.2 μM on TaC12 cells and 5.3 and 5.2 μM on BoMac cells, respectively. Thus, with therapeutic indexes of 11 and 18, they represent promising leads for further development of antitheilerial chemotherapeutics. PMID:26976863

  7. Genetic Screen Reveals Link between the Maternal Effect Sterile Gene mes-1 and Pseudomonas aeruginosa-induced Neurodegeneration in Caenorhabditis elegans.

    PubMed

    Wu, Qiuli; Cao, Xiou; Yan, Dong; Wang, Dayong; Aballay, Alejandro

    2015-12-01

    Increasing evidence indicates that immune responses to microbial infections may contribute to neurodegenerative diseases. Here, we show that Pseudomonas aeruginosa infection of Caenorhabditis elegans causes a number of neural changes that are hallmarks of neurodegeneration. Using an unbiased genetic screen to identify genes involved in the control of P. aeruginosa-induced neurodegeneration, we identified mes-1, which encodes a receptor tyrosine kinase-like protein that is required for unequal cell divisions in the early embryonic germ line. We showed that sterile but not fertile mes-1 animals were resistant to neurodegeneration induced by P. aeruginosa infection. Similar results were observed using animals carrying a mutation in the maternal effect gene pgl-1, which is required for postembryonic germ line development, and the germ line-deficient strains glp-1 and glp-4. Additional studies indicated that the FOXO transcription factor DAF-16 is required for resistance to P. aeruginosa-induced neurodegeneration in germ line-deficient strains. Thus, our results demonstrate that P. aeruginosa infection results in neurodegeneration phenotypes in C. elegans that are controlled by the germ line in a cell-nonautonomous manner.

  8. Genetic Screen Reveals Link between the Maternal Effect Sterile Gene mes-1 and Pseudomonas aeruginosa-induced Neurodegeneration in Caenorhabditis elegans.

    PubMed

    Wu, Qiuli; Cao, Xiou; Yan, Dong; Wang, Dayong; Aballay, Alejandro

    2015-12-01

    Increasing evidence indicates that immune responses to microbial infections may contribute to neurodegenerative diseases. Here, we show that Pseudomonas aeruginosa infection of Caenorhabditis elegans causes a number of neural changes that are hallmarks of neurodegeneration. Using an unbiased genetic screen to identify genes involved in the control of P. aeruginosa-induced neurodegeneration, we identified mes-1, which encodes a receptor tyrosine kinase-like protein that is required for unequal cell divisions in the early embryonic germ line. We showed that sterile but not fertile mes-1 animals were resistant to neurodegeneration induced by P. aeruginosa infection. Similar results were observed using animals carrying a mutation in the maternal effect gene pgl-1, which is required for postembryonic germ line development, and the germ line-deficient strains glp-1 and glp-4. Additional studies indicated that the FOXO transcription factor DAF-16 is required for resistance to P. aeruginosa-induced neurodegeneration in germ line-deficient strains. Thus, our results demonstrate that P. aeruginosa infection results in neurodegeneration phenotypes in C. elegans that are controlled by the germ line in a cell-nonautonomous manner. PMID:26475858

  9. Combinatorial drug screening and molecular profiling reveal diverse mechanisms of intrinsic and adaptive resistance to BRAF inhibition in V600E BRAF mutant melanomas

    PubMed Central

    Roller, Devin G.; Capaldo, Brian; Bekiranov, Stefan; Mackey, Aaron J.; Conaway, Mark R.; Petricoin, Emanuel F.; Gioeli, Daniel; Weber, Michael J.

    2016-01-01

    Over half of BRAFV600E melanomas display intrinsic resistance to BRAF inhibitors, in part due to adaptive signaling responses. In this communication we ask whether BRAFV600E melanomas share common adaptive responses to BRAF inhibition that can provide clinically relevant targets for drug combinations. We screened a panel of 12 treatment-naïve BRAFV600E melanoma cell lines with MAP Kinase pathway inhibitors in pairwise combination with 58 signaling inhibitors, assaying for synergistic cytotoxicity. We found enormous diversity in the drug combinations that showed synergy, with no two cell lines having an identical profile. Although the 6 lines most resistant to BRAF inhibition showed synergistic benefit from combination with lapatinib, the signaling mechanisms by which this combination generated synergistic cytotoxicity differed between the cell lines. We conclude that adaptive responses to inhibition of the primary oncogenic driver (BRAFV600E) are determined not only by the primary oncogenic driver but also by diverse secondary genetic and epigenetic changes (“back-seat drivers”) and hence optimal drug combinations will be variable. Because upregulation of receptor tyrosine kinases is a major source of drug resistance arising from diverse adaptive responses, we propose that inhibitors of these receptors may have substantial clinical utility in combination with inhibitors of the MAP Kinase pathway. PMID:26673621

  10. Influence of Salmonella enterica Serovar Typhimurium ssrB on Colonization of Eastern Oysters (Crassostrea virginica) as Revealed by a Promoter Probe Screen

    PubMed Central

    Cox, Clayton E.; Wright, Anita C.; McClelland, Michael

    2015-01-01

    Although Salmonella has been isolated from 7.4 to 8.6% of domestic raw oysters, representing a significant risk for food-borne illness, little is known about the factors that influence their initial colonization by Salmonella. This study tested the hypothesis that specific regulatory changes enable a portion of the invading Salmonella population to colonize oysters. An in vivo promoter probe library screen identified 19 unique regions as regulated during colonization. The mutants in the nearest corresponding downstream genes were tested for colonization defects in oysters. Only one mutation, in ssrB, resulted in a significantly reduced ability to colonize oysters compared to that of wild-type Salmonella. Because ssrB regulates Salmonella pathogenicity island 2 (SPI-2)-dependent infections in vertebrate macrophages, the possibility that ssrB mediated colonization of oyster hemocytes in a similar manner was examined. However, no difference in hemocyte colonization was observed. The complementary hypothesis that signal exchange between Salmonella and the oyster's native microbial community aids colonization was also tested. Signals that triggered responses in quorum sensing (QS) reporters were shown to be produced by oyster-associated bacteria and present in oyster tissue. However, no evidence for signal exchange was observed in vivo. The sdiA reporter responded to salinity, suggesting that SdiA may also have a role in environmental sensing. Overall, this study suggests the initial colonization of live oysters by Salmonella is controlled by a limited number of regulators, including ssrB. PMID:26497459

  11. Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1) influenza virus reveals unexpected antigenically important regions.

    PubMed

    Xu, Wanghui; Han, Lu; Lin, Zhanglin

    2011-01-01

    The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify "hot spots" or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins. PMID:21437206

  12. A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.

    PubMed

    Wang, Peng; Alvarez-Perez, Juan-Carlos; Felsenfeld, Dan P; Liu, Hongtao; Sivendran, Sharmila; Bender, Aaron; Kumar, Anil; Sanchez, Roberto; Scott, Donald K; Garcia-Ocaña, Adolfo; Stewart, Andrew F

    2015-04-01

    Types 1 and 2 diabetes affect some 380 million people worldwide. Both ultimately result from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with a peak percentage (∼2%) engaged in the cell cycle in the first year of life. In embryonic life and after early childhood, beta cell replication is barely detectable. Whereas beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts. Hence, there remains an urgent need for antidiabetic therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, using a high-throughput small-molecule screen (HTS), we find that analogs of the small molecule harmine function as a new class of human beta cell mitogenic compounds. We also define dual-specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine and the nuclear factors of activated T cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation and differentiation. Using three different mouse and human islet in vivo-based models, we show that harmine is able to induce beta cell proliferation, increase islet mass and improve glycemic control. These observations suggest that harmine analogs may have unique therapeutic promise for human diabetes therapy. Enhancing the potency and beta cell specificity of these compounds are important future challenges. PMID:25751815

  13. Function of a plant stress-induced gene, HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic.

    PubMed

    Brands, Alex; Ho, Tuan-hua David

    2002-11-01

    Expression of the barley (Hordeum vulgare) HVA22 gene is induced by environmental stresses, such as dehydration, salinity, and extreme temperatures, and by a plant stress hormone, abscisic acid. Genes sharing high level of sequence similarities with HVA22 exist in diverse eukaryotic organisms, including animals, plants, and fungi, but not in any prokaryotic organisms. The yeast (Saccharomyces cerevisiae) HVA22 homolog, Yop1p, has been shown to interact with the GTPase-interacting protein, Yip1p. Deletion of YOP1 led to only a modest reduction of the stationary phase titer at 37C. A synthetic enhancement mutant screen was performed in the yop1 deletion background to identify genes interacting with YOP1. The open reading frame YOR165W (renamed SEY1 for synthetic enhancement of YOP1) was identified as a YOP1-dependent complementation gene. The yeast SEY1 is a homolog of the Arabidopsis RHD3 gene whose mutations cause the accumulation of transport vesicles near the tips of defective root hairs. The yeast double mutant of yop1 and sey1 is defective in vesicular traffic as evidenced by the accumulation of transport vesicles and the decrease in invertase secretion. Based on these observations, we suggest that Yop1p/HVA22 regulates vesicular traffic in stressed cells either to facilitate membrane turnover, or to decrease unnecessary secretion.

  14. Screening of a kinase library reveals novel pro-senescence kinases and their common NF-κB-dependent transcriptional program.

    PubMed

    Ferrand, Mylène; Kirsh, Olivier; Griveau, Audrey; Vindrieux, David; Martin, Nadine; Defossez, Pierre-Antoine; Bernard, David

    2015-11-01

    Cellular senescence results in proliferation arrest and acquisition of hallmarks such as the Senescence-Associated Secretory Phenotype (SASP). Senescence is involved in regulating numerous physio-pathological responses, including embryonic development, cancer, and several aging-related diseases. Only a few kinases, centered on the RAS signaling pathway, have been identified as inducing premature senescence. About possible other senescence-regulating kinases and signaling pathways, practically little is known. By screening a library of activated kinases, we identified 33 kinases whose constitutive expression decreases cell proliferation and induces expression of senescence markers; p16 and SASP components. Focusing on some kinases showing the strongest pro-senescence effects, we observed that they all induce expression of SASP-component genes through activation of an NF-κB-dependent transcriptional program. Furthermore, inhibition of the p53 or Rb pathway failed to prevent the SASP-inducing effect of pro-senescence kinases. Inhibition of the NF-κB, p53, or Rb pathway proved insufficient to prevent kinase-triggered cell cycle arrest. We have thus identified a repertoire of novel pro-senescence kinases and pathways. These results will open new perspectives in the understanding on the role of cellular senescence in various physio-pathological responses. PMID:26583757

  15. RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans

    PubMed Central

    Ossareh-Nazari, Batool; Katsiarimpa, Anthi; Merlet, Jorge; Pintard, Lionel

    2016-01-01

    Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans. Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint. PMID:27543292

  16. A systematic screen for dominant second-site modifiers of Merlin/NF2 phenotypes reveals an interaction with blistered/DSRF and scribbler.

    PubMed Central

    LaJeunesse, D R; McCartney, B M; Fehon, R G

    2001-01-01

    Merlin, the Drosophila homologue of the human tumor suppressor gene Neurofibromatosis 2 (NF2), is required for the regulation of cell proliferation and differentiation. To better understand the cellular functions of the NF2 gene product, Merlin, recent work has concentrated on identifying proteins with which it interacts either physically or functionally. In this article, we describe genetic screens designed to isolate second-site modifiers of Merlin phenotypes from which we have identified five multiallelic complementation groups that modify both loss-of-function and dominant-negative Merlin phenotypes. Three of these groups, Group IIa/scribbler (also known as brakeless), Group IIc/blistered, and Group IId/net, are known genes, while two appear to be novel. In addition, two genes, Group IIa/scribbler and Group IIc/blistered, alter Merlin subcellular localization in epithelial and neuronal tissues, suggesting that they regulate Merlin trafficking or function. Furthermore, we show that mutations in scribbler and blistered display second-site noncomplementation with one another. These results suggest that Merlin, blistered, and scribbler function together in a common pathway to regulate Drosophila wing epithelial development. PMID:11404331

  17. A genome-wide screen in Saccharomyces cerevisiae reveals a critical role for the mitochondria in the toxicity of a trichothecene mycotoxin

    PubMed Central

    McLaughlin, John E.; Bin-Umer, Mohamed Anwar; Tortora, Andrew; Mendez, Natasha; McCormick, Susan; Tumer, Nilgun E.

    2009-01-01

    Trichothecene mycotoxins synthesized by Fusarium species are potent inhibitors of eukaryotic translation. They are encountered in both the environment and in food, posing a threat to human and animal health. They have diverse roles in the cell that are not limited to the inhibition of protein synthesis. To understand the trichothecene mechanism of action, we screened the yeast knockout library to identify genes whose deletion confers resistance to trichothecin (Tcin). The largest group of resistant strains affected mitochondrial function, suggesting a role for fully active mitochondria in trichothecene toxicity. Tcin inhibited mitochondrial translation in the wild-type strain to a greater extent than in the most resistant strains, implicating mitochondrial translation as a previously unrecognized site of action. The Tcin-resistant strains were cross-resistant to anisomycin and chloramphenicol, suggesting that Tcin targets the peptidyltransferase center of mitochondrial ribosomes. Tcin-induced cell death was partially rescued by mutants that regulate mitochondrial fusion and maintenance of the tubular morphology of mitochondria. Treatment of yeast cells with Tcin led to the fragmentation of the tubular mitochondrial network, supporting a role for Tcin in disruption of mitochondrial membrane morphology. These results provide genome-wide insight into the mode of action of trichothecene mycotoxins and uncover a critical role for mitochondrial translation and membrane maintenance in their toxicity. PMID:20007368

  18. [Lethal intoxication with arsenic using prepared butter].

    PubMed

    Weller, Jens-Peter; Larsch, Klaus-Peter; Teske, Jörg; Tröger, Hans Dieter

    2008-01-01

    The present case report deals with a lethal intoxication with arsenic mixed into butter. It describes the course of events over about two days on the basis of the statements by the persons involved, the clinical findings after the belated hospitalisation of the victim, the results of the first pathological autopsy, the forensic autopsy performed after exhumation and the results of the chemical-toxicological investigations. The results are discussed in relation to the later confession of the female perpetrator and her statements regarding a previous unsuccessful murder attempt by poisoning. It also presents the judgement pronounced by the court and the reasons given for it.

  19. Lethal predators: psychopathic, sadistic, and sane.

    PubMed

    Ochberg, Frank M; Brantley, Alan C; Hare, R D; Houk, Peter D; Ianni, Robert; James, Earl; O'Toole, Mary Ellen; Saathoff, Gregory

    2003-01-01

    The violent criminals defined in this article are a small, exceptionally dangerous group of offenders designated by the authors as "lethal predators." They have a history of sexual predation, have killed at least once, and are mentally abnormal but legally sane. They are highly likely to keep killing as long as they are free. Laws permitting civil commitment of dangerous and mentally abnormal sexual predators after they have completed criminal prison sentences have been upheld by the U.S. Supreme Court. Such laws can provide a legal means of keeping these highly dangerous killers confined so they cannot kill again.

  20. Lethal mobilization of DDT by cowbirds

    USGS Publications Warehouse

    Van Velzen, A.C.; Stiles, W.B.; Stickel, L.F.

    1972-01-01

    This study is an experimental demonstration of lethal mobilization of DDT by brown-headed cowbirds (Molothrus ater) and the effects of food deprivation on the distribution and loss of DDT, DDD, and DDE. The principal experimental group consisted of 20 birds fed a dietary dosage of 100 ppm of DDT for 13 days. After 2 days of full rations of untreated food, they were subjected to food restriction. Food was reduced to 43 percent of normal. Seven of the 20 birds died within 4 days. No birds died in the three control groups, treated as follows: ( 1 ) 20 birds fed 100 ppm DDT for 13 days and full rations of untreated food thereafter, (2) 20 birds fed only untreated food but subjected to food restriction, and (3) 20 birds fed full rations of untreated food throughout. In a pilot study, birds were fed 100, 200, or 300 ppm of DDT and subjected to two periods of food restriction, the first of these immediately after dosage ceased and the second 4 months later. DDT-dosed birds from all dosage levels died in each period of food restriction. Before the weight loss that accompanied food restriction, the brains of DDT-dosed birds had concentrations of DDT and DDD that were far below the lethal range. Concentrations increased rapidly to lethal levels. In these birds, DDT in carcasses decreased while DDD increased. DDT-dosed birds that died during food restriction lost 16 percent of their total body burden of DDT + DDD + DDE, 21 percent of their weight, and 81 percent of their fat. The DDT-dosed birds that were subjected to food restriction but survived lost a significantly greater proportion of their body burden of residues than similarly dosed birds not subjected to weight loss. Brain levels of DDT and DDD in birds that died during food restriction soon after dosage did not differ significantly from brain levels of birds that died in a period of food restriction 4 months after dosage. Concentrations of DDE were significantly higher in the latter group, although they were lower

  1. A kinase inhibitor screen reveals protein kinase C-dependent endocytic recycling of ErbB2 in breast cancer cells.

    PubMed

    Bailey, Tameka A; Luan, Haitao; Tom, Eric; Bielecki, Timothy Alan; Mohapatra, Bhopal; Ahmad, Gulzar; George, Manju; Kelly, David L; Natarajan, Amarnath; Raja, Srikumar M; Band, Vimla; Band, Hamid

    2014-10-31

    ErbB2 overexpression drives oncogenesis in 20-30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca(2+)-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling.

  2. A high-throughput kinome screen reveals serum/glucocorticoid-regulated kinase 1 as a therapeutic target for NF2-deficient meningiomas

    PubMed Central

    Beauchamp, Roberta L.; James, Marianne F.; DeSouza, Patrick A.; Wagh, Vilas; Zhao, Wen-Ning; Jordan, Justin T.; Stemmer-Rachamimov, Anat; Plotkin, Scott R.; Gusella, James F.; Haggarty, Stephen J.; Ramesh, Vijaya

    2015-01-01

    Meningiomas are the most common primary intracranial adult tumor. All Neurofibromatosis 2 (NF2)-associated meningiomas and ~60% of sporadic meningiomas show loss of NF2 tumor suppressor protein. There are no effective medical therapies for progressive and recurrent meningiomas. Our previous work demonstrated aberrant activation of mTORC1 signaling that led to ongoing clinical trials with rapamycin analogs for NF2 and sporadic meningioma patients. Here we performed a high-throughput kinome screen to identify kinases responsible for mTORC1 pathway activation in NF2-deficient meningioma cells. Among the emerging top candidates were the mTORC2-specific target serum/glucocorticoid-regulated kinase 1 (SGK1) and p21-activated kinase 1 (PAK1). In NF2-deficient meningioma cells, inhibition of SGK1 rescues mTORC1 activation, and SGK1 activation is sensitive to dual mTORC1/2 inhibitor AZD2014, but not to rapamycin. PAK1 inhibition also leads to attenuated mTORC1 but not mTORC2 signaling, suggesting that mTORC2/SGK1 and Rac1/PAK1 pathways are independently responsible for mTORC1 activation in NF2-deficient meningiomas. Using CRISPR-Cas9 genome editing, we generated isogenic human arachnoidal cell lines (ACs), the origin cell type for meningiomas, expressing or lacking NF2. NF2-null CRISPR ACs recapitulate the signaling of NF2-deficient meningioma cells. Interestingly, we observe increased SGK1 transcription and protein expression in NF2-CRISPR ACs and in primary NF2-negative meningioma lines. Moreover, we demonstrate that the dual mTORC1/mTORC2 inhibitor, AZD2014 is superior to rapamycin and PAK inhibitor FRAX597 in blocking proliferation of meningioma cells. Importantly, AZD2014 is currently in use in several clinical trials of cancer. Therefore, we believe that AZD2014 may provide therapeutic advantage over rapalogs for recurrent and progressive meningiomas. PMID:26219339

  3. Isotope-Assisted Screening for Iron-Containing Metabolites Reveals a High Degree of Diversity among Known and Unknown Siderophores Produced by Trichoderma spp.

    PubMed Central

    Lehner, Sylvia M.; Atanasova, Lea; Neumann, Nora K. N.; Krska, Rudolf; Lemmens, Marc; Druzhinina, Irina S.

    2013-01-01

    Due to low iron availability under environmental conditions, many microorganisms excrete iron-chelating agents (siderophores) to cover their iron demands. A novel screening approach for the detection of siderophores using liquid chromatography coupled to high-resolution tandem mass spectrometry was developed to study the production of extracellular siderophores of 10 wild-type Trichoderma strains. For annotation of siderophores, an in-house library comprising 422 known microbial siderophores was established. After 96 h of cultivation, 18 different iron chelators were detected. Four of those (dimerum acid, fusigen, coprogen, and ferricrocin) were identified by measuring authentic standards. cis-Fusarinine, fusarinine A and B, and des-diserylglycylferrirhodin were annotated based on high-accuracy mass spectral analysis. In total, at least 10 novel iron-containing metabolites of the hydroxamate type were found. On average Trichoderma spp. produced 12 to 14 siderophores, with 6 common to all species tested. The highest number (15) of siderophores was detected for the most common environmental opportunistic and strongly fungicidic species, Trichoderma harzianum, which, however, did not have any unique compounds. The tropical species T. reesei had the most distinctive pattern, producing one unique siderophore (cis-fusarinine) and three others that were present only in T. harzianum and not in other species. The diversity of siderophores did not directly correlate with the antifungal potential of the species tested. Our data suggest that the high diversity of siderophores produced by Trichoderma spp. might be the result of further modifications of the nonribosomal peptide synthetase (NRPS) products and not due to diverse NRPS-encoding genes. PMID:23064341

  4. TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response

    PubMed Central

    Rozanov, Dmitri; Cheltsov, Anton; Sergienko, Eduard; Vasile, Stefan; Golubkov, Vladislav; Aleshin, Alexander E.; Levin, Trevor; Traer, Elie; Hann, Byron; Freimuth, Julia; Alexeev, Nikita; Alekseyev, Max A.; Budko, Sergey P; Bächinger, Hans Peter; Spellman, Paul

    2015-01-01

    A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model. PMID:26075913

  5. Screening a wide host-range, waste-water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes.

    PubMed

    Li, Youguo; Wexler, Margaret; Richardson, David J; Bond, Philip L; Johnston, Andrew W B

    2005-12-01

    A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors. PMID:16309391

  6. Adapted Lethality: What We Can Learn from Guinea Pig-Adapted Ebola Virus Infection Model

    PubMed Central

    Cheresiz, S. V.; Semenova, E. A.; Chepurnov, A. A.

    2016-01-01

    Establishment of small animal models of Ebola virus (EBOV) infection is important both for the study of genetic determinants involved in the complex pathology of EBOV disease and for the preliminary screening of antivirals, production of therapeutic heterologic immunoglobulins, and experimental vaccine development. Since the wild-type EBOV is avirulent in rodents, the adaptation series of passages in these animals are required for the virulence/lethality to emerge in these models. Here, we provide an overview of our several adaptation series in guinea pigs, which resulted in the establishment of guinea pig-adapted EBOV (GPA-EBOV) variants different in their characteristics, while uniformly lethal for the infected animals, and compare the virologic, genetic, pathomorphologic, and immunologic findings with those obtained in the adaptation experiments of the other research groups. PMID:26989413

  7. An Immature Myeloid/Myeloid-Suppressor Cell Response Associated with Necrotizing Inflammation Mediates Lethal Pulmonary Tularemia.

    PubMed

    Periasamy, Sivakumar; Avram, Dorina; McCabe, Amanda; MacNamara, Katherine C; Sellati, Timothy J; Harton, Jonathan A

    2016-03-01

    Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but the mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that in contrast to overwhelming bacterial burden or cytokine production, an overt innate cellular response to Ft drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature and die, leading to subsequent necrotic lung damage, loss of pulmonary function, and host death that is partially dependent upon immature Ly6G+ cells. Acceleration of this process may account for the rapid lethality seen with Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection. PMID:27015566

  8. An Immature Myeloid/Myeloid-Suppressor Cell Response Associated with Necrotizing Inflammation Mediates Lethal Pulmonary Tularemia.

    PubMed

    Periasamy, Sivakumar; Avram, Dorina; McCabe, Amanda; MacNamara, Katherine C; Sellati, Timothy J; Harton, Jonathan A

    2016-03-01

    Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but the mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that in contrast to overwhelming bacterial burden or cytokine production, an overt innate cellular response to Ft drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature and die, leading to subsequent necrotic lung damage, loss of pulmonary function, and host death that is partially dependent upon immature Ly6G+ cells. Acceleration of this process may account for the rapid lethality seen with Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection.

  9. An Immature Myeloid/Myeloid-Suppressor Cell Response Associated with Necrotizing Inflammation Mediates Lethal Pulmonary Tularemia

    PubMed Central

    Periasamy, Sivakumar; Avram, Dorina; McCabe, Amanda; MacNamara, Katherine C.; Sellati, Timothy J.; Harton, Jonathan A.

    2016-01-01

    Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but the mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that in contrast to overwhelming bacterial burden or cytokine production, an overt innate cellular response to Ft drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature and die, leading to subsequent necrotic lung damage, loss of pulmonary function, and host death that is partially dependent upon immature Ly6G+ cells. Acceleration of this process may account for the rapid lethality seen with Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection. PMID:27015566

  10. Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function

    PubMed Central

    Langer, Marybeth; Duggan, Elizabeth Stewart; Booth, John Leland; Patel, Vineet Indrajit; Zander, Ryan A.; Silasi-Mansat, Robert; Ramani, Vijay; Veres, Tibor Zoltan; Prenzler, Frauke; Sewald, Katherina; Williams, Daniel M.; Coggeshall, Kenneth Mark; Awasthi, Shanjana; Lupu, Florea; Burian, Dennis; Ballard, Jimmy Dale; Braun, Armin

    2012-01-01

    The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness. PMID:23027535

  11. Issues surrounding lethal injection as a means of capital punishment.

    PubMed

    Romanelli, Frank; Whisman, Tyler; Fink, Joseph L

    2008-12-01

    Lethal injection as a method of state-sanctioned capital punishment was initially proposed in the United States in 1977 and used for the first time in 1982. Most lethal injection protocols use a sequential drug combination of sodium thiopental, pancuronium bromide, and potassium chloride. Lethal injection was originally introduced as a more humane form of execution compared with existing mechanical methods such as electrocution, toxic gassing, hanging, or firing squad. Lethal injection has not, however, been without controversy. Several states are considering whether lethal injection meets constitutional scrutiny forbidding cruel and unusual punishment. Recently in the case of Ralph Baze and Thomas C. Bowling, Petitioners, v John D. Rees, Commissioner, Kentucky Department of Corrections et al, the United States Supreme Court upheld the constitutionality of the lethal injection protocol as carried out in the Commonwealth of Kentucky. Most of the debate has surrounded the dosing and procedures used in lethal injection and whether the drug combinations and measures for administering the drugs truly produce a timely, pain-free, and fail-safe death. Many have also raised issues regarding the "medicalization" of execution and the ethics of health care professionals' participation in any part of the lethal injection process. As a result of all these issues, the future of lethal injection as a means of execution in the United States is under significant scrutiny. Outcomes of ongoing legislative and judicial reviews might result in cessation of lethal injection in totality or in alterations involving specific drug combinations or administration procedures.

  12. Differential screening of mutated SOD1 transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons.

    PubMed

    Dupuis, L; de Tapia, M; René, F; Lutz-Bucher, B; Gordon, J W; Mercken, L; Pradier, L; Loeffler, J P

    2000-08-01

    In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.

  13. mRNA Expression Signature of Gleason Grade Predicts Lethal Prostate Cancer

    PubMed Central

    Penney, Kathryn L.; Sinnott, Jennifer A.; Fall, Katja; Pawitan, Yudi; Hoshida, Yujin; Kraft, Peter; Stark, Jennifer R.; Fiorentino, Michelangelo; Perner, Sven; Finn, Stephen; Calza, Stefano; Flavin, Richard; Freedman, Matthew L.; Setlur, Sunita; Sesso, Howard D.; Andersson, Swen-Olof; Martin, Neil; Kantoff, Philip W.; Johansson, Jan-Erik; Adami, Hans-Olov; Rubin, Mark A.; Loda, Massimo; Golub, Todd R.; Andrén, Ove; Stampfer, Meir J.; Mucci, Lorelei A.

    2011-01-01

    Purpose Prostate-specific antigen screening has led to enormous overtreatment of prostate cancer because of the inability to distinguish potentially lethal disease at diagnosis. We reasoned that by identifying an mRNA signature of Gleason grade, the best predictor of prognosis, we could improve prediction of lethal disease among men with moderate Gleason 7 tumors, the most common grade, and the most indeterminate in terms of prognosis. Patients and Methods Using the complementary DNA–mediated annealing, selection, extension, and ligation assay, we measured the mRNA expression of 6,100 genes in prostate tumor tissue in the Swedish Watchful Waiting cohort (n = 358) and Physicians' Health Study (PHS; n = 109). We developed an mRNA signature of Gleason grade comparing individuals with Gleason ≤ 6 to those with Gleason ≥ 8 tumors and applied the model among patients with Gleason 7 to discriminate lethal cases. Results We built a 157-gene signature using the Swedish data that predicted Gleason with low misclassification (area under the curve [AUC] = 0.91); when this signature was tested in the PHS, the discriminatory ability remained high (AUC = 0.94). In men with Gleason 7 tumors, who were excluded from the model building, the signature significantly improved the prediction of lethal disease beyond knowing whether the Gleason score was 4 + 3 or 3 + 4 (P = .006). Conclusion Our expression signature and the genes identified may improve our understanding of the de-differentiation process of prostate tumors. Additionally, the signature may have clinical applications among men with Gleason 7, by further estimating their risk of lethal prostate cancer and thereby guiding therapy decisions to improve outcomes and reduce overtreatment. PMID:21537050

  14. Two different forms of lethal chondrodysplasias caused by COL2A1 gene mutations

    SciTech Connect

    Winterpacht, A.; Hilbert, K.; Schwarze, U.

    1994-09-01

    Two bone dysplasia families seem to be due to mutations in the type II procollagen gene (COL2A1): the so-called spondyloepiphyseal dysplasia congenita (SEDC) group with achondrogenesis II, hypochondrogenesis, SEDC, osteoarthrosis and the Stickler-Kniest pattern that include different forms of Kniest and Stickler dysplasia. Both groups comprise a clinical spectrum ranging from lethal to mild. COL2A1-mutations have been identified in lethal forms of the SEDC family but not in lethal forms of the Stickler/Kniest group. We now report a COL2A-1 mutation in an additional case of hypochondrogenesis (patient S) and in a lethal case of Kniest dysplasia (patient B). We amplified all 54 exons of the COL2A1 gene in both patients and screened the PCR products for mutations by SSCP analysis and sequencing. In patient B, we identified an 18 bp deletion in exon 34 which removes 6 amino acids from the mature protein. In patient S, we were able to identify a two base pair exchange (GG to AT) in exon 31, which leads to the very unusual conversion of Gly to Ile. To our knowledge, this is the first report of a Gly to Ile conversion in the COL2A1 gene, and the first report of a COL2A1 gene mutation in a lethal form of Kniest dysplasia. On the basis of the known COL2A1 gene mutations and the genotype-phenotype correlations established so far, we provide molecular data (an in frame deletion in patient B and a Gly conversion in patient S) that support their clinical classification as Kniest dysplasia and hypochondrogenesis, respectively.

  15. Antenatal diagnosis of lethal skeletal dysplasias.

    PubMed

    Tretter, A E; Saunders, R C; Meyers, C M; Dungan, J S; Grumbach, K; Sun, C C; Campbell, A B; Wulfsberg, E A

    1998-02-17

    Lethal skeletal dysplasias (LSD) are a heterogeneous group of rare but important genetic disorders characterized by abnormal growth and development of bone and cartilage. We describe the diagnosis and outcome of 29 cases of lethal skeletal dysplasias evaluated between January 1989 and December 1996 at the University of Maryland Medical Center and the Ultrasound Institute of Baltimore. Two cases presented at delivery with no prenatal care while the remaining 27 cases were identified by antenatal sonography. Final diagnoses included thanatophoric dysplasia (14), osteogenesis imperfecta, type II (6), achondrogenesis (2), short rib syndromes (3), campomelic syndrome (2), atelosteogenesis (1), and no evidence of a skeletal dysplasia (1). Twenty out of 27 pregnancies were terminated with an average at detection of 21.6 weeks. The other 7 pregnancies that went on to deliver had an average age at detection of 29.2 weeks. Fetal abnormalities in the terminated pregnancies were identified at a significantly earlier gestational age (P = 0.0016) than the pregnancies that continued. While the identification of LSD by sonography was excellent (26/27), only 13/27 (48%) were given an accurate specific antenatal diagnosis. In 8/14 (57%) cases with an inaccurate or nonspecific diagnosis there was a significant or crucial change in the genetic counseling. Thus, while antenatal sonography is an excellent method for discovering LSD, clinical examination, radiographs, and autopsy are mandatory for making a specific diagnosis. PMID:9489797

  16. Suicide Intent and Accurate Expectations of Lethality: Predictors of Medical Lethality of Suicide Attempts

    ERIC Educational Resources Information Center

    Brown, Gregory K.; Henriques, Gregg R.; Sosdjan, Daniella; Beck, Aaron T.

    2004-01-01

    The degree of intent to commit suicide and the severity of self-injury were examined in individuals (N = 180) who had recently attempted suicide. Although a minimal association was found between the degree of suicide intent and the degree of lethality of the attempt, the accuracy of expectations about the likelihood of dying was found to moderate…

  17. Gonadosomatic mosaicism for lethal mutations in Drosophila lethal mutations disturbing larval development

    SciTech Connect

    Ivanov, A.I.; Sakharova, N.Yu.

    1988-11-01

    Phenogenetic analysis of autonomous lethal mutations obtained by the method of gonadosomatic mosaicism which manifested during larval stages, established that the nuclei of hypodermal cells, salivary glands suprapharyngeal ganglion, pharynx, esophagus, gizzard, and hindgut are the derivatives of the same nucleus (from the first two nuclei of cleavage) as the nuclei of the cells of the imaginal-somatic tissues.

  18. A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4

    PubMed Central

    Williams, Corey L.; Pieczynski, Jay N.; Roszczynialski, Kelly N.; Covington, Jannese E.; Malarkey, Erik B.; Yoder, Bradley K.

    2016-01-01

    Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit

  19. Suppressor Screen and Phenotype Analyses Revealed an Emerging Role of the Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 in Compensated Cell Enlargement.

    PubMed

    Katano, Mana; Takahashi, Kazuki; Hirano, Tomonari; Kazama, Yusuke; Abe, Tomoko; Tsukaya, Hirokazu; Ferjani, Ali

    2016-01-01

    Efficient use of seed nutrient reserves is crucial for germination and establishment of plant seedlings. Mobilizing seed oil reserves in Arabidopsis involves β-oxidation, the glyoxylate cycle, and gluconeogenesis, which provide essential energy and the carbon skeletons needed to sustain seedling growth until photoautotrophy is acquired. We demonstrated that H(+)-PPase activity is required for gluconeogenesis. Lack of H(+)-PPase in fugu5 mutants increases cytosolic pyrophosphate (PPi) levels, which partially reduces sucrose synthesis de novo and inhibits cell division. In contrast, post-mitotic cell expansion in cotyledons was unusually enhanced, a phenotype called compensation. Therefore, it appears that PPi inhibits several cellular functions, including cell cycling, to trigger compensated cell enlargement (CCE). Here, we mutagenized fugu5-1 seeds with (12)C(6+) heavy-ion irradiation and screened mutations that restrain CCE to gain insight into the genetic pathway(s) involved in CCE. We isolated A#3-1, in which cell size was severely reduced, but cell number remained similar to that of original fugu5-1. Moreover, cell number decreased in A#3-1 single mutant (A#3-1sm), similar to that of fugu5-1, but cell size was almost equal to that of the wild type. Surprisingly, A#3-1 mutation did not affect CCE in other compensation exhibiting mutant backgrounds, such as an3-4 and fugu2-1/fas1-6. Subsequent map-based cloning combined with genome sequencing and HRM curve analysis identified enoyl-CoA hydratase 2 (ECH2) as the causal gene of A#3-1. The above phenotypes were consistently observed in the ech2-1 allele and supplying sucrose restored the morphological and cellular phenotypes in fugu5-1, ech2-1, A#3-1sm, fugu5-1 ech2-1, and A#3-1; fugu5-1. Taken together, these results suggest that defects in either H(+)-PPase or ECH2 compromise cell proliferation due to defects in mobilizing seed storage lipids. In contrast, ECH2 alone likely promotes CCE during the post

  20. Loss-of-function mutations in a glutathione S-transferase suppress the prune-Killer of prune lethal interaction.

    PubMed

    Provost, Elayne; Hersperger, Grafton; Timmons, Lisa; Ho, Wen Qi; Hersperger, Evelyn; Alcazar, Rosa; Shearn, Allen

    2006-01-01

    The prune gene of Drosophila melanogaster is predicted to encode a phosphodiesterase. Null alleles of prune are viable but cause an eye-color phenotype. The abnormal wing discs gene encodes a nucleoside diphosphate kinase. Killer of prune is a missense mutation in the abnormal wing discs gene. Although it has no phenotype by itself even when homozygous, Killer of prune when heterozygous causes lethality in the absence of prune gene function. A screen for suppressors of transgenic Killer of prune led to the recovery of three mutations, all of which are in the same gene. As heterozygotes these mutations are dominant suppressors of the prune-Killer of prune lethal interaction; as homozygotes these mutations cause early larval lethality and the absence of imaginal discs. These alleles are loss-of-function mutations in CG10065, a gene that is predicted to encode a protein with several zinc finger domains and glutathione S-transferase activity. PMID:16143620

  1. Screening of a Fosmid Library of Marine Environmental Genomic DNA Fragments Reveals Four Clones Related to Members of the Order Planctomycetales

    PubMed Central

    Vergin, Kevin L.; Urbach, Ena; Stein, Jeffery L.; DeLong, Edward F.; Lanoil, Brian D.; Giovannoni, Stephen J.

    1998-01-01

    A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591–599, 1996) yielded four clones with 16S rRNA genes from the order Planctomycetales. Three of the clones belong to the Pirellula group and one clone belongs to the Planctomyces group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes. PMID:9687477

  2. Disease screening of three breeding populations of adult exhibition budgerigars (Melopsittacus undulatus) in New Zealand reveals a high prevalence of a novel polyomavirus and avian malaria infection.

    PubMed

    Baron, Hamish R; Howe, Laryssa; Varsani, Arvind; Doneley, Robert J T

    2014-03-01

    Disease surveillance is vital to the management of New Zealand's endemic and threatened avian species. Three infectious agents that are potential threats to New Zealand's endemic birds include avian polyomavirus (APV), beak and feather disease virus (BFDV), and avian malaria. All three agents have been reported in New Zealand; however, possible reservoir populations have not been identified. In this communication, we report the first study of APV, BFDV, and avian malaria in introduced adult exhibition budgerigars (Melopsittacus undulatus) in New Zealand. Blood samples were collected from 90 living adult budgerigars from three breeding locations in the North Island of New Zealand. An overall APV prevalence of 22% was determined using a broad-spectrum nested PCR that amplified the major capsid protein VP1 gene of polyomavirus. Phylogenetic analysis of the VP1 gene revealed a unique isolate of APV, which had a sequence divergence of 32% to previously reported budgerigar fledgling disease strains and 33% to the recently reported New Zealand finch isolate. All of the budgerigars sampled were found to be PCR negative for BFDV, and an overall prevalence of 30% was detected by PCR for avian malaria. Sequencing revealed the presence of ubiquitous malarial strains and also the potentially destructive Plasmodium relictum strain. The results of this study suggest that both APV and avian malaria are present in New Zealand adult budgerigars, and our study highlights the need for further studies to determine whether these pathogens in captive bird populations may be a threat or spill over into New Zealand's endemic and threatened avifauna and whether prevention and control methods need to be implemented. PMID:24758122

  3. Low-coverage exome sequencing screen in formalin-fixed paraffin-embedded tumors reveals evidence of exposure to carcinogenic aristolochic acid

    PubMed Central

    Castells, Xavier; Karanović, Sandra; Ardin, Maude; Tomić, Karla; Xylinas, Evanguelos; Durand, Geoffroy; Villar, Stephanie; Forey, Nathalie; Le Calvez-Kelm, Florence; Voegele, Catherine; Karlović, Krešimir; Mišić, Maja; Dittrich, Damir; Dolgalev, Igor; McKay, James; Shariat, Shahrokh F.; Sidorenko, Viktoria S.; Fernandes, Andrea; Heguy, Adriana; Dickman, Kathleen G.; Olivier, Magali; Grollman, Arthur P.; Jelaković, Bojan; Zavadil, Jiri

    2015-01-01

    Background Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urological cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA sequencing studies using fresh frozen tumors. Methods Here we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multi-sample detection of the AA mutational signature, and demonstrate its utility in 17 formalin-fixed paraffin-embedded urothelial tumors obtained from 15 patients with endemic nephropathy, an environmental form of aristolochic acid nephropathy. Results LC-WES identified the AA signature, alongside signatures of age and APOBEC enzyme activity, in 15 samples sequenced at the average per-base coverage of ~10x. Analysis at 3–9x coverage revealed the signature in 91% of the positive samples. The exome-wide distribution of the predominant A>T transversions exhibited a stochastic pattern whereas 83 cancer driver genes were enriched for recurrent non-synonymous A>T mutations. In two patients, pairs of tumors from different parts of the urinary tract, including the bladder, harbored overlapping mutation patterns, suggesting tumor dissemination via cell seeding. Conclusion LC-WES analysis of archived tumor tissues is a reliable method applicable to investigations of both the exposure to AA and its biologic effects in human carcinomas. Impact By detecting cancers associated with AA exposure in high-risk populations, LC-WES can support future molecular epidemiology studies and provide evidence-base for relevant preventive measures. PMID:26383547

  4. Synthetic lethal approaches for assessing combinatorial efficacy of chemotherapeutic drugs.

    PubMed

    Jackson, Rebecca A; Chen, Ee Sin

    2016-06-01

    The recent advances in pharmacogenomics have made personalized medicine no longer a pipedream but a precise and powerful way to tailor individualized cancer treatment strategies. Cancer is a devastating disease, and contemporary chemotherapeutic strategies now integrate several agents in the treatment of some types of cancer, with the intent to block more than one target simultaneously. This constitutes the premise of synthetic lethality, an attractive therapeutic strategy already demonstrating clinical success in patients with breast and ovarian cancers. Synthetic lethal combinations offer the potential to also target the hitherto "undruggable" mutations that have challenged the cancer field for decades. However, synthetic lethality in clinical cancer therapy is very much still in its infancy, and selecting the most appropriate combinations-or synthetic lethal pairs-is not always an intuitive process. Here, we review some of the recent progress in identifying synthetic lethal combinations and their potential for therapy and highlight some of the tools through which synthetic lethal pairs are identified.

  5. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ-ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation.

    PubMed

    Jobe, Timothy O; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A; Mendoza-Cózatl, David G; Schroeder, Julian I

    2012-06-01

    Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M(2) seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione depletion

  6. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ -ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation

    PubMed Central

    Jobe, Timothy O.; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

    2015-01-01

    Summary Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M2 seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione

  7. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ-ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation.

    PubMed

    Jobe, Timothy O; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A; Mendoza-Cózatl, David G; Schroeder, Julian I

    2012-06-01

    Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M(2) seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione depletion

  8. High-throughput screening of a large collection of non-conventional yeasts reveals their potential for aroma formation in food fermentation.

    PubMed

    Gamero, Amparo; Quintilla, Raquel; Groenewald, Marizeth; Alkema, Wynand; Boekhout, Teun; Hazelwood, Lucie

    2016-12-01

    Saccharomyces yeast species are currently the most important yeasts involved in industrial-scale food fermentations. However, there are hundreds of other yeast species poorly studied that are highly promising for flavour development, some of which have also been identified in traditional food fermentations. This work explores natural yeast biodiversity in terms of aroma formation, with a particular focus on aromas relevant for industrial fermentations such as wine and beer. Several non-Saccharomyces species produce important aroma compounds such as fusel alcohols derived from the Ehrlich pathway, acetate esters and ethyl esters in significantly higher quantities than the well-known Saccharomyces species. These species are Starmera caribaea, Hanseniaspora guilliermondii, Galactomyces geotrichum, Saccharomycopsis vini and Ambrosiozyma monospora. Certain species revealed a strain-dependent flavour profile while other species were very homogenous in their flavour profiles. Finally, characterization of a selected number of yeast species using valine or leucine as sole nitrogen sources indicates that the mechanisms of regulation of the expression of the Ehrlich pathway exist amongst non-conventional yeast species. PMID:27554157

  9. Targeted DNA Methylation Screen in the Mouse Mammary Genome Reveals a Parity-Induced Hypermethylation of Igf1r That Persists Long after Parturition.

    PubMed

    Katz, Tiffany A; Liao, Serena G; Palmieri, Vincent J; Dearth, Robert K; Pathiraja, Thushangi N; Huo, Zhiguang; Shaw, Patricia; Small, Sarah; Davidson, Nancy E; Peters, David G; Tseng, George C; Oesterreich, Steffi; Lee, Adrian V

    2015-10-01

    The most effective natural prevention against breast cancer is an early first full-term pregnancy. Understanding how the protective effect is elicited will inform the development of new prevention strategies. To better understand the role of epigenetics in long-term protection, we investigated parity-induced DNA methylation in the mammary gland. FVB mice were bred or remained nulliparous and mammary glands harvested immediately after involution (early) or 6.5 months following involution (late), allowing identification of both transient and persistent changes. Targeted DNA methylation (109 Mb of Ensemble regulatory features) analysis was performed using the SureSelectXT Mouse Methyl-seq assay and massively parallel sequencing. Two hundred sixty-nine genes were hypermethylated and 128 hypomethylated persistently at both the early and late time points. Pathway analysis of the persistently differentially methylated genes revealed Igf1r to be central to one of the top identified signaling networks, and Igf1r itself was one of the most significantly hypermethylated genes. Hypermethylation of Igf1r in the parous mammary gland was associated with a reduction of Igf1r mRNA expression. These data suggest that the IGF pathway is regulated at multiple levels during pregnancy and that its modification might be critical in the protective role of pregnancy. This supports the approach of lowering IGF action for prevention of breast cancer, a concept that is currently being tested clinically.

  10. Screening sourdough samples for gliadin-degrading activity revealed Lactobacillus casei strains able to individually metabolize the coeliac-disease-related 33-mer peptide.

    PubMed

    Alvarez-Sieiro, Patricia; Redruello, Begoña; Ladero, Victor; Martín, Maria Cruz; Fernández, María; Alvarez, Miguel A

    2016-05-01

    A selective culture medium containing acid-hydrolyzed gliadins as the sole nitrogen source was used in the search for sourdough-indigenous lactic acid bacteria (LAB) with gliadin-metabolizing activity. Twenty gliadin-degrading LAB strains were isolated from 10 sourdoughs made in different ways and from different geographical regions. Fifteen of the 20 isolated strains were identified as Lactobacillus casei, a species usually reported as subdominant in sourdough populations. The other 5 gliadin-degrading strains belonged to the more commonly encountered sourdough species Leuconostoc mesenteroides and Lactobacillus plantarum. All these strains were shown to be safe in terms of their resistance to antimicrobial agents. When individually incubated with the α2-gliadin-derived immunotoxic 33-mer peptide (97.5 ppm), half of the L. casei strains metabolized at least 50% of it within 24 h. One strain metabolized 82% of the 33-mer peptide within 8 h and made it fully disappear within 12 h. These results reveal for the first time the presence in sourdough of proteolytic L. casei strains with the capacity to individually metabolize the coeliac-disease-related 33-mer peptide.

  11. Screening sourdough samples for gliadin-degrading activity revealed Lactobacillus casei strains able to individually metabolize the coeliac-disease-related 33-mer peptide.

    PubMed

    Alvarez-Sieiro, Patricia; Redruello, Begoña; Ladero, Victor; Martín, Maria Cruz; Fernández, María; Alvarez, Miguel A

    2016-05-01

    A selective culture medium containing acid-hydrolyzed gliadins as the sole nitrogen source was used in the search for sourdough-indigenous lactic acid bacteria (LAB) with gliadin-metabolizing activity. Twenty gliadin-degrading LAB strains were isolated from 10 sourdoughs made in different ways and from different geographical regions. Fifteen of the 20 isolated strains were identified as Lactobacillus casei, a species usually reported as subdominant in sourdough populations. The other 5 gliadin-degrading strains belonged to the more commonly encountered sourdough species Leuconostoc mesenteroides and Lactobacillus plantarum. All these strains were shown to be safe in terms of their resistance to antimicrobial agents. When individually incubated with the α2-gliadin-derived immunotoxic 33-mer peptide (97.5 ppm), half of the L. casei strains metabolized at least 50% of it within 24 h. One strain metabolized 82% of the 33-mer peptide within 8 h and made it fully disappear within 12 h. These results reveal for the first time the presence in sourdough of proteolytic L. casei strains with the capacity to individually metabolize the coeliac-disease-related 33-mer peptide. PMID:27021684

  12. Small molecule screening reveals a transcription-independent pro-survival function of androgen receptor in castration-resistant prostate cancer.

    PubMed

    Narizhneva, Natalia V; Tararova, Natalia D; Ryabokon, Petro; Shyshynova, Inna; Prokvolit, Anatoly; Komarov, Pavel G; Purmal, Andrei A; Gudkov, Andrei V; Gurova, Katerina V

    2009-12-15

    In prostate cancer (PCa) patients, initial responsiveness to androgen deprivation therapy is frequently followed by relapse due to development of treatment-resistant androgen-independent PCa. This is typically associated with acquisition of mutations in AR that allow activity as a transcription factor in the absence of ligand, indicating that androgen-independent PCa remains dependent on AR function. Our strategy to effectively target AR in androgen-independent PCa involved using a cell-based readout to isolate small molecules that inhibit AR transactivation function through mechanisms other than modulation of ligand binding. A number of the identified inhibitors were toxic to AR-expressing PCa cells regardless of their androgen dependence. Among these, some only suppressed PCa cell growth (ARTIS), while others induced cell death (ARTIK). ARTIK, but not ARTIS, compounds caused disappearance of AR protein from treated cells. siRNA against AR behaved like ARTIK compounds, while a dominant negative AR mutant that prevents AR-mediated transactivation but does not eliminate the protein showed only a growth suppressive effect. These observations reveal a transcription-independent function of AR that is essential for PCa cell viability and, therefore, is an ideal target for anti-PCa treatment. Indeed, several of the identified AR inhibitors demonstrated in vivo efficacy in mouse models of PCa and are candidates for pharmacologic optimization.

  13. Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development.

    PubMed

    Posnien, Nico; Koniszewski, Nikolaus Dieter Bernhard; Hein, Hendrikje Jeannette; Bucher, Gregor

    2011-12-01

    Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly.

  14. High-throughput screening of a large collection of non-conventional yeasts reveals their potential for aroma formation in food fermentation.

    PubMed

    Gamero, Amparo; Quintilla, Raquel; Groenewald, Marizeth; Alkema, Wynand; Boekhout, Teun; Hazelwood, Lucie

    2016-12-01

    Saccharomyces yeast species are currently the most important yeasts involved in industrial-scale food fermentations. However, there are hundreds of other yeast species poorly studied that are highly promising for flavour development, some of which have also been identified in traditional food fermentations. This work explores natural yeast biodiversity in terms of aroma formation, with a particular focus on aromas relevant for industrial fermentations such as wine and beer. Several non-Saccharomyces species produce important aroma compounds such as fusel alcohols derived from the Ehrlich pathway, acetate esters and ethyl esters in significantly higher quantities than the well-known Saccharomyces species. These species are Starmera caribaea, Hanseniaspora guilliermondii, Galactomyces geotrichum, Saccharomycopsis vini and Ambrosiozyma monospora. Certain species revealed a strain-dependent flavour profile while other species were very homogenous in their flavour profiles. Finally, characterization of a selected number of yeast species using valine or leucine as sole nitrogen sources indicates that the mechanisms of regulation of the expression of the Ehrlich pathway exist amongst non-conventional yeast species.

  15. Lethal photosensitization of biofilm-grown bacteria

    NASA Astrophysics Data System (ADS)

    Wilson, Michael

    1997-12-01

    Antibacterial agents are increasingly being used for the prophylaxis and treatment of oral diseases. As these agents can be rendered ineffective by resistance development in the target organisms there is a need to develop alternative antimicrobial approaches. Light-activated antimicrobial agents release singlet oxygen and free radicals which can kill adjacent bacteria and a wide range of cariogenic and periodontopathogenic bacteria has been shown to be susceptible to such agents. In the oral cavity these organisms are present as biofilms (dental plaques) which are less susceptible to traditional antimicrobial agents than bacterial suspensions. The results of these studies have shown that biofilm-grown oral bacteria are also susceptible to lethal photosensitization although the light energy doses required are grater than those needed to kill the organisms when they are grown as aqueous suspensions.

  16. Alleged lethal sorcery in East Timor.

    PubMed

    Pollanen, Michael S

    2004-01-01

    A wide range of cultural and social perspectives exists on the concept of sudden and unexpected death. In countries, without a formal system of death investigation, sudden death is shrouded in mysticism often based on traditional belief systems. This cultural perspective on sudden death is often at variance with medical and forensic concepts and may include explanations such as sorcery, magic, and voodoo. In this case report, the postmortem findings in an alleged victim of lethal 'black magic', known as ema halo by the indigenous people of East Timor, is described. The alleged victim died suddenly in front of witnesses. At autopsy, marked dilation of a bicuspid aortic valve with annuloaortic ectasia and a sinus of Valsalva aneurysm was found after exhumation of the body. The findings mitigated the local belief in witchcraft and established a natural manner of death.

  17. Ants defend aphids against lethal disease.

    PubMed

    Nielsen, Charlotte; Agrawal, Anurag A; Hajek, Ann E

    2010-04-23

    Social insects defend their own colonies and some species also protect their mutualist partners. In mutualisms with aphids, ants typically feed on honeydew produced by aphids and, in turn guard and shelter aphid colonies from insect natural enemies. Here we report that Formica podzolica ants tending milkweed aphids, Aphis asclepiadis, protect aphid colonies from lethal fungal infections caused by an obligate aphid pathogen, Pandora neoaphidis. In field experiments, bodies of fungal-killed aphids were quickly removed from ant-tended aphid colonies. Ant workers were also able to detect infective conidia on the cuticle of living aphids and responded by either removing or grooming these aphids. Our results extend the long-standing view of ants as mutualists and protectors of aphids by demonstrating focused sanitizing and quarantining behaviour that may lead to reduced disease transmission in aphid colonies.

  18. Collagen defects in lethal perinatal osteogenesis imperfecta.

    PubMed

    Bateman, J F; Chan, D; Mascara, T; Rogers, J G; Cole, W G

    1986-12-15

    Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.

  19. Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins.

    PubMed

    Silin, Vitalii; Kasianowicz, John J; Michelman-Ribeiro, Ariel; Panchal, Rekha G; Bavari, Sina; Robertson, Joseph W F

    2016-01-01

    Tethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer. We characterize the sensor and its properties with electrochemical impedance spectroscopy and surface plasmon resonance. The sensor shows a sensitivity similar to ELISA and can also be used to rapidly screen for molecules that bind to the toxins and potentially inhibit their lethal effects. PMID:27348008

  20. Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins

    PubMed Central

    Silin, Vitalii; Kasianowicz, John J.; Michelman-Ribeiro, Ariel; Panchal, Rekha G.; Bavari, Sina; Robertson, Joseph W. F.

    2016-01-01

    Tethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer. We characterize the sensor and its properties with electrochemical impedance spectroscopy and surface plasmon resonance. The sensor shows a sensitivity similar to ELISA and can also be used to rapidly screen for molecules that bind to the toxins and potentially inhibit their lethal effects. PMID:27348008

  1. ALC/50/ values for some polymeric materials. [Apparent Lethal Concentration fire toxicity

    NASA Technical Reports Server (NTRS)

    Hilado, C. J.; Cumming, H. J.; Schneider, J. E.; Kourtides, D. A.; Parker, J. A.

    1978-01-01

    Apparent lethal concentrations for 50 per cent of the test animals within a 30-min exposure period (ALC/50/) were determined for seventeen samples of polymeric materials, using the screening test method. The materials evaluated included resin-glass composites, film composites, and miscellaneous resins. ALC(50) values, based on weight of original sample charged, ranged from 24 to 110 mg/l. Modified phenolic resins seemed to exhibit less toxicity than the baseline epoxy resins. Among the film composites evaluated, only flame modified polyvinyl fluoride appeared to exhibit less toxicity than the baseline polyvinyl fluoride film.

  2. Tetracycline-suppressible female lethality and sterility in the Mexican fruit fly, Anastrepha ludens.

    PubMed

    Schetelig, M F; Targovska, A; Meza, J S; Bourtzis, K; Handler, A M

    2016-08-01

    The sterile insect technique (SIT) involves the mass release of sterile males to suppress insect pest populations. SIT has been improved for larval pests by the development of strains for female-specific tetracycline-suppressible (Tet-off) embryonic lethal systems for male-only populations. Here we describe the extension of this approach to the Mexican fruit fly, Anastrepha ludens, using a Tet-off driver construct with the Tet-transactivator (tTA) under embryo-specific Anastrepha suspensa serendipity α (As-sry-α) promoter regulation. In the absence of tetracycline, tTA acts upon a Tet-response element linked to the pro-apoptotic cell death gene lethal effector, head involuation defective (hid), from A. ludens (Alhid(Ala2) ) that contains a sex-specific intron splicing cassette, resulting in female-specific expression of the lethal effector. Parental adults double-homozygous for the driver/effector vectors were expected to yield male-only progeny when reared on Tet-free diet, but a complete lack of oviposited eggs resulted for each of the three strains tested. Ovary dissection revealed nonvitellogenic oocytes in all strains that was reversible by feeding females tetracycline for 5 days after eclosion, resulting in male-only adults in one strain. Presumably the sry-α promoter exhibits prezygotic maternal expression as well as zygotic embryonic expression in A. ludens, resulting in a Tet-off sterility effect in addition to female-specific lethality. PMID:27135433

  3. Tetracycline-suppressible female lethality and sterility in the Mexican fruit fly, Anastrepha ludens.

    PubMed

    Schetelig, M F; Targovska, A; Meza, J S; Bourtzis, K; Handler, A M

    2016-08-01

    The sterile insect technique (SIT) involves the mass release of sterile males to suppress insect pest populations. SIT has been improved for larval pests by the development of strains for female-specific tetracycline-suppressible (Tet-off) embryonic lethal systems for male-only populations. Here we describe the extension of this approach to the Mexican fruit fly, Anastrepha ludens, using a Tet-off driver construct with the Tet-transactivator (tTA) under embryo-specific Anastrepha suspensa serendipity α (As-sry-α) promoter regulation. In the absence of tetracycline, tTA acts upon a Tet-response element linked to the pro-apoptotic cell death gene lethal effector, head involuation defective (hid), from A. ludens (Alhid(Ala2) ) that contains a sex-specific intron splicing cassette, resulting in female-specific expression of the lethal effector. Parental adults double-homozygous for the driver/effector vectors were expected to yield male-only progeny when reared on Tet-free diet, but a complete lack of oviposited eggs resulted for each of the three strains tested. Ovary dissection revealed nonvitellogenic oocytes in all strains that was reversible by feeding females tetracycline for 5 days after eclosion, resulting in male-only adults in one strain. Presumably the sry-α promoter exhibits prezygotic maternal expression as well as zygotic embryonic expression in A. ludens, resulting in a Tet-off sterility effect in addition to female-specific lethality.

  4. Airport Screening

    MedlinePlus

    ... 2011 Photo courtesy of Dan Paluska/Flickr Denver Airport Security Screening Introduction With air travel regaining popularity and increased secu- rity measures, airport security screening has become an area of interest for ...

  5. Health Screening

    MedlinePlus

    Screenings are tests that look for diseases before you have symptoms. Screening tests can find diseases early, when they're easier ... Overweight and obesity Prostate cancer in men Which tests you need depends on your age, your sex, ...

  6. MRSA Screening

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? MRSA Screening Share this page: Was this page helpful? Formal name: Methicillin resistant Staphylococcus aureus Screening Related tests: Wound Culture At a Glance ...

  7. Chronic exposure of corals to fine sediments: lethal and sub-lethal impacts.

    PubMed

    Flores, Florita; Hoogenboom, Mia O; Smith, Luke D; Cooper, Timothy F; Abrego, David; Negri, Andrew P

    2012-01-01

    Understanding the sedimentation and turbidity thresholds for corals is critical in assessing the potential impacts of dredging projects in tropical marine systems. In this study, we exposed two species of coral sampled from offshore locations to six levels of total suspended solids (TSS) for 16 weeks in the laboratory, including a 4 week recovery period. Dose-response relationships were developed to quantify the lethal and sub-lethal thresholds of sedimentation and turbidity for the corals. The sediment treatments affected the horizontal foliaceous species (Montipora aequituberculata) more than the upright branching species (Acropora millepora). The lowest sediment treatments that caused full colony mortality were 30 mg l(-1) TSS (25 mg cm(-2) day(-1)) for M. aequituberculata and 100 mg l(-1) TSS (83 mg cm(-2) day(-1)) for A. millepora after 12 weeks. Coral mortality generally took longer than 4 weeks and was closely related to sediment accumulation on the surface of the corals. While measurements of damage to photosystem II in the symbionts and reductions in lipid content and growth indicated sub-lethal responses in surviving corals, the most reliable predictor of coral mortality in this experiment was long-term sediment accumulation on coral tissue. PMID:22662225

  8. Chronic Exposure of Corals to Fine Sediments: Lethal and Sub-Lethal Impacts

    PubMed Central

    Flores, Florita; Hoogenboom, Mia O.; Smith, Luke D.; Cooper, Timothy F.; Abrego, David; Negri, Andrew P.

    2012-01-01

    Understanding the sedimentation and turbidity thresholds for corals is critical in assessing the potential impacts of dredging projects in tropical marine systems. In this study, we exposed two species of coral sampled from offshore locations to six levels of total suspended solids (TSS) for 16 weeks in the laboratory, including a 4 week recovery period. Dose-response relationships were developed to quantify the lethal and sub-lethal thresholds of sedimentation and turbidity for the corals. The sediment treatments affected the horizontal foliaceous species (Montipora aequituberculata) more than the upright branching species (Acropora millepora). The lowest sediment treatments that caused full colony mortality were 30 mg l−1 TSS (25 mg cm−2 day−1) for M. aequituberculata and 100 mg l−1 TSS (83 mg cm−2 day−1) for A. millepora after 12 weeks. Coral mortality generally took longer than 4 weeks and was closely related to sediment accumulation on the surface of the corals. While measurements of damage to photosystem II in the symbionts and reductions in lipid content and growth indicated sub-lethal responses in surviving corals, the most reliable predictor of coral mortality in this experiment was long-term sediment accumulation on coral tissue. PMID:22662225

  9. CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

    PubMed Central

    Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J.; Ashworth, Alan

    2016-01-01

    Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

  10. CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

    PubMed

    Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J; Ashworth, Alan

    2016-01-01

    Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

  11. Whole-Transcriptome Shotgun Sequencing (RNA-seq) Screen Reveals Upregulation of Cellobiose and Motility Operons of Lactobacillus ruminis L5 during Growth on Tetrasaccharides Derived from Barley β-Glucan

    PubMed Central

    Lawley, Blair; Sims, Ian M.

    2013-01-01

    Lactobacillus ruminis is an inhabitant of human bowels and bovine rumens. None of 10 isolates (three from bovine rumen, seven from human feces) of L. ruminis that were tested could utilize barley β-glucan for growth. Seven of the strains of L. ruminis were, however, able to utilize tetrasaccharides (3-O-β-cellotriosyl-d-glucose [LDP4] or 4-O-β-laminaribiosyl-d-cellobiose [CDP4]) present in β-glucan hydrolysates for growth. The tetrasaccharides were generated by the use of lichenase or cellulase, respectively. To learn more about the utilization of tetrasaccharides by L. ruminis, whole-transcriptome shotgun sequencing (RNA-seq) was tested as a transcriptional screen to detect altered gene expression when an autochthonous human strain (L5) was grown in medium containing CDP4. RNA-seq results were confirmed and extended by reverse transcription-quantitative PCR assays of selected genes in two upregulated operons when cells were grown as batch cultures in medium containing either CDP4 or LDP4. The cellobiose utilization operon had increased transcription, particularly in early growth phase, whereas the chemotaxis/motility operon was upregulated in late growth phase. Phenotypic changes were seen in relation to upregulation of chemotaxis/flagellar operons: flagella were rarely seen by electron microscopy on glucose-grown cells but cells cultured in tetrasaccharide medium were commonly flagellated. Chemotactic movement toward tetrasaccharides was demonstrated in capillary cultures. L. ruminis utilized 3-O-β-cellotriosyl-d-glucose released by β-glucan hydrolysis due to bowel commensal Coprococcus sp., indicating that cross feeding of tetrasaccharide between bacteria could occur. Therefore, the RNA-seq screen and subsequent experiments had utility in revealing foraging attributes of gut commensal Lactobacillus ruminis. PMID:23851085

  12. Towards a compendium of essential genes – From model organisms to synthetic lethality in cancer cells

    PubMed Central

    Zhan, Tianzuo; Boutros, Michael

    2016-01-01

    Abstract Essential genes are defined by their requirement to sustain life in cells or whole organisms. The systematic identification of essential gene sets not only allows insights into the fundamental building blocks of life, but may also provide novel therapeutic targets in oncology. The discovery of essential genes has been tightly linked to the development and deployment of various screening technologies. Here, we describe how gene essentiality was addressed in different eukaryotic model organisms, covering a range of organisms from yeast to mouse. We describe how increasing knowledge of evolutionarily divergent genomes facilitate identification of gene essentiality across species. Finally, the impact of gene essentiality and synthetic lethality on cancer research and the clinical translation of screening results are highlighted. PMID:26627871

  13. Lethal body burdens of polar narcotics: Chlorophenols

    SciTech Connect

    Wezel, A.P. van; Punte, S.S.; Opperhuizen, A.

    1995-09-01

    The goal of the present study was to measure in fathead minnow (Pimephales promelas) the lethal body burden (LBB) of three chlorophenols that are known as polar narcotic chemicals. The LBBs of the chlorophenols were compared to LBBs of nonpolar narcotic chemicals to consider if the two classes of narcotic chemicals differ on a body burden level. The LBB of the most acidic chlorophenol was measured at two different levels of pH exposure to determine the influence of the degree of ionization on the magnitude of the LBB. Both n-octanol/water partition coefficients and n-hexane/water partition coefficients of the chlorophenols were determined at different pH levels to consider the influence of ionization on the partition coefficient and to determine the importance of a polar group in the organic phase on the partitioning behavior. Partitioning to n-octanol and n-hexane was used as input in a model to simulate the equilibrium partitioning between hydrophobic and nonhydrophobic and target and nontarget compartments in the fish.

  14. Tumor clone dynamics in lethal prostate cancer.

    PubMed

    Carreira, Suzanne; Romanel, Alessandro; Goodall, Jane; Grist, Emily; Ferraldeschi, Roberta; Miranda, Susana; Prandi, Davide; Lorente, David; Frenel, Jean-Sebastien; Pezaro, Carmel; Omlin, Aurelius; Rodrigues, Daniel Nava; Flohr, Penelope; Tunariu, Nina; S de Bono, Johann; Demichelis, Francesca; Attard, Gerhardt

    2014-09-17

    It is unclear whether a single clone metastasizes and remains dominant over the course of lethal prostate cancer. We describe the clonal architectural heterogeneity at different stages of disease progression by sequencing serial plasma and tumor samples from 16 ERG-positive patients. By characterizing the clonality of commonly occurring deletions at 21q22, 8p21, and 10q23, we identified multiple independent clones in metastatic disease that are differentially represented in tissue and circulation. To exemplify the clinical utility of our studies, we then showed a temporal association between clinical progression and emergence of androgen receptor (AR) mutations activated by glucocorticoids in about 20% of patients progressing on abiraterone and prednisolone or dexamethasone. Resistant clones showed a complex dynamic with temporal and spatial heterogeneity, suggesting distinct mechanisms of resistance at different sites that emerged and regressed depending on treatment selection pressure. This introduces a management paradigm requiring sequential monitoring of advanced prostate cancer patients with plasma and tumor biopsies to ensure early discontinuation of agents when they become potential disease drivers.

  15. Targeting cancer using KAT inhibitors to mimic lethal knockouts.

    PubMed

    Brown, James A L; Bourke, Emer; Eriksson, Leif A; Kerin, Michael J

    2016-08-15

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  16. Targeting cancer using KAT inhibitors to mimic lethal knockouts

    PubMed Central

    Brown, James A.L.; Bourke, Emer; Eriksson, Leif A.; Kerin, Michael J.

    2016-01-01

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  17. Colon cancer screening

    MedlinePlus

    Screening for colon cancer; Colonoscopy - screening; Sigmoidoscopy - screening; Virtual colonoscopy - screening; Fecal immunochemical test; Stool DNA test; sDNA test; Colorectal cancer - screening; Rectal ...

  18. [Detection of the functionally active domains in the molecule of the lethal factor of the anthrax exotoxin].

    PubMed

    Noskov, A N; Kravchenko, T B; Noskova, V P

    1996-01-01

    Three functional domains were revealed in the molecule of the lethal factor of B. anthracis. They are located in the linear structure of the molecula as follows: the associative domain occupies the area from Lys39 to Met242, the stabilizing domain from Leu517 to Lys614, and the effector domain still further to the COOH-terminal Lys mino acid.

  19. Bacillus anthracis cell wall produces injurious inflammation but paradoxically decreases the lethality of anthrax lethal toxin in a rat model

    PubMed Central

    Cui, Xizhong; Su, Junwu; Li, Yan; Shiloach, Joseph; Solomon, Steven; Kaufman, Jeanne B.; Mani, Haresh; Fitz, Yvonne; Weng, Jia; Altaweel, Laith; Besch, Virginia; Eichacker, Peter Q.

    2012-01-01

    Objectives The in vivo inflammatory effects of the Bacillus anthracis cell wall are unknown. We therefore investigated these effects in rats and, for comparison, those of known inflammatory stimulants, Staphylococcus aureus cell wall or lipopolysaccharide (LPS). Method and Results Sprague–Dawley rats (n = 103) were challenged with increasing B. anthracis cell wall doses (10, 20, 40, 80, or 160 mg/kg) or diluent (control) as a bolus or 24-h infusion. The three highest bolus doses were lethal (20–64% lethality rates) as were the two highest infused doses (13% with each). Comparisons among lethal or nonlethal doses on other measured parameters were not significantly different, and these were combined for analysis. Over the 24 h after challenge initiation with lethal bolus or infusion, compared to controls, ten inflammatory cytokines and NO levels were increased and circulating neutrophils and platelets decreased (P ≤ 0.05). Changes with lethal doses were greater than changes with nonlethal doses (P ≤ 0.01). Lethal bolus or infusion doses produced hypotension or hypoxemia, respectively (P ≤ 0.05). The effects with B. anthracis cell wall were similar to those of S. aureus cell wall or LPS. However, paradoxically administration of B. anthracis cell wall or LPS decreased the lethality of concurrently administered B. anthracis lethal toxin (P < 0.0001 and 0.04, respectively). Conclusion B. anthracis cell wall has the potential to produce inflammatory injury during anthrax infection clinically. However, understanding why cell wall or LPS paradoxically reduced lethality with lethal toxin may help understand this toxin’s pathogenic effects. PMID:19756496

  20. Human synthetic lethal inference as potential anti-cancer target gene detection

    PubMed Central

    2009-01-01

    Background Two genes are called synthetic lethal (SL) if mutation of either alone is not lethal, but mutation of both leads to death or a significant decrease in organism's fitness. The detection of SL gene pairs constitutes a promising alternative for anti-cancer therapy. As cancer cells exhibit a large number of mutations, the identification of these mutated genes' SL partners may provide specific anti-cancer drug candidates, with minor perturbations to the healthy cells. Since existent SL data is mainly restricted to yeast screenings, the road towards human SL candidates is limited to inference methods. Results In the present work, we use phylogenetic analysis and database manipulation (BioGRID for interactions, Ensembl and NCBI for homology, Gene Ontology for GO attributes) in order to reconstruct the phylogenetically-inferred SL gene network for human. In addition, available data on cancer mutated genes (COSMIC and Cancer Gene Census databases) as well as on existent approved drugs (DrugBank database) supports our selection of cancer-therapy candidates. Conclusions Our work provides a complementary alternative to the current methods for drug discovering and gene target identification in anti-cancer research. Novel SL screening analysis and the use of highly curated databases would contribute to improve the results of this methodology. PMID:20015360

  1. Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib.

    PubMed

    Miller, Rowan E; Brough, Rachel; Bajrami, Ilirjana; Williamson, Chris T; McDade, Simon; Campbell, James; Kigozi, Asha; Rafiq, Rumana; Pemberton, Helen; Natrajan, Rachel; Joel, Josephine; Astley, Holly; Mahoney, Claire; Moore, Jonathan D; Torrance, Chris; Gordan, John D; Webber, James T; Levin, Rebecca S; Shokat, Kevan M; Bandyopadhyay, Sourav; Lord, Christopher J; Ashworth, Alan

    2016-07-01

    New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR.

  2. Hairy polyp can be lethal even when small in size.

    PubMed

    Koike, Yuhki; Uchida, Keiichi; Inoue, Mikihiro; Ohtsu, Kazuya; Tanaka, Takaaki; Otake, Kohei; Tanaka, Koji; Kusunoki, Masato

    2013-06-01

    A case of sudden cardiopulmonary arrest in a 3-month-old girl is presented. The patient had barely recovered from hypoxic encephalopathy when she presented with repeated respiratory distress. Computed tomography and endoscopic analysis revealed a shiny polyp in the lateral wall of the nasopharynx, and this polyp was suspected to be the main cause of respiratory distress. After referral to our hospital, surgical removal was performed, and the histopathological diagnosis was hairy polyp. Hairy polyp is a rare congenital benign tumor that sometimes induces respiratory distress. This polyp can potentially induce a life-threatening event. In a systematic review of 40 reported cases, polyps of ≤ 3.0 cm in diameter have a higher risk of respiratory distress than do those >3.0 cm in diameter (P = 0.01). Small hairy polyps may be lethal because of delayed diagnosis. To locate small hairy polyps, physicians should not hesitate to perform further examination because there is the possibility of oversight with only physical examination.

  3. Gill lesions in the major carp, Labeo rohita exposed to lethal and sublethal concentrations of tannery effluent.

    PubMed

    Dhanapakiam, P; Sampoorani, V; Kavitha, M; Ramasamy, V K; Chandrakala, A; Aruna, K C

    2004-07-01

    The major carp, Labeo rohita were exposed to (0.873%) lethal and sublethal (0.073%) concentrations of tannery effluent for 24h and 40 days respectively under static bioassay condition. The surface architecture of gill revealed severe damages such as : fusion and clumping in the middle and distal parts of the primary lamellae, swelling and deterioration of the cells. The interlamellar space was filled either with hyperplastic epithelial or mucous cells. Secondary lamellae lost their identity and appeared finger like in structure in the lethal concentration and necrosis was observed in the primary and secondary epithelium. Swelling of primary and secondary epithelial cells was evident in sublethal concentration.

  4. Are high-lethality suicide attempters with bipolar disorder a distinct phenotype?

    PubMed

    Oquendo, Maria A; Carballo, Juan Jose; Rajouria, Namita; Currier, Dianne; Tin, Adrienne; Merville, Jessica; Galfalvy, Hanga C; Sher, Leo; Grunebaum, Michael F; Burke, Ainsley K; Mann, J John

    2009-01-01

    Because Bipolar Disorder (BD) individuals making highly lethal suicide attempts have greater injury burden and risk for suicide, early identification is critical. BD patients were classified as high- or low-lethality attempters. High-lethality attempts required inpatient medical treatment. Mixed effects logistic regression models and permutation analyses examined correlations between lethality, number, and order of attempts. High-lethality attempters reported greater suicidal intent and more previous attempts. Multiple attempters showed no pattern of incremental lethality increase with subsequent attempts, but individuals with early high-lethality attempts more often made high-lethality attempts later. A subset of high-lethality attempters make only high-lethality attempts. However, presence of previous low-lethality attempts does not indicate that risk for more lethal, possibly successful, attempts is reduced.

  5. Low survival of mice following lethal gamma-irradiation after administration of inhibitors of prostaglandin synthesis.

    PubMed

    Hofer, M; Pospísil, M; Tkadlecek, L; Viklická, S; Pipalová, I; Holá, J

    1992-01-01

    An impairment of the survival of mice subjected to whole-body gamma-irradiation with a lethal dose of 10 Gy and treated with a repeated postirradiation administration of prostaglandin synthesis inhibitors (PGSIs), indomethacin or diclofenac, was observed. Morphological examination of the gastrointestinal tract and the estimation of blood loss into its lumen in animals treated with diclofenac did not show serious damage such as haemorrhages or perforation, but revealed structural injury to the intestinal mucosa indicating inflammatory processes. The lesions found are supposed to be connected with increased intestinal permeability which leads to endotoxin escape from the gut and a subsequent increased mortality rate of irradiated animals. It may be concluded that PGSIs are not suitable for the management of radiation sickness after an exposure to lethal doses of ionizing radiation.

  6. Ethical language and decision-making for prenatally diagnosed lethal malformations

    PubMed Central

    Wilkinson, Dominic; de Crespigny, Lachlan; Xafis, Vicki

    2014-01-01

    Summary In clinical practice, and in the medical literature, severe congenital malformations such as trisomy 18, anencephaly, and renal agenesis are frequently referred to as ‘lethal’ or as ‘incompatible with life’. However, there is no agreement about a definition of lethal malformations, nor which conditions should be included in this category. Review of outcomes for malformations commonly designated ‘lethal’ reveals that prolonged survival is possible, even if rare. This article analyses the concept of lethal malformations and compares it to the problematic concept of ‘futility’. We recommend avoiding the term ‘lethal’ and suggest that counseling should focus on salient prognostic features instead. For conditions with a high chance of early death or profound impairment in survivors despite treatment, perinatal and neonatal palliative care would be ethical. However, active obstetric and neonatal management, if desired, may also sometimes be appropriate. PMID:25200733

  7. A Targetable GATA2-IGF2 Axis Confers Aggressiveness in Lethal Prostate Cancer

    PubMed Central

    Vidal, Samuel J.; Rodriguez-Bravo, Veronica; Quinn, S. Aidan; Rodriguez-Barrueco, Ruth; Lujambio, Amaia; Williams, Estrelania; Sun, Xiaochen; de la Iglesia-Vicente, Janis; Lee, Albert; Readhead, Ben; Chen, Xintong; Galsky, Matthew; Esteve, Berta; Petrylak, Daniel P.; Dudley, Joel T.; Rabadan, Raul; Silva, Jose M.; Hoshida, Yujin; Lowe, Scott W.; Cordon-Cardo, Carlos; Domingo-Domenech, Josep

    2015-01-01

    SUMMARY Elucidating the determinants of aggressiveness in lethal prostate cancer may stimulate therapeutic strategies that improve clinical outcomes. We used experimental models and clinical databases to identify GATA2 as a regulator of chemotherapy resistance and tumorigenicity in this context. Mechanistically, direct upregulation of the growth hormone IGF2 emerged as a mediator of the aggressive properties regulated by GATA2. IGF2 in turn activated IGF1R and INSR as well as a downstream polykinase program. The characterization of this axis prompted a combination strategy whereby dual IGF1R/INSR inhibition restored the efficacy of chemotherapy and improved survival in preclinical models. These studies reveal a GATA2-IGF2 aggressiveness axis in lethal prostate cancer and identify a therapeutic opportunity in this challenging disease. PMID:25670080

  8. Ectopic expression of Cripto-1 in transgenic mouse embryos causes hemorrhages, fatal cardiac defects and embryonic lethality

    PubMed Central

    Lin, Xiaolin; Zhao, Wentao; Jia, Junshuang; Lin, Taoyan; Xiao, Gaofang; Wang, Shengchun; Lin, Xia; Liu, Yu; Chen, Li; Qin, Yujuan; Li, Jing; Zhang, Tingting; Hao, Weichao; Chen, Bangzhu; Xie, Raoying; Cheng, Yushuang; Xu, Kang; Yao, Kaitai; Huang, Wenhua; Xiao, Dong; Sun, Yan

    2016-01-01

    Targeted disruption of Cripto-1 in mice caused embryonic lethality at E7.5, whereas we unexpectedly found that ectopic Cripto-1 expression in mouse embryos also led to embryonic lethality, which prompted us to characterize the causes and mechanisms underlying embryonic death due to ectopic Cripto-1 expression. RCLG/EIIa-Cre embryos displayed complex phenotypes between embryonic day 14.5 (E14.5) and E17.5, including fatal hemorrhages (E14.5-E15.5), embryo resorption (E14.5-E17.5), pale body surface (E14.5-E16.5) and no abnormal appearance (E14.5-E16.5). Macroscopic and histological examination revealed that ectopic expression of Cripto-1 transgene in RCLG/EIIa-Cre embryos resulted in lethal cardiac defects, as evidenced by cardiac malformations, myocardial thinning, failed assembly of striated myofibrils and lack of heartbeat. In addition, Cripto-1 transgene activation beginning after E8.5 also caused the aforementioned lethal cardiac defects in mouse embryos. Furthermore, ectopic Cripto-1 expression in embryonic hearts reduced the expression of cardiac transcription factors, which is at least partially responsible for the aforementioned lethal cardiac defects. Our results suggest that hemorrhages and cardiac abnormalities are two important lethal factors in Cripto-1 transgenic mice. Taken together, these findings are the first to demonstrate that sustained Cripto-1 transgene expression after E11.5 causes fatal hemorrhages and lethal cardiac defects, leading to embryonic death at E14.5-17.5. PMID:27687577

  9. Protective Effects of Hong Shan Capsule against Lethal Total-Body Irradiation-Induced Damage in Wistar Rats.

    PubMed

    Li, Jianzhong; Xu, Jing; Xu, Weiheng; Qi, Yang; Lu, Yiming; Qiu, Lei; Hu, Zhenlin; Chu, Zhiyong; Chai, Yifeng; Zhang, Junping

    2015-08-12

    Hong Shan Capsule (HSC), a crude drug of 11 medicinal herbs, was used in clinical practice for the treatment of radiation injuries in China. In this study, we investigated its protection in rats against acute lethal total-body irradiation (TBI). Pre-administration of HSC reduced the radiation sickness characteristics, while increasing the 30-day survival of the irradiated rats. Administration of HSC also reduced the radiation sickness characteristics and increased the 30-day survival of mice after exposure to lethal TBI. Ultrastructural observation illustrated that the pretreatment of rats with HSC significantly attenuated the TBI-induced morphological changes in the different organs of irradiated rats. Gene expression profiles revealed the dramatic effect of HSC on alterations of gene expression caused by lethal TBI. Pretreatment with HSC prevented differential expression of 66% (1398 genes) of 2126 genes differentially expressed in response to TBI. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 32 pathways, such as pathways in cancer and the mitogen-activated protein kinase (MAPK) signaling pathway. Our analysis indicated that the pretreatment of rats with HSC modulated these pathways induced by lethal TBI, such as multiple MAPK pathways, suggesting that pretreatment with HSC might provide protective effects on lethal TBI mainly or partially through the modulation of these pathways. Our data suggest that HSC has the potential to be used as an effective therapeutic or radio-protective agent to minimize irradiation damage.

  10. Protective Effects of Hong Shan Capsule against Lethal Total-Body Irradiation-Induced Damage in Wistar Rats

    PubMed Central

    Li, Jianzhong; Xu, Jing; Xu, Weiheng; Qi, Yang; Lu, Yiming; Qiu, Lei; Hu, Zhenlin; Chu, Zhiyong; Chai, Yifeng; Zhang, Junping

    2015-01-01

    Hong Shan Capsule (HSC), a crude drug of 11 medicinal herbs, was used in clinical practice for the treatment of radiation injuries in China. In this study, we investigated its protection in rats against acute lethal total-body irradiation (TBI). Pre-administration of HSC reduced the radiation sickness characteristics, while increasing the 30-day survival of the irradiated rats. Administration of HSC also reduced the radiation sickness characteristics and increased the 30-day survival of mice after exposure to lethal TBI. Ultrastructural observation illustrated that the pretreatment of rats with HSC significantly attenuated the TBI-induced morphological changes in the different organs of irradiated rats. Gene expression profiles revealed the dramatic effect of HSC on alterations of gene expression caused by lethal TBI. Pretreatment with HSC prevented differential expression of 66% (1398 genes) of 2126 genes differentially expressed in response to TBI. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 32 pathways, such as pathways in cancer and the mitogen-activated protein kinase (MAPK) signaling pathway. Our analysis indicated that the pretreatment of rats with HSC modulated these pathways induced by lethal TBI, such as multiple MAPK pathways, suggesting that pretreatment with HSC might provide protective effects on lethal TBI mainly or partially through the modulation of these pathways. Our data suggest that HSC has the potential to be used as an effective therapeutic or radio-protective agent to minimize irradiation damage. PMID:26274957

  11. Lethal mass mimicking myxoma in the heart.

    PubMed

    Soo, Wern Miin; Pang, Yin Huei; Poh, Kian-Keong

    2014-10-01

    A 70-year-old man presented in advanced heart failure with jaundice. Transthoracic echocardiography revealed a 21 × 24-mm mass in the left atrium attached to the posterior mitral valve leaflet. Surgical excision was attempted, but the tumor had infiltrated the entire left atrial wall and was deemed too extensive to be resectable. Histology confirmed a high-grade pleomorphic sarcoma with malignant fibrous histiocytoma-like features. Liver biopsy revealed a high-grade liver sarcoma.

  12. Proteome-wide screening reveals immunodominance in the CD8 T cell response against classical swine fever virus with antigen-specificity dependent on MHC class I haplotype expression.

    PubMed

    Franzoni, Giulia; Kurkure, Nitin V; Essler, Sabine E; Pedrera, Miriam; Everett, Helen E; Bodman-Smith, Kikki B; Crooke, Helen R; Graham, Simon P

    2013-01-01

    Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core₂₄₁₋₂₅₅ PESRKKLEKALLAWA and NS3₁₉₀₂₋₁₉₁₂ VEYSFIFLDEY, or minimal length antigenic peptides: E2₉₉₆₋₁₀₀₃ YEPRDSYF, NS2₁₂₂₃₋₁₂₃₀ STVTGIFL and NS5A₃₀₇₀₋₃₀₇₈ RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS₁₂₂₃₋₁₂₃₀ STVTGIFL and NS3₁₉₀₂₋₁₉₁₂ VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides

  13. A rational approach to prenatal screening and intervention.

    PubMed

    Yagel, S; Anteby, E

    1998-05-01

    Improved testing procedures now allow prenatal screening for a wide range of congenital defects, including cystic fibrosis and muscular dystrophy. An emerging technique also allows diagnosis of congenital anomalies during the pre-implantation stage of in vitro fertilization. Thus, clinicians need an established criteria to use as a guide when counseling parents about what prenatal testing is possible, feasible, and desirable. For example, there are limits to prenatal testing for conditions like mutations in the breast cancer gene because affected individuals do not necessary develop the condition and a cure may be found by the time the condition develops. It is even questionable if parents should be given this information until the child reaches an appropriate age. One approach to development of guidelines is to classify congenital abnormalities according to severity, age of onset, and type (structural-functional versus mental). This system reveals anomalies that are clearly lethal, lead to moderate or severe disability with little or no prospect of improvement or cure, are characterized by early onset, and/or involve obvious mental retardation. This approach is particularly relevant in cases of trisomy 21, and progressively invasive screening techniques are available to detect this most common pattern of malformation in humans. PMID:9647530

  14. Screens for piwi suppressors in Drosophila identify dosage-dependent regulators of germline stem cell division.

    PubMed Central

    Smulders-Srinivasan, Tora K; Lin, Haifan

    2003-01-01

    The Drosophila piwi gene is the founding member of the only known family of genes whose function in stem cell maintenance is highly conserved in both animal and plant kingdoms. piwi mutants fail to maintain germline stem cells in both male and female gonads. The identification of piwi-interacting genes is essential for understanding how stem cell divisions are regulated by piwi-mediated mechanisms. To search for such genes, we screened the Drosophila third chromosome ( approximately 36% of the euchromatic genome) for suppressor mutations of piwi2 and identified six strong and three weak piwi suppressor genes/sequences. These genes/sequences interact negatively with piwi in a dosage-sensitive manner. Two of the strong suppressors represent known genes--serendipity-delta and similar, both encoding transcription factors. These findings reveal that the genetic regulation of germline stem cell division involves dosage-sensitive mechanisms and that such mechanisms exist at the transcriptional level. In addition, we identified three other types of piwi interactors. The first type consists of deficiencies that dominantly interact with piwi2 to cause male sterility, implying that dosage-sensitive regulation also exists in the male germline. The other two types are deficiencies that cause lethality and female-specific lethality in a piwi2 mutant background, revealing the zygotic function of piwi in somatic development. PMID:14704180

  15. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  16. An F1 genetic screen for maternal-effect mutations affecting embryonic pattern formation in Drosophila melanogaster.

    PubMed Central

    Luschnig, Stefan; Moussian, Bernard; Krauss, Jana; Desjeux, Isabelle; Perkovic, Josip; Nüsslein-Volhard, Christiane

    2004-01-01

    Large-scale screens for female-sterile mutations have revealed genes required maternally for establishment of the body axes in the Drosophila embryo. Although it is likely that the majority of components involved in axis formation have been identified by this approach, certain genes have escaped detection. This may be due to (1) incomplete saturation of the screens for female-sterile mutations and (2) genes with essential functions in zygotic development that mutate to lethality, precluding their identification as female-sterile mutations. To overcome these limitations, we performed a genetic mosaic screen aimed at identifying new maternal genes required for early embryonic patterning, including zygotically required ones. Using the Flp-FRT technique and a visible germline clone marker, we developed a system that allows efficient screening for maternal-effect phenotypes after only one generation of breeding, rather than after the three generations required for classic female-sterile screens. We identified 232 mutants showing various defects in embryonic pattern or morphogenesis. The mutants were ordered into 10 different phenotypic classes. A total of 174 mutants were assigned to 86 complementation groups with two alleles on average. Mutations in 45 complementation groups represent most previously known maternal genes, while 41 complementation groups represent new loci, including several involved in dorsoventral, anterior-posterior, and terminal patterning. PMID:15166158

  17. Lethality of sortase depletion in Actinomyces oris caused by excessive membrane accumulation of a surface glycoprotein.

    PubMed

    Wu, Chenggang; Huang, I-Hsiu; Chang, Chungyu; Reardon-Robinson, Melissa Elizabeth; Das, Asis; Ton-That, Hung

    2014-12-01

    Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane-localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalysed by sortase SrtA. Significantly, this novel phenomenon of glyco-stress provides convenient cell-based assays for developing a new class of inhibitors against Gram-positive pathogens.

  18. Lethality of Sortase Depletion in Actinomyces oris Caused by Excessive Membrane Accumulation of a Surface Glycoprotein

    PubMed Central

    Wu, Chenggang; Huang, I-Hsiu; Chang, Chungyu; Reardon-Robinson, Melissa Elizabeth; Das, Asis; Ton-That, Hung

    2014-01-01

    Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harboring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane-localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalyzed by sortase SrtA. Significantly, this novel phenomenon of glyco-stress provides convenient cell-based assays for developing a new class of inhibitors against Gram-positive pathogens. PMID:25230351

  19. Metabolic Response of Escherichia coli upon Treatment with Hypochlorite at Sub-Lethal Concentrations

    PubMed Central

    Winter, Jeannette; Eisenreich, Wolfgang

    2015-01-01

    Hypochlorite is a reactive oxygen species that is worldwide as an antibacterial disinfectant. Hypochlorite exposure is known to cause oxidative damage to DNA and proteins. As a response to these effects, the metabolite profiles of organisms treated with sub-lethal doses of hypochlorite are assumed to be severely modified; however, the nature of these changes is hardly understood. Therefore, using nuclear magnetic resonance spectroscopy and gas chromatography-coupled mass spectrometry, we analyzed the time-dependent impact of hypochlorite exposure with a sub-lethal concentration (50 µM) on the metabolite profile of the Escherichia coli strain MG1655. Principle component analysis clearly distinguished between the metabolite profiles of bacteria treated for 0, 5,10, 20, 40, or 60 min. Major changes in the relative amounts of fatty acids, acetic acid, and formic acid occurred within the first 5 min. Comparative gas chromatography-coupled mass spectrometry analyses revealed that the amounts of free methionine and alanine were significantly decreased in the treated cells, demonstrating their susceptibility to hypochlorite exposure. The concentrations of succinate, urea, orotic acid, 2-aminobutyric acid, and 2-hydroxybutyric acid were also severely affected, indicating general changes in the metabolic network by hypochlorite. However, most metabolite levels relaxed to the reference values of untreated cells after 40–60 min, reflecting the capability of E. coli to rapidly adapt to environmental stress factors such as the presence of sub-lethal oxidant levels. PMID:25932918

  20. Defining the origins of electron transfer at screen-printed graphene-like and graphite electrodes: MoO2 nanowire fabrication on edge plane sites reveals electrochemical insights.

    PubMed

    Rowley-Neale, Samuel J; Brownson, Dale A C; Banks, Craig E

    2016-08-18

    Molybdenum (di)oxide (MoO2) nanowires are fabricated onto graphene-like and graphite screen-printed electrodes (SPEs) for the first time, revealing crucial insights into the electrochemical properties of carbon/graphitic based materials. Distinctive patterns observed in the electrochemical process of nanowire decoration show that electron transfer occurs predominantly on edge plane sites when utilising SPEs fabricated/comprised of graphitic materials. Nanowire fabrication along the edge plane sites (and on edge plane like-sites/defects) of graphene/graphite is confirmed with Cyclic Voltammetry, Scanning Electron Microscopy (SEM) and Raman Spectroscopy. Comparison of the heterogeneous electron transfer (HET) rate constants (k°) at unmodified and nanowire coated SPEs show a reduction in the electrochemical reactivity of SPEs when the edge plane sites are effectively blocked/coated with MoO2. Throughout the process, the basal plane sites of the graphene/graphite electrodes remain relatively uncovered; except when the available edge plane sites have been utilised, in which case MoO2 deposition grows from the edge sites covering the entire surface of the electrode. This work clearly illustrates the distinct electron transfer properties of edge and basal plane sites on graphitic materials, indicating favourable electrochemical reactivity at the edge planes in contrast to limited reactivity at the basal plane sites. In addition to providing fundamental insights into the electron transfer properties of graphite and graphene-like SPEs, the reported simple, scalable, and cost effective formation of unique and intriguing MoO2 nanowires realised herein is of significant interest for use in both academic and commercial applications. PMID:27487988

  1. Defining the origins of electron transfer at screen-printed graphene-like and graphite electrodes: MoO2 nanowire fabrication on edge plane sites reveals electrochemical insights.

    PubMed

    Rowley-Neale, Samuel J; Brownson, Dale A C; Banks, Craig E

    2016-08-18

    Molybdenum (di)oxide (MoO2) nanowires are fabricated onto graphene-like and graphite screen-printed electrodes (SPEs) for the first time, revealing crucial insights into the electrochemical properties of carbon/graphitic based materials. Distinctive patterns observed in the electrochemical process of nanowire decoration show that electron transfer occurs predominantly on edge plane sites when utilising SPEs fabricated/comprised of graphitic materials. Nanowire fabrication along the edge plane sites (and on edge plane like-sites/defects) of graphene/graphite is confirmed with Cyclic Voltammetry, Scanning Electron Microscopy (SEM) and Raman Spectroscopy. Comparison of the heterogeneous electron transfer (HET) rate constants (k°) at unmodified and nanowire coated SPEs show a reduction in the electrochemical reactivity of SPEs when the edge plane sites are effectively blocked/coated with MoO2. Throughout the process, the basal plane sites of the graphene/graphite electrodes remain relatively uncovered; except when the available edge plane sites have been utilised, in which case MoO2 deposition grows from the edge sites covering the entire surface of the electrode. This work clearly illustrates the distinct electron transfer properties of edge and basal plane sites on graphitic materials, indicating favourable electrochemical reactivity at the edge planes in contrast to limited reactivity at the basal plane sites. In addition to providing fundamental insights into the electron transfer properties of graphite and graphene-like SPEs, the reported simple, scalable, and cost effective formation of unique and intriguing MoO2 nanowires realised herein is of significant interest for use in both academic and commercial applications.

  2. Streptococcal screen

    MedlinePlus

    A negative strep screen most often means group A streptococcus is not present. It is unlikely that you have strep throat. If your provider still thinks that you may have strep throat, a throat culture will be done.

  3. Hypertension screening

    NASA Technical Reports Server (NTRS)

    Foulke, J. M.

    1975-01-01

    An attempt was made to measure the response to an announcement of hypertension screening at the Goddard Space Center, to compare the results to those of previous statistics. Education and patient awareness of the problem were stressed.

  4. Developmental Screening

    MedlinePlus

    Learn More about Your Child’s Development: Developmental Monitoring and Screening Taking a first step, waving “bye-bye,” and pointing to something interesting are all developmental milestones, ...

  5. TORCH screen

    MedlinePlus

    ... in a newborn. TORCH stands for toxoplasmosis , rubella , cytomegalovirus, herpes simplex, and HIV, but it can also ... to screen infants for infections such as toxoplasmosis, cytomegalovirus, herpes simplex, syphilis and others. These infections may ...

  6. Get Screened

    MedlinePlus

    ... Get Ready 3 of 4 sections Take Action: Cost and Insurance What about cost? Depending on your insurance plan, you may be able to get screening tests at no cost to you. Most insurance plans, including Medicaid and ...

  7. Newborn Screening

    MedlinePlus

    ... Pulse Oximetry Screening for CCHDs Sickle Cell Disease Laboratory SCID Quality Assurance Training and Resources For Lab Professionals Data and Reports Laboratory Reports National Birth Defects Prevention Network (NBDPN) Resources ...

  8. Chemotherapy agents and the induction of late lethal defects.

    PubMed

    Seymour, C B; Mothersill, C

    1991-01-01

    Radiation is now known to be capable of producing both lethal and non lethal lesions in a variety of mammalian cells which may not be expressed for several cell divisions after the initial insult. The mechanism by which such an effect occurs is unknown. Because of the possible implications for cancer treatment, if such an effect also occurred following chemotherapy exposure, cells were exposed to various cytotoxic chemotherapy agents with known and well characterised effects on DNA or other areas of cell function or structure. The results indicate that late lethal defects are not detectable after treatment with appropriate ranges of doses of cisplatinum, vincristine, BCNU or adriamycin, but that they are induced by Bleomycin and to a lesser extent by 5-Fluorouracil. Bleomycin is known to cause strand breaks and is regarded as a radiomimetic agent. 5-Fluorouracil may act by preventing efficient and faithful synthesis of DNA, allowing mutations to become integrated into the genome. The occurrence of lethal mutations with both these agents supports previous suggestions that error-prone repair of DNA base sequence abnormalities may be fundamental to the process of late lethal damage production in mammalian cells. The cloning efficiency of cells which survived exposure to Vincristine or BCNU over a wide dose range was found to be significantly increased; this may represent an adaptive response to the drugs.

  9. Perinatal lethal osteogenesis imperfecta in a Thai newborn: the autopsy and histopathogical findings.

    PubMed

    Himakhun, Wanwisa; Rojnueangnit, Kitiwan; Prachukthum, Sariya

    2012-01-01

    Osteogenesis imperfecta (OI) is an inherited disorder of type I collagen synthesis with an estimate incidence of I in 100,000 live births. Among all types, OI type II is the most severe type with perinatal death. The authors describes a male neonate with characteristic features of osteogenesis imperfect type II, including short crumpling limbs, beaded ribs, poorly bony ossification and blue sclera. Autopsy with histological study revealed not only multiple fractures, but pulmonary hypoplasia and intracerebral hemorrhages were also present. Both are the leading causes of death in the lethal type OI patients. PMID:23964465

  10. Acute oral toxicity and brine shrimp lethality of Elaeis guineensis Jacq., (oil palm leaf) methanol extract.

    PubMed

    Syahmi, Abdul Rani Muhamad; Vijayarathna, Soundararajan; Sasidharan, Sreenivasan; Latha, Lachimanan Yoga; Kwan, Yuet Ping; Lau, Yee Ling; Shin, Lai Ngit; Chen, Yeng

    2010-11-01

    Elaeis guineensis (Arecaceae) is widely used in West African traditional medicine for treating various ailments. An evaluation on the toxicity of extracts of this plant is crucial to support the therapeutic claims. The acute oral toxicity and brine shrimp lethality of a methanolic extract of this plant was tested. Oral administration of crude extract at the highest dose of 5,000 mg/kg resulted in no mortalities or evidence of adverse effects, implying that E. guineensis is nontoxic. Normal behavioral pattern, clinical signs and histology of vital organs confirm this evidence. The E. guineensis extracts screened for toxicity against brine shrimp had 50% lethal concentration (LC₅₀) values of more than 1.0 mg/mL (9.00 and 3.87 mg/mL, at 6 and 24 h, respectively), confirming that the extract was not toxic. Maximum mortalities occurred at 100 mg/mL concentration while the least mortalities happened to be at 0.195 mg/mL concentration. The results of both tests confirm that E. guineensis is nontoxic and hence safe for commercial utilization. PMID:21072022

  11. Acute oral toxicity and brine shrimp lethality of Elaeis guineensis Jacq., (oil palm leaf) methanol extract.

    PubMed

    Syahmi, Abdul Rani Muhamad; Vijayarathna, Soundararajan; Sasidharan, Sreenivasan; Latha, Lachimanan Yoga; Kwan, Yuet Ping; Lau, Yee Ling; Shin, Lai Ngit; Chen, Yeng

    2010-11-10

    Elaeis guineensis (Arecaceae) is widely used in West African traditional medicine for treating various ailments. An evaluation on the toxicity of extracts of this plant is crucial to support the therapeutic claims. The acute oral toxicity and brine shrimp lethality of a methanolic extract of this plant was tested. Oral administration of crude extract at the highest dose of 5,000 mg/kg resulted in no mortalities or evidence of adverse effects, implying that E. guineensis is nontoxic. Normal behavioral pattern, clinical signs and histology of vital organs confirm this evidence. The E. guineensis extracts screened for toxicity against brine shrimp had 50% lethal concentration (LC₅₀) values of more than 1.0 mg/mL (9.00 and 3.87 mg/mL, at 6 and 24 h, respectively), confirming that the extract was not toxic. Maximum mortalities occurred at 100 mg/mL concentration while the least mortalities happened to be at 0.195 mg/mL concentration. The results of both tests confirm that E. guineensis is nontoxic and hence safe for commercial utilization.

  12. Lethal effects of short-wavelength visible light on insects

    NASA Astrophysics Data System (ADS)

    Hori, Masatoshi; Shibuya, Kazuki; Sato, Mitsunari; Saito, Yoshino

    2014-12-01

    We investigated the lethal effects of visible light on insects by using light-emitting diodes (LEDs). The toxic effects of ultraviolet (UV) light, particularly shortwave (i.e., UVB and UVC) light, on organisms are well known. However, the effects of irradiation with visible light remain unclear, although shorter wavelengths are known to be more lethal. Irradiation with visible light is not thought to cause mortality in complex animals including insects. Here, however, we found that irradiation with short-wavelength visible (blue) light killed eggs, larvae, pupae, and adults of Drosophila melanogaster. Blue light was also lethal to mosquitoes and flour beetles, but the effective wavelength at which mortality occurred differed among the insect species. Our findings suggest that highly toxic wavelengths of visible light are species-specific in insects, and that shorter wavelengths are not always more toxic. For some animals, such as insects, blue light is more harmful than UV light.

  13. Lethal effects of short-wavelength visible light on insects.

    PubMed

    Hori, Masatoshi; Shibuya, Kazuki; Sato, Mitsunari; Saito, Yoshino

    2014-12-09

    We investigated the lethal effects of visible light on insects by using light-emitting diodes (LEDs). The toxic effects of ultraviolet (UV) light, particularly shortwave (i.e., UVB and UVC) light, on organisms are well known. However, the effects of irradiation with visible light remain unclear, although shorter wavelengths are known to be more lethal. Irradiation with visible light is not thought to cause mortality in complex animals including insects. Here, however, we found that irradiation with short-wavelength visible (blue) light killed eggs, larvae, pupae, and adults of Drosophila melanogaster. Blue light was also lethal to mosquitoes and flour beetles, but the effective wavelength at which mortality occurred differed among the insect species. Our findings suggest that highly toxic wavelengths of visible light are species-specific in insects, and that shorter wavelengths are not always more toxic. For some animals, such as insects, blue light is more harmful than UV light.

  14. Isolation of avian paramyxovirus 1 from a patient with a lethal case of pneumonia.

    PubMed

    Goebel, Scott J; Taylor, Jill; Barr, Bradd C; Kiehn, Timothy E; Castro-Malaspina, Hugo R; Hedvat, Cyrus V; Rush-Wilson, Kim A; Kelly, Cassandra D; Davis, Stephen W; Samsonoff, William A; Hurst, Kelley R; Behr, Melissa J; Masters, Paul S

    2007-11-01

    An unknown virus was isolated from a lung biopsy sample and multiple other samples from a patient who developed a lethal case of pneumonia following a peripheral blood stem cell transplant. A random PCR-based molecular screening method was used to identify the infectious agent as avian paramyxovirus 1 (APMV-1; a group encompassing Newcastle disease virus), which is a highly contagious poultry pathogen that has only rarely been found in human infections. Immunohistochemical analysis confirmed the presence of APMV-1 antigen in sloughed alveolar cells in lung tissue from autopsy. Sequence from the human isolate showed that it was most closely related to virulent pigeon strains of APMV-1. This is the most completely documented case of a systemic human infection caused by APMV-1 and is the first report of an association between this virus and a fatal disease in a human.

  15. Building high-resolution synthetic lethal networks: a 'Google map' of the cancer cell.

    PubMed

    Paul, James M; Templeton, Shaina D; Baharani, Akanksha; Freywald, Andrew; Vizeacoumar, Franco J

    2014-12-01

    The most commonly used therapies for cancer involve delivering high doses of radiation or toxic chemicals to the patient that also cause substantial damage to normal tissue. To overcome this, researchers have recently resorted to a basic biological concept called 'synthetic lethality' (SL) that takes advantage of interactions between gene pairs. The identification of SL interactions is of considerable therapeutic interest because if a particular gene is SL with a tumor-causing mutation, then the targeting that gene carries therapeutic advantages. Mapping these interactions in the context of human cancer cells could hold the key to effective, targeted cancer treatments. In this review, we cover the recent advances that aim to identify these SL interactions using unbiased genetic screens.

  16. Cyclopeptide toxins of lethal amanitas: Compositions, distribution and phylogenetic implication.

    PubMed

    Tang, Shanshan; Zhou, Qian; He, Zhengmi; Luo, Tao; Zhang, Ping; Cai, Qing; Yang, Zhuliang; Chen, Jia; Chen, Zuohong

    2016-09-15

    Lethal amanitas (Amanita sect. Phalloideae) are responsible for 90% of all fatal mushroom poisonings. Since 2000, more than ten new lethal Amanita species have been discovered and some of them had caused severe mushroom poisonings in China. However, the contents and distribution of cyclopeptides in these lethal mushrooms remain poorly known. In this study, the diversity of major cyclopeptide toxins in seven Amanita species from Eastern Asia and three species from Europe and North America were systematically analyzed, and a new approach to inferring phylogenetic relationships using cyclopeptide profile was evaluated for the first time. The results showed that there were diversities of the cyclopeptides among lethal Amanita species, and cyclopeptides from Amanita rimosa and Amanita fuligineoides were reported for the first time. The amounts of amatoxins in East Asian Amanita species were significantly higher than those in European and North American species. The analysis of distribution of amatoxins and phallotoxins in various Amanita species demonstrated that the content of phallotoxins was higher than that of amatoxins in Amanita phalloides and Amanita virosa. In contrast, the content of phallotoxins was significantly lower than that of amatoxins in all East Asian lethal Amanita species tested. However, the distribution of amatoxins and phallotoxins in different tissues showed the same tendency. Eight cyclopeptides and three unknown compounds were identified using cyclopeptide standards and high-resolution MS. Based on the cyclopeptide profiles, phylogenetic relationships of lethal amanitas were inferred through a dendrogram generated by UPGMA method. The results showed high similarity to the phylogeny established previously based on the multi-locus DNA sequences. PMID:27476461

  17. Functional screen analysis reveals miR-26b and miR-128 as central regulators of pituitary somatomammotrophic tumor growth through activation of the PTEN-AKT pathway.

    PubMed

    Palumbo, T; Faucz, F R; Azevedo, M; Xekouki, P; Iliopoulos, D; Stratakis, C A

    2013-03-28

    MicroRNAs (miRNAs) have been involved in the pathogenesis of different types of cancer; however, their function in pituitary tumorigenesis remains poorly understood. Cyclic-AMP-dependent protein kinase-defective pituitaries occasionally form aggressive growth-hormone (GH)-producing pituitary tumors in the background of hyperplasia caused by haploinsufficiency of the protein kinase's main regulatory subunit, PRKAR1A. The molecular basis for this development remains unknown. We have identified a 17-miRNA signature of pituitary tumors formed in the background of hyperplasia (caused in half of the cases by PRKAR1A-mutations). We selected two miRNAs on the basis of their functional screen analysis: inhibition of miR-26b expression and upregulation of miR-128 suppressed the colony formation ability and invasiveness of pituitary tumor cells. Furthermore, we identified that miR-26b and miR-128 affected pituitary tumor cell behavior through regulation of their direct targets, PTEN and BMI1, respectively. In addition, we found that miR-128 through BMI1 direct binding on the PTEN promoter affected PTEN expression levels and AKT activity in the pituitary tumor cells. Our in vivo data revealed that inhibition of miR-26b and overexpression of miR-128 could suppress pituitary GH3 tumor growth in xenografts. Taken together, we have identified a miRNA signature for GH-producing pituitary tumors and found that miR-26b and miR-128 regulate the activity of the PTEN-AKT pathway in these tumors. This is the first suggestion of the possible involvement of miRNAs regulating the PTEN-AKT pathway in GH-producing pituitary tumor formation in the context of hyperplasia or due to germline PRKAR1A defects. MiR-26b suppression and miR-128 upregulation could have therapeutic potential in GH-producing pituitary tumor patients. PMID:22614013

  18. Lethal toxicity of cadmium to Cyprinus carpio and Tilapia aurea

    SciTech Connect

    Not Available

    1986-09-01

    There have been several studies of the lethal toxicity of cadmium to freshwater fishes, but further information is required on a number of points. For example, the shallow slope which is characteristic of the cadmium toxicity curve makes interspecific comparisons difficult. There also is a paucity of information on cadmium toxicity to non-Salmonid European species. As part of a study of the water quality requirements of cultured fish species in the Mediterranean, the authors report on the lethal toxicity of cadmium to two such species, the common carp Cyprinus carpio, and Tilapia aurea, for which little information has previously been reported.

  19. Lethal neuroleptic malignant syndrome due to amisulpride.

    PubMed

    Musshoff, Frank; Doberentz, Elke; Madea, Burkhard

    2013-06-01

    A 42-year old-man was found lying in his bed having seizures. Later he became unconscious and hypotonic developing mydriasis as well as rigidity. The body core temperature (rectal temperature) was above 42 °C. Blood pH was decreased during treatment, and his general condition deteriorated. The patient developed gasping respiration, ventricular fibrillation, and died. During autopsy and histological investigation cerebral and pulmonary edema were noted together with general congestion of the internal organs. Further observations included contraction bands of myocytes, a contracted spleen, fibrosis of the liver, and gall stones. Toxicological analyses of peripheral blood revealed the following results: amisulpride 4.65 mg/l, biperiden 0.12 mg/l, imipramine 0.33 mg/l, and desipramine 0.68 mg/l. An amisulpride-induced neuroleptic malignant syndrome was therefore diagnosed as the patho-physiological mechanism leading to death.

  20. Lethal liquorice lollies (liquorice abuse causing pseudohyperaldosteronism).

    PubMed

    Robles, B J Flores; Sandoval, A R Hurtarte; Dardon, J D Penate; Blas, C Alonso

    2013-09-19

    A 47-year-old woman was admitted to the emergency department with a history of asthenia, periorbital and lower limbs oedema, associated with hypokalaemia and increased blood pressure levels. Metabolic and renal causes were initially investigated as thyroid disease, Cushing syndrome and tubulopathies were excluded during the first week of admission. However, further questioning of the patient, revealed that she had been consuming several sachets of raw liquorice lollies (ignored amount) obtained from a herbalist a month ago. Based on the history and clinical findings, liquorice poisoning was highly suspected; an apparent mineralocorticoid excess secondary to ingestion of liquorice. Afterwards, levels of aldosterone and plasma renin activity were measured and found low 3 weeks later; therefore, our clinical suspicion was established. During the patient's stay at the hospital, liquorice was stopped and potassium supplements were started. Subsequently, a week after, the patient fully recovered without any significant sequelae.

  1. Novel Lethal Form of Congenital Hypopituitarism Associated With the First Recessive LHX4 Mutation

    PubMed Central

    Gregory, L. C.; Humayun, K. N.; Turton, J. P. G.; McCabe, M. J.; Rhodes, S. J.

    2015-01-01

    Background: LHX4 encodes a member of the LIM-homeodomain family of transcription factors that is required for normal development of the pituitary gland. To date, only incompletely penetrant heterozygous mutations in LHX4 have been described in patients with variable combined pituitary hormone deficiencies. Objective/Hypothesis: To report a unique family with a novel recessive variant in LHX4 associated with a lethal form of congenital hypopituitarism that was identified through screening a total of 97 patients. Method: We screened 97 unrelated patients with combined pituitary hormone deficiency, including 65% with an ectopic posterior pituitary, for variants in the LHX4 gene using Sanger sequencing. Control databases (1000 Genomes, dbSNP, Exome Variant Server, ExAC Browser) were consulted upon identification of variants. Results: We identified the first novel homozygous missense variant (c.377C>T, p.T126M) in two deceased male patients of Pakistani origin with severe panhypopituitarism associated with anterior pituitary aplasia and posterior pituitary ectopia. Both were born small for gestational age with a small phallus, undescended testes, and mid-facial hypoplasia. The parents' first-born child was a female with mid-facial hypoplasia (DNA was unavailable). Despite rapid commencement of hydrocortisone and T4 in the brothers, all three children died within the first week of life. The LHX4(p.T126M) variant is located within the LIM2 domain, in a highly conserved location. The absence of homozygosity for the variant in over 65 000 controls suggests that it is likely to be responsible for the phenotype. Conclusion: We report, for the first time to our knowledge, a novel homozygous mutation in LHX4 associated with a lethal phenotype, implying that recessive mutations in LHX4 may be incompatible with life. PMID:25871839

  2. Screening for cancer

    SciTech Connect

    Miller, A.B.

    1985-01-01

    This book contains three sections: Fundamentals of Screening, Screening Tests, and Screening for Specific Cancer Sites. Each section consists of several chapters. Some of the chapter titles are: Principles of Screening and of the Evaluation of Screening Programs; Economic Aspects of Screening; Cervical Cytology; Screening Tests for Bladder Cancer; Fecal Occult Blood Testing; Screening for Cancer of the Cervix; Screening for Gastric Cancer; and Screening for Oral Cancer.

  3. Determinants of the lethality of climate-related disasters in the Caribbean Community (CARICOM): a cross-country analysis.

    PubMed

    Andrewin, Aisha N; Rodriguez-Llanes, Jose M; Guha-Sapir, Debarati

    2015-07-08

    Floods and storms are climate-related hazards posing high mortality risk to Caribbean Community (CARICOM) nations. However risk factors for their lethality remain untested. We conducted an ecological study investigating risk factors for flood and storm lethality in CARICOM nations for the period 1980-2012. Lethality--deaths versus no deaths per disaster event- was the outcome. We examined biophysical and social vulnerability proxies and a decadal effect as predictors. We developed our regression model via multivariate analysis using a generalized logistic regression model with quasi-binomial distribution; removal of multi-collinear variables and backward elimination. Robustness was checked through subset analysis. We found significant positive associations between lethality, percentage of total land dedicated to agriculture (odds ratio [OR] 1.032; 95% CI: 1.013-1.053) and percentage urban population (OR 1.029, 95% CI 1.003-1.057). Deaths were more likely in the 2000-2012 period versus 1980-1989 (OR 3.708, 95% CI 1.615-8.737). Robustness checks revealed similar coefficients and directions of association. Population health in CARICOM nations is being increasingly impacted by climate-related disasters connected to increasing urbanization and land use patterns. Our findings support the evidence base for setting sustainable development goals (SDG).

  4. Determinants of the lethality of climate-related disasters in the Caribbean Community (CARICOM): a cross-country analysis

    NASA Astrophysics Data System (ADS)

    Andrewin, Aisha N.; Rodriguez-Llanes, Jose M.; Guha-Sapir, Debarati

    2015-07-01

    Floods and storms are climate-related hazards posing high mortality risk to Caribbean Community (CARICOM) nations. However risk factors for their lethality remain untested. We conducted an ecological study investigating risk factors for flood and storm lethality in CARICOM nations for the period 1980-2012. Lethality - deaths versus no deaths per disaster event- was the outcome. We examined biophysical and social vulnerability proxies and a decadal effect as predictors. We developed our regression model via multivariate analysis using a generalized logistic regression model with quasi-binomial distribution; removal of multi-collinear variables and backward elimination. Robustness was checked through subset analysis. We found significant positive associations between lethality, percentage of total land dedicated to agriculture (odds ratio [OR] 1.032; 95% CI: 1.013-1.053) and percentage urban population (OR 1.029, 95% CI 1.003-1.057). Deaths were more likely in the 2000-2012 period versus 1980-1989 (OR 3.708, 95% CI 1.615-8.737). Robustness checks revealed similar coefficients and directions of association. Population health in CARICOM nations is being increasingly impacted by climate-related disasters connected to increasing urbanization and land use patterns. Our findings support the evidence base for setting sustainable development goals (SDG).

  5. Determinants of the lethality of climate-related disasters in the Caribbean Community (CARICOM): a cross-country analysis

    PubMed Central

    Andrewin, Aisha N.; Rodriguez-Llanes, Jose M.; Guha-Sapir, Debarati

    2015-01-01

    Floods and storms are climate-related hazards posing high mortality risk to Caribbean Community (CARICOM) nations. However risk factors for their lethality remain untested. We conducted an ecological study investigating risk factors for flood and storm lethality in CARICOM nations for the period 1980–2012. Lethality - deaths versus no deaths per disaster event- was the outcome. We examined biophysical and social vulnerability proxies and a decadal effect as predictors. We developed our regression model via multivariate analysis using a generalized logistic regression model with quasi-binomial distribution; removal of multi-collinear variables and backward elimination. Robustness was checked through subset analysis. We found significant positive associations between lethality, percentage of total land dedicated to agriculture (odds ratio [OR] 1.032; 95% CI: 1.013–1.053) and percentage urban population (OR 1.029, 95% CI 1.003–1.057). Deaths were more likely in the 2000–2012 period versus 1980–1989 (OR 3.708, 95% CI 1.615–8.737). Robustness checks revealed similar coefficients and directions of association. Population health in CARICOM nations is being increasingly impacted by climate-related disasters connected to increasing urbanization and land use patterns. Our findings support the evidence base for setting sustainable development goals (SDG). PMID:26153115

  6. Phytochemical screening and anticonvulsant studies of ethyl acetate fraction of Globimetula braunii on laboratory animals

    PubMed Central

    Aliyu, Musa Mumammad; Musa, Abdullahi Isma'il; Kamal, Muhammad Ja'afar; Mohammed, Magaji Garba

    2014-01-01

    Objective To investigate the phytochemical properties and the anticonvulsant potential of the ethyl acetate soluble fraction of ethanol leaf extract of Globimetula braunii, a plant used in ethnomedicine for the treatment of epilepsy. Methods The phytochemical screening was carried out using standard protocol while the anticonvulsant activity was studied using maximal electroshock test in chicks, pentylenetetrazole and 4-aminopyridine-induced seizures in mice. Results The preliminary phytochemical screening carried out on the crude ethanol extract revealed the presence of saponins, carbohydrates, flavonoids, tannins, anthraquinones and steroids. Similarly, tannins, flavonoids and steroids/terpenes were found to be present in the ethyl acetate fraction. In the pharmacological screening, 150 mg/kg of the fraction protected 83.33% of animals against pentylenetetrazole-induced seizure in mice whereas sodium valproate a standard anti-epileptic drug offered 100% protection. In the 4-aminopyridine-induced seizure model, the fraction produced a significant (P<0.05) increase in the mean onset of seizure in unprotected animals. The fraction did not exhibit a significant activity against maximal electroshock convulsion. The median lethal dose of the fraction was found to be 1 261.91 mg/kg. Conclusions These results suggest that the ethyl acetate fraction of Globimetula braunii leaves extract possesses psychoactive compound that may be useful in the management of petit mal epilepsy and lend credence to the ethnomedical use of the plant in the management of epilepsy. PMID:25182552

  7. Hearing Screening

    ERIC Educational Resources Information Center

    Johnson-Curiskis, Nanette

    2012-01-01

    Hearing levels are threatened by modern life--headsets for music, rock concerts, traffic noises, etc. It is crucial we know our hearing levels so that we can draw attention to potential problems. This exercise requires that students receive a hearing screening for their benefit as well as for making the connection of hearing to listening.

  8. On quantifying nonthermal effects on the lethality of pressure-assisted heat preservation processes.

    PubMed

    Peleg, Micha; Corradini, Maria G; Normand, Mark D

    2012-01-01

    Direct experimental identification and quantification of the pressure contribution to a pressure-assisted sterilization process efficacy is difficult. However, dynamic kinetic models of thermal inactivation can be used to assess the lethality of a purely thermal process having the same temperature profile. Thus, a pressure-assisted process' temperature record can be used to generate a corresponding purely thermal survival curve with parameters determined in conventional heating experiments. Comparison of the actual final survival ratio with that calculated for the purely thermal process would reveal whether the hydrostatic pressure had synergistic or antagonistic effect on bacterial spores survival. The effect would be manifested in the number of log cycles subtracted or added to the survival ratio, and in the length of time at the holding temperature needed to produce the final survival ratio of the combined process. A set of combined treatments would reveal how the temperature and pressure profiles affect the pressure's influence on the process' lethality to either vegetative cells or spores. The need to withdraw samples during the thermal and combined processes would be avoided if the thermal survival parameters could be calculated by the "three endpoints method," which does not require the entire survival curve determination. Currently however, this method is limited to thermal inactivation patterns characterized by up to 3 survival parameters, the Weibull-Log logistic (WeLL) model, for example.

  9. Small Molecule Inhibitors of Anthrax Lethal Factor Toxin

    PubMed Central

    Williams, John D.; Khan, Atiyya R.; Cardinale, Steven C.; Butler, Michelle M.; Bowlin, Terry L.; Peet, Norton P.

    2014-01-01

    This manuscript describes the preparation of new small molecule inhibitors of Bacillus anthracis lethal factor. Our starting point was the symmetrical, bis-quinolinyl compound 1 (NSC 12155). Optimization of one half of this molecule led to new LF inhibitors that were desymmetrized to afford more drug-like compounds. PMID:24290062

  10. Dominant-lethal mutations and heritable translocations in mice

    SciTech Connect

    Generoso, W.M.

    1983-01-01

    Chromosome aberrations are a major component of radiation or chemically induced genetic damage in mammalian germ cells. The types of aberration produced are dependent upon the mutagen used and the germ-cell stage treated. For example, in male meiotic and postmeiotic germ cells certain alkylating chemicals induce both dominant-lethal mutations and heritable translocations while others induce primarily dominant-lethal mutations. Production of these two endpoints appears to be determined by the stability of alkylation products with the chromosomes. If the reaction products are intact in the male chromosomes at the time of sperm entry, they may be repaired in fertilized eggs. If repair is not effected and the alkylation products persist to the time of pronuclear chromosome replication, they lead to chromatid-type aberrations and eventually to dominant-lethality. The production of heritable translocations, on the other hand, requires a transformation of unstable alkylation products into suitable intermediate lesions. The process by which these lesions are converted into chromosome exchange within the male genome takes place after sperm enters the egg but prior to the time of pronuclear chromosome replication (i.e., chromosome-type). Thus, dominant-lethal mutations result from both chromatid- and chromosome-type aberrations while heritable translocations result primarily from the latter type. DNA target sites associated with the production of these two endpoints are discussed.

  11. Subcutaneous wounding postirradiation reduces radiation lethality in mice.

    PubMed

    Garrett, Joy; Orschell, Christie M; Mendonca, Marc S; Bigsby, Robert M; Dynlacht, Joseph R

    2014-06-01

    The detonation of an improvised nuclear device during a radiological terrorist attack could result in the exposure of thousands of civilians and first responders to lethal or potentially lethal doses of ionizing radiation (IR). There is a major effort in the United States to develop phamacological mitigators of radiation lethality that would be effective particularly if administered after irradiation. We show here that giving female C57BL/6 mice a subcutaneous surgical incision after whole body exposure to an LD50/30 X-ray dose protects against radiation lethality and increases survival from 50% to over 90% (P = 0.0001). The increase in survival, at least in part, appears to be due to enhanced recovery of hematopoiesis, notably red blood cells, neutrophils and platelets. While a definitive mechanism has yet to be elucidated, we propose that this approach may be used to identify potentially novel mechanisms and pathways that could aid in the development of novel pharmacological radiation countermeasures. PMID:24811864

  12. The "Lethal Chamber": Further Evidence of the Euthanasia Option.

    ERIC Educational Resources Information Center

    Elks, Martin A.

    1993-01-01

    Historical discussions of the euthanasia or "lethal chamber" option in relation to people with mental retardation are presented. The paper concludes that eugenic beliefs in the primacy of heredity over environment and the positive role of natural selection may have condoned the poor conditions characteristic of large, segregated institutions and…

  13. The Prevalence, Lethality and Intent of Suicide Attempts among Adolescents.

    ERIC Educational Resources Information Center

    Andrews, Judy A.; Lewinsohn, Peter M.

    Although suicide is the second leading cause of death among adolescents in the United States, little is known about the prevalence or characteristics of suicide attempts among adolescents. Data from 1,710 adolescents attending 9 high schools in 5 communities were examined to determine the prevalence of suicide attempts and the lethality and intent…

  14. An overview of the future of non-lethal weapons.

    PubMed

    Alexander, J B

    2001-01-01

    During the past decade, vast changes have occurred in the geopolitical landscape and the nature of the types of conflicts in which technologically developed countries have been involved. While the threat of conventional war remains, forces have been more frequently deployed in situations that require great restraint. Adversaries are often likely to be elusive and commingled with noncombatants. There has been some shift in public opinion away from tolerance of collateral casualties. Therefore there is a need to be able to apply force while limiting casualties. Non-lethal weapons provide part of the solution. Among the changes that will influence the future have been studies by the US and NATO concerning the use of non-lethal weapons, coincidental with increased funding for their development and testing. New concepts and policies have recently been formalized. Surprisingly, the most strident objections to the implementation of non-lethal weapons have come from organizations that are ostensibly designed to protect non-combatants. These arguments are specious and, while technically and academically challenging, actually serve to foster an environment that will result in the deaths of many more innocent civilians. They misconstrue technology with human intent. The reasons for use of force will not abate. Alternatives to bombs, missiles, tanks and artillery must therefore be found. Non-lethal weapons are not a panacea but do offer the best hope of minimizing casualties while allowing nations or alliances the means to use force in protection of national or regional interests.

  15. Moving ahead on harnessing synthetic lethality to fight cancer.

    PubMed

    Jerby-Arnon, Livnat; Ruppin, Eytan

    2015-01-01

    We have recently developed a data-mining pipeline that comprehensively identifies cancer unique susceptibilities, following the concept of Synthetic Lethality (SL). The approach enables, for the first time, to identify and harness genome-scale SL-networks to accurately predict gene essentiality, drug response, and clinical prognosis in cancer.

  16. An overview of the future of non-lethal weapons.

    PubMed

    Alexander, J B

    2001-01-01

    During the past decade, vast changes have occurred in the geopolitical landscape and the nature of the types of conflicts in which technologically developed countries have been involved. While the threat of conventional war remains, forces have been more frequently deployed in situations that require great restraint. Adversaries are often likely to be elusive and commingled with noncombatants. There has been some shift in public opinion away from tolerance of collateral casualties. Therefore there is a need to be able to apply force while limiting casualties. Non-lethal weapons provide part of the solution. Among the changes that will influence the future have been studies by the US and NATO concerning the use of non-lethal weapons, coincidental with increased funding for their development and testing. New concepts and policies have recently been formalized. Surprisingly, the most strident objections to the implementation of non-lethal weapons have come from organizations that are ostensibly designed to protect non-combatants. These arguments are specious and, while technically and academically challenging, actually serve to foster an environment that will result in the deaths of many more innocent civilians. They misconstrue technology with human intent. The reasons for use of force will not abate. Alternatives to bombs, missiles, tanks and artillery must therefore be found. Non-lethal weapons are not a panacea but do offer the best hope of minimizing casualties while allowing nations or alliances the means to use force in protection of national or regional interests. PMID:11578037

  17. 40 CFR 798.5450 - Rodent dominant lethal assay.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... result, one of which is a statistically significant dose-related increase in the number of dominant... a statistically significant dose-related increase in the number of dominant lethals or a... of the uteri are examined to determine the numbers of implants and live and dead embryos....

  18. 40 CFR 798.5450 - Rodent dominant lethal assay.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... result, one of which is a statistically significant dose-related increase in the number of dominant... a statistically significant dose-related increase in the number of dominant lethals or a... of the uteri are examined to determine the numbers of implants and live and dead embryos....

  19. The Lethal "Femme Fatale" in the Noir Tradition.

    ERIC Educational Resources Information Center

    Boozer, Jack

    2000-01-01

    Traces the lethal seductress through Hollywood's "noir" history from "Double Indemnity" (1944) to "The Last Seduction" (1996). Examines how this figure largely abjures traditional romance and passive domesticity, choosing instead to apply her sexuality to homicidal plots toward greed. Argues that her narrative positioning serves as a barometer of…

  20. Conditional lethality strains for the biological control of Anastrepha species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pro-apoptotic cell death genes are promising candidates for biologically-based autocidal control of pest insects as demonstrated by tetracycline (tet)-suppressible systems for conditional embryonic lethality in Drosophila melanogaster (Dm) and the medfly, Ceratitis capitata (Cc). However, for medfly...

  1. Resveratrol Antagonizes Antimicrobial Lethality and Stimulates Recovery of Bacterial Mutants

    PubMed Central

    Liu, Yuanli; Zhou, Jinan; Qu, Yilin; Yang, Xinguang; Shi, Guojing; Wang, Xiuhong; Hong, Yuzhi; Drlica, Karl; Zhao, Xilin

    2016-01-01

    Reactive oxygen species (ROS; superoxide, peroxide, and hydroxyl radical) are thought to contribute to the rapid bactericidal activity of diverse antimicrobial agents. The possibility has been raised that consumption of antioxidants in food may interfere with the lethal action of antimicrobials. Whether nutritional supplements containing antioxidant activity are also likely to interfere with antimicrobial lethality is unknown. To examine this possibility, resveratrol, a popular antioxidant dietary supplement, was added to cultures of Escherichia coli and Staphylococcus aureus that were then treated with antimicrobial and assayed for bacterial survival and the recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Resveratrol, at concentrations likely to be present during human consumption, caused a 2- to 3-fold reduction in killing during a 2-hr treatment with moxifloxacin or kanamycin. At higher, but still subinhibitory concentrations, resveratrol reduced antimicrobial lethality by more than 3 orders of magnitude. Resveratrol also reduced the increase in reactive oxygen species (ROS) characteristic of treatment with quinolone (oxolinic acid). These data support the general idea that the lethal activity of some antimicrobials involves ROS. Surprisingly, subinhibitory concentrations of resveratrol promoted (2- to 6-fold) the recovery of rifampicin-resistant mutants arising from the action of ciprofloxacin, kanamycin, or daptomycin. This result is consistent with resveratrol reducing ROS to sublethal levels that are still mutagenic, while the absence of resveratrol allows ROS levels to high enough to kill mutagenized cells. Suppression of antimicrobial lethality and promotion of mutant recovery by resveratrol suggests that the antioxidant may contribute to the emergence of resistance to several antimicrobials, especially if new derivatives and/or formulations of resveratrol markedly increase bioavailability. PMID:27045517

  2. A multivariate model of stakeholder preference for lethal cat management.

    PubMed

    Wald, Dara M; Jacobson, Susan K

    2014-01-01

    Identifying stakeholder beliefs and attitudes is critical for resolving management conflicts. Debate over outdoor cat management is often described as a conflict between two groups, environmental advocates and animal welfare advocates, but little is known about the variables predicting differences among these critical stakeholder groups. We administered a mail survey to randomly selected stakeholders representing both of these groups (n=1,596) in Florida, where contention over the management of outdoor cats has been widespread. We used a structural equation model to evaluate stakeholder intention to support non-lethal management. The cognitive hierarchy model predicted that values influenced beliefs, which predicted general and specific attitudes, which in turn, influenced behavioral intentions. We posited that specific attitudes would mediate the effect of general attitudes, beliefs, and values on management support. Model fit statistics suggested that the final model fit the data well (CFI=0.94, RMSEA=0.062). The final model explained 74% of the variance in management support, and positive attitudes toward lethal management (humaneness) had the largest direct effect on management support. Specific attitudes toward lethal management and general attitudes toward outdoor cats mediated the relationship between positive (p<0.05) and negative cat-related impact beliefs (p<0.05) and support for management. These results supported the specificity hypothesis and the use of the cognitive hierarchy to assess stakeholder intention to support non-lethal cat management. Our findings suggest that stakeholders can simultaneously perceive both positive and negative beliefs about outdoor cats, which influence attitudes toward and support for non-lethal management.

  3. A Multivariate Model of Stakeholder Preference for Lethal Cat Management

    PubMed Central

    Wald, Dara M.; Jacobson, Susan K.

    2014-01-01

    Identifying stakeholder beliefs and attitudes is critical for resolving management conflicts. Debate over outdoor cat management is often described as a conflict between two groups, environmental advocates and animal welfare advocates, but little is known about the variables predicting differences among these critical stakeholder groups. We administered a mail survey to randomly selected stakeholders representing both of these groups (n = 1,596) in Florida, where contention over the management of outdoor cats has been widespread. We used a structural equation model to evaluate stakeholder intention to support non-lethal management. The cognitive hierarchy model predicted that values influenced beliefs, which predicted general and specific attitudes, which in turn, influenced behavioral intentions. We posited that specific attitudes would mediate the effect of general attitudes, beliefs, and values on management support. Model fit statistics suggested that the final model fit the data well (CFI = 0.94, RMSEA = 0.062). The final model explained 74% of the variance in management support, and positive attitudes toward lethal management (humaneness) had the largest direct effect on management support. Specific attitudes toward lethal management and general attitudes toward outdoor cats mediated the relationship between positive (p<0.05) and negative cat-related impact beliefs (p<0.05) and support for management. These results supported the specificity hypothesis and the use of the cognitive hierarchy to assess stakeholder intention to support non-lethal cat management. Our findings suggest that stakeholders can simultaneously perceive both positive and negative beliefs about outdoor cats, which influence attitudes toward and support for non-lethal management. PMID:24736744

  4. Vision Screening

    NASA Technical Reports Server (NTRS)

    1993-01-01

    The Visi Screen OSS-C, marketed by Vision Research Corporation, incorporates image processing technology originally developed by Marshall Space Flight Center. Its advantage in eye screening is speed. Because it requires no response from a subject, it can be used to detect eye problems in very young children. An electronic flash from a 35 millimeter camera sends light into a child's eyes, which is reflected back to the camera lens. The photorefractor then analyzes the retinal reflexes generated and produces an image of the child's eyes, which enables a trained observer to identify any defects. The device is used by pediatricians, day care centers and civic organizations that concentrate on children with special needs.

  5. Podophyllum hexandrum-Mediated Survival Protection and Restoration of Other Cellular Injuries in Lethally Irradiated Mice.

    PubMed

    Sankhwar, Sanghmitra; Gupta, Manju Lata; Gupta, Vanita; Verma, Savita; Suri, Krishna Avtar; Devi, Memita; Sharma, Punita; Khan, Ehsan Ahmed; Alam, M Sarwar

    2011-01-01

    This study aims at the development of a safe and effective formulation to counter the effects of lethal irradiation. The sub-fraction (G-001M), prepared from Podophyllum hexandrum has rendered high degree of survival (>90%) at a dose of 6 mg kg(-1) body weight (intramuscular) in lethally irradiated mice. Therapeutic dose of G-001M, at about 20 times lower concentration than its LD(100), has revealed a DRF of 1.62. Comet assay studies in peripheral blood leukocytes have reflected that, treatment of G-001M before irradiation has significantly reduced DNA tail length (P < .001) and DNA damage score (P < .001), as compared to radiation-only group. Spleen cell counts in irradiated animals had declined drastically at the very first day of exposure, and the fall continued till the 5th day (P < .001). In the treated irradiated groups, there was a steep reduction in the counts initially, but this phase did not prolong. More than 60% decline in thymocytes of irradiated group animals was registered at 5 h of irradiation when compared with controls, and the fall progressed further downwards with the similar pace till 5th day of exposure (P < .001). At later intervals, thymus was found fully regressed. In G-001M pre-treated irradiated groups also, thymocytes decreased till the 5th day but thereafter rejuvenated and within 30 days of treatment the values were close to normal. Current studies have explicitly indicated that, G-001M in very small doses has not only rendered high survivability in lethally irradiated mice, but also protected their cellular DNA, besides supporting fast replenishment of the immune system.

  6. Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia indica Linn.

    PubMed

    Apu, As; Muhit, Ma; Tareq, Sm; Pathan, Ah; Jamaluddin, Atm; Ahmed, M

    2010-01-01

    The crude methanolic extract of Dillenia indica Linn. (Dilleniaceae) leaves has been investigated for the evaluation of antimicrobial and cytotoxic activities. Organic solvent (n-hexane, carbon tetrachloride and chloroform) fractions of methanolic extract and methanolic fraction (aqueous) were screened for their antimicrobial activity by disc diffusion method. Besides, the fractions were screened for cytotoxic activity using brine shrimp (Artemia salina) lethality bioassay. Among the four fractions tested, n-hexane, carbon tetrachloride, and chloroform fractions showed moderate antibacterial and antifungal activity compared to standard antibiotic, kanamycin. The average zone of inhibition was ranged from 6 to 8 mm at a concentration of 400 µg/disc. But the aqueous fraction was found to be insensitive to microbial growth. Compared to vincristine sulfate (with LC(50) of 0.52 µg/ ml), n-hexane and chloroform fractions demonstrated a significant cytotoxic activity (having LC(50) of 1.94 µg/ml and 2.13 µg/ml, respectively). The LC(50) values of the carbon tetrachloride and aqueous fraction were 4.46 µg/ml and 5.13 µg/ ml, respectively. The study confirms the moderate antimicrobial and potent cytotoxic activities of Dillenia indica leaves extract and therefore demands the isolation of active principles and thorough bioassay. PMID:21331191

  7. Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia indica Linn.

    PubMed

    Apu, As; Muhit, Ma; Tareq, Sm; Pathan, Ah; Jamaluddin, Atm; Ahmed, M

    2010-01-01

    The crude methanolic extract of Dillenia indica Linn. (Dilleniaceae) leaves has been investigated for the evaluation of antimicrobial and cytotoxic activities. Organic solvent (n-hexane, carbon tetrachloride and chloroform) fractions of methanolic extract and methanolic fraction (aqueous) were screened for their antimicrobial activity by disc diffusion method. Besides, the fractions were screened for cytotoxic activity using brine shrimp (Artemia salina) lethality bioassay. Among the four fractions tested, n-hexane, carbon tetrachloride, and chloroform fractions showed moderate antibacterial and antifungal activity compared to standard antibiotic, kanamycin. The average zone of inhibition was ranged from 6 to 8 mm at a concentration of 400 µg/disc. But the aqueous fraction was found to be insensitive to microbial growth. Compared to vincristine sulfate (with LC(50) of 0.52 µg/ ml), n-hexane and chloroform fractions demonstrated a significant cytotoxic activity (having LC(50) of 1.94 µg/ml and 2.13 µg/ml, respectively). The LC(50) values of the carbon tetrachloride and aqueous fraction were 4.46 µg/ml and 5.13 µg/ ml, respectively. The study confirms the moderate antimicrobial and potent cytotoxic activities of Dillenia indica leaves extract and therefore demands the isolation of active principles and thorough bioassay.

  8. Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia indica Linn

    PubMed Central

    Apu, AS; Muhit, MA; Tareq, SM; Pathan, AH; Jamaluddin, ATM; Ahmed, M

    2010-01-01

    The crude methanolic extract of Dillenia indica Linn. (Dilleniaceae) leaves has been investigated for the evaluation of antimicrobial and cytotoxic activities. Organic solvent (n-hexane, carbon tetrachloride and chloroform) fractions of methanolic extract and methanolic fraction (aqueous) were screened for their antimicrobial activity by disc diffusion method. Besides, the fractions were screened for cytotoxic activity using brine shrimp (Artemia salina) lethality bioassay. Among the four fractions tested, n-hexane, carbon tetrachloride, and chloroform fractions showed moderate antibacterial and antifungal activity compared to standard antibiotic, kanamycin. The average zone of inhibition was ranged from 6 to 8 mm at a concentration of 400 µg/disc. But the aqueous fraction was found to be insensitive to microbial growth. Compared to vincristine sulfate (with LC50 of 0.52 µg/ ml), n-hexane and chloroform fractions demonstrated a significant cytotoxic activity (having LC50 of 1.94 µg/ml and 2.13 µg/ml, respectively). The LC50 values of the carbon tetrachloride and aqueous fraction were 4.46 µg/ml and 5.13 µg/ ml, respectively. The study confirms the moderate antimicrobial and potent cytotoxic activities of Dillenia indica leaves extract and therefore demands the isolation of active principles and thorough bioassay. PMID:21331191

  9. New "lethal highs": a case of a deadly cocktail of GHB and Mephedrone.

    PubMed

    Aromatario, Mariarosaria; Bottoni, Edoardo; Santoni, Mariangela; Ciallella, Costantino

    2012-11-30

    Drug scenes within several countries have changed in recent years to incorporate a range of licit psychoactive products collectively known as "legal highs": these in combination with substances already in use can cause major health problems and even death. Consumption of Gamma-hydroxybutyric acid (GHB) spread from 1980s to 2000s, when several nations have enacted laws that have made it illegal. Cases of GHB-caused or related deaths are often described in association with alcohol or traditional drugs (heroin, cocaine, amphetamine); few cases of acute lethal toxicity due to Mephedrone have been recently reported; we describe the first case of fatality due to concomitant consumption of GHB and Mephedrone. A 43 years old man died during a drugs-based party: the two substances were not detected at toxicological screening, but were identified by further analysis on the basis of circumstantial data reported by a survivor. Through our work we aim to bring to the attention in the emerging role of new drugs of abuse, and highlight problems in identifying these drugs with immunoassay screening test commonly used. PMID:23088826

  10. Genetic studies: dominant lethal study, sex linked recessive lethal, ames mutagenicity, and heritable translocation test of thermal processed, frozen, electron irradiated, and gamma irradiated chicken. Final report Jun 76-Aug 83

    SciTech Connect

    Sullivan, D.; Lusskin, R.M.; Thomson, G.M.; Kuzdas, C.D.; Ronning, D.C.

    1983-01-01

    Four samples of chicken meat identified as the frozen control, thermally processed, gamma sterilized (5.9 Mrad), and electron sterilized (5.9 MeV), along with negative and positive controls, were evaluated for genetic activity. The samples were evaluated for ability to induce dominant lethal mutations in spermatid and spermatozoan stages of spermatogenesis in mice fed 35 percent chicken meat. The test meat samples were not observed to have an effect on the incidence of the dominant lethal mutations. However, the positive control failed to give a positive response. The meat samples were investigated for mutagenic activity employing Drosophila melanogaster in the sex linked recessive lethal test. The samples were determined to be nonmutagenic in this test and the positive control gave a significant response. Reduced production of offspring in cultures of Drosophila reared on gamma irradiated chicken which could not be overcome by the addition of vitamins was observed. Pre-incubation tests with and without added mutagens revealed that in no case was a positive result observed in the Ames test from chicken meat without an added mutagen. The manner in which chicken meat was processed had no effect upon the response to the Ames test. A heritable translocation test in mice failed to reveal any cytological evidence of translocation heterozygosity in any of the chicken-containing diets.

  11. Strategy for enhanced transgenic strain development for embryonic conditionnal lethality in Anastrepha suspensa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here the first reproductive sterility system for the tephritid pest, Anastrepha suspensa, is presented, based on lethality primarily in embryos heterozygous for a lethal conditional transgene combination. The tetracycline-suppressible system uses the cellularization-specific A. suspensa serendipity...

  12. Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus

    PubMed Central

    Walther, Elisabeth; Xu, Zhongli; Richter, Martina; Kirchmair, Johannes; Grienke, Ulrike; Rollinger, Judith M.; Krumbholz, Andi; Saluz, Hans P.; Pfister, Wolfgang; Sauerbrei, Andreas; Schmidtke, Michaela

    2016-01-01

    Secondary infections with Streptococcus pneumoniae cause severe pneumonia and enhance lethality during influenza epidemics and pandemics. Structural and functional similarities with viral neuraminidase (NA) suggest that the highly prevalent pneumococcal NAs, NanA and NanB, might contribute to this lethal synergism by supporting viral replication and that dual acting NA inhibitors (NAIs) will disrupt it. To verify this hypothesis, NanA and NanB were expressed in E. coli. After confirming their activity in enzyme assays, in vitro models with influenza virus A/Jena/8178/09 (Jena/8178) and the recombinant NanA or NanB (rNanA and rNanB) were established in A549 and MDCK cells to mimic the role of these pneumococcal NAs during co-infection. Studies on the influence of both NAs on viral receptor expression, spread, and yield revealed a distinct effect of NanA and NanB on viral replication in these in vitro models. Both enzymes were able to support Jena/8178 replication at certain concentrations. This synergism was disrupted by the NAIs oseltamivir, DANA, katsumadain A, and artocarpin exerting an inhibitory effect on viral NA and NanA. Interestingly, katsumadain A and artocarpin inhibited rNanA and rNanB similarly. Zanamivir did not show activity. These results demonstrate a key role of pneumococcal NAs in the lethal synergism with influenza viruses and reveal opportunities for its effective disruption. PMID:27047471

  13. A framework for the assessment of non-lethal weapons.

    PubMed

    Rappert, Brian

    2004-01-01

    In many government, police and military circles, attention is being given to so-called 'non-lethal' weapons as means of reducing many of the negative effects directly or indirectly associated with the use of force. Despite the purported ability of the adoption of such weaponry to lessen grounds for contention and concern, past experience suggests the need for scepticism regarding the purported benefits. Rather than relying on poorly substantiated claims, comprehensive procedures are needed to ensure the appropriateness of force options. This article outlines some of the institutional structures required for 'carefully evaluating' and 'carefully controlling' non-lethal weapons, with a discussion of the perennial tensions associated with ensuring the relative 'acceptability' of the use of force. PMID:15015546

  14. Variability of platyspondylic lethal chondrodysplasia: another case report.

    PubMed

    Nishimura, G; Iwasawa, T; Fukuzawa, R; Hirabayashi, Y; Ito, T

    1998-07-01

    We report the radiological and histological findings of another case of platyspondylic lethal chondrodysplasia. The patient was a girl, who died of respiratory failure at 18 days of age. The radiological changes comprised moderate platyspondyly with ovoid-shaped vertebral bodies, broad and short ilia, rhizomelic shortening and mild bowing of the long bones (particularly of the humeri), relatively long short tubular bones, and retarded epiphyseal ossification and ragged metaphyses, which were most similar to those of a mild variant of this entity, the Luton type. However, the histological findings of cartilage, including hypercellularity of the reserve zone with round resting chondrocytes, relatively normal column formation of the proliferative and hypertrophic zones, and incorporation of hypertrophic cartilage with a columnar arrangement into metaphyseal bony trabeculae, resemble those of a severe variant of this entity, the Torrance type. Our observation provides an insight into the phenotypic variabilities of platyspondylic lethal chondrodysplasia. PMID:9689993

  15. Medical ethics, cultural values, and physician participation in lethal injection.

    PubMed

    Boehnlein, J K; Parker, R M; Arnold, R M; Bosk, C F; Sparr, L F

    1995-01-01

    Capital punishment by lethal injection has been discussed in the literature, but there has been no consideration of the sociocultural foundations of the ethical issues related to medical aspects of capital punishment. Lethal injection represents the inappropriate medicalization of a complex social issue whereby medical skills and procedures are used in ways that contradict established medical practice. Although physicians are socialized to their healing role during medical education and training, their behavior is influenced by social and cultural values that both precede and coexist with their professional life. Because of this dynamic interplay between professional and sociocultural values, physicians can neither exempt themselves from societal debate by merely invoking professional ethics, nor can they define their professional role exclusively in terms of societal values that potentially diminish personal and collective professional responsibility. It is essential that physicians have a broad historical perspective on the development of the profession's standards and values in order to deal effectively with present and future complex ethical issues.

  16. Lethal congenital contracture syndrome: further delineation and genetic aspects.

    PubMed Central

    Vuopala, K; Herva, R

    1994-01-01

    In a national morphology based study of lethal arthrogryposis between 1979 and 1992, 40 fetuses and infants with lethal congenital contracture syndrome (LCCS, McKusick 253310) were found in Finland. The incidence of LCCS in Finland was 1:19,000 births. There were 20 affected males and 20 affected females in 26 families. In 16 cases the pregnancy was terminated after the prenatal diagnosis of total akinesia and fetal hydrops on ultrasound. There were 19 stillborn infants and five were born showing signs of life, but died within one hour. The segregation analyses yielded 0.45 affected by the "singles" method and 0.34 by the "sib" method. The birthplaces of the grandparents were located in the sparsely populated north east of Finland. This finding supports the existence of an autosomal recessive LCCS gene in Finland, particularly in the north eastern part. Images PMID:7966188

  17. Assessing lethal and sub-lethal effects of trichlorfon on different trophic levels.

    PubMed

    Coelho, Sónia; Oliveira, Rhaul; Pereira, Susana; Musso, Carolina; Domingues, Inês; Bhujel, Ram C; Soares, Amadeu M V M; Nogueira, António J A

    2011-06-01

    Trichlorfon (TCF) is one of the most used veterinary pharmaceuticals not only to fight infestations but also as a preventive measure worldwide. The high concentrations used generate concerns about environmental and human health. In this work we assessed the acute toxicity of this compound to non-target organisms belonging to different trophic levels: Danio rerio (early life stages and adults), Daphnia magna and algae (Pseudokirchneriella subcapitata and Chlorella vulgaris), and studied the potential of the biomarkers cholinesterase (ChE), glutathione-S-transferase (GST), lactate dehydrogenase (LDH) and catalase (CAT) to assess sub-lethal effects of trichlorfon in zebrafish and daphnids. The fish embryo test followed the OECD draft guideline FET and was based on the exposure of newly fertilized eggs to 0, 2.5, 5.0, 10, 20, 40, 80 and 160 mg/L of TCF for 5 days; the fish acute test followed the OECD guideline 203 and was based on the exposure of adult fish to 0, 2.5, 5, 10, 20, 40, 60 and 80 mg/L of TCF for 4 days; Daphnia sp. immobilization assay followed the OECD guideline 202 and was based on the exposure of juvenile daphnids to 0, 0.1, 0.3, 0.5, 0.7, 0.9, 1 and 2 μg/L of TCF for 2 days and the algae growth inhibition assay followed the OECD guideline 201 and was based on the exposure of the two species to 0, 1, 3.2, 10, 32, 100 and 300 mg/L of TCF for 4 days. Biomarker levels were measured after 96 h exposure to TCF in zebrafish early life stages and adults and after 48 h exposure in D. magna. Tested organisms seem to have dissimilar sensitivities towards TCF exposure. D. magna (48 h-LC(50)=0.29 μg/L) was the most sensitive organism, followed by early life stages and adults of zebrafish (96 h-LC(50)=25.4 and 28.8 mg/L, respectively) and finally by the algae P. subcapitata (96 h-LC(50)=274.5 mg/L) and C. vulgaris (no effect observed). As daphnids are a source of food for organisms of higher trophic levels, the impairment on its population is prone to have

  18. Rapid, Optimized Interactomic Screening

    PubMed Central

    Hakhverdyan, Zhanna; Domanski, Michal; Hough, Loren; Oroskar, Asha A.; Oroskar, Anil R.; Keegan, Sarah; Dilworth, David J.; Molloy, Kelly R.; Sherman, Vadim; Aitchison, John D.; Fenyö, David; Chait, Brian T.; Jensen, Torben Heick; Rout, Michael P.; LaCava, John

    2015-01-01

    We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles even for well-studied proteins. Our approach is robust, economical and automatable, providing an inroad to the rigorous, systematic dissection of cellular interactomes. PMID:25938370

  19. Identification of a Novel RNA Virus Lethal to Tilapia

    PubMed Central

    Eyngor, Marina; Zamostiano, Rachel; Kembou Tsofack, Japhette Esther; Berkowitz, Asaf; Bercovier, Hillel; Tinman, Simon; Lev, Menachem; Hurvitz, Avshalom; Galeotti, Marco; Eldar, Avi

    2014-01-01

    Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide. PMID:25232154

  20. Autopsy observations in lethal short-rib polydactyly syndromes.

    PubMed

    Okiro, Patricia; Wainwright, Helen; Spranger, Jürgen; Beighton, Peter

    2015-01-01

    The short rib-polydactyly syndromes are a heterogeneous group of lethal autosomal recessive disorders (SRP I-IV), which result from cellular ciliary dysfunction during embryogenesis. Diagnosis is conventionally based on radiographic imaging. Since 1976, postmortem investigations of 5 affected fetuses or stillbirths have been undertaken and the visceral abnormalities have been documented. These anomalies are discussed in the context of prenatal differential diagnosis and prognostication following imaging in pregnancy and at autopsy following miscarriage or stillbirth.

  1. Spondyloepiphyseal dysplasia congenita. A cause of lethal neonatal dwarfism.

    PubMed

    Macpherson, R I; Wood, B P

    1980-07-01

    Spondyloepiphyseal dysplasia congenita is a form of primary short dwarfism, that is manifest at birth generally has not been regarded as a cause of lethal neonatal dwarfism. Seven neonates with severe dwarfism are presented. The first survived the newborn period, but the other six were early neonatal deaths. All displayed the clinical and radiologic features of spondyloepiphyseal dysplasia congenita. The striking similarities between spondyloepiphyseal dysplasia congenita and achondrogenesis type 2 are discussed. PMID:6773018

  2. [Lethal achondrogenesis: a review of 56 cases (author's transl)].

    PubMed

    Schulte, M J; Lenz, W; Vogel, M

    1978-07-01

    54 cases with lethal achondrogenesis from the literature as well as two own cases are reviewed and analyzed with regard to the following characteristics: sex, hydramnios, breech presentation, duration of pregnancy, length and weight at birth, head circumference, length of upper and lower extremities, clinical and radiological data, age of mother and father at time of birth, familial occurrence and consanguinity of parents, histological, histochemical and electronmicroscopic tissue examination. PMID:353375

  3. Disruption of the Rice Plastid Ribosomal Protein S20 Leads to Chloroplast Developmental Defects and Seedling Lethality

    PubMed Central

    Gong, Xiaodi; Jiang, Quan; Xu, Jianlong; Zhang, Jianhui; Teng, Sheng; Lin, Dongzhi; Dong, Yanjun

    2013-01-01

    Plastid ribosomal proteins (PRPs) are essential for ribosome biogenesis, plastid protein biosynthesis, chloroplast differentiation, and early chloroplast development. This study identifies the first rice PRP mutant, asl1 (albino seedling lethality1), which exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered chlorophyll (Chl) content and chloroplast development. Map-based cloning revealed that ASL1 encodes PRP S20 (PRPS20), which localizes to the chloroplast. ASL1 showed tissue-specific expression, as it was highly expressed in plumule and young seedlings but expressed at much lower levels in other tissues. In addition, ASL1 expression was regulated by light. The transcript levels of nuclear genes for Chl biosynthesis and chloroplast development were strongly affected in asl1 mutants; transcripts of some plastid genes for photosynthesis were undetectable. Our findings indicate that nuclear-encoded PRPS20 plays an important role in chloroplast development in rice. PMID:23979931

  4. 28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Use of less-than-lethal weapons... Use of less-than-lethal weapons, including chemical agents. (a) The Warden may authorize the use of less-than-lethal weapons, including those containing chemical agents, only when the situation is...

  5. 28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Use of less-than-lethal weapons... Use of less-than-lethal weapons, including chemical agents. (a) The Warden may authorize the use of less-than-lethal weapons, including those containing chemical agents, only when the situation is...

  6. 28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Use of less-than-lethal weapons... Use of less-than-lethal weapons, including chemical agents. (a) The Warden may authorize the use of less-than-lethal weapons, including those containing chemical agents, only when the situation is...

  7. 28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Use of less-than-lethal weapons... Use of less-than-lethal weapons, including chemical agents.