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 Central

    Kim, Bogyou; Wang, Shangzi; Lee, Ji Min; Jeong, Yunju; Ahn, TaeJin; Son, Dae-Soon; Park, Hye Won; Yoo, Hyeon-seok; Song, Yun-Jeong; Lee, Eunjin; Oh, Young Mi; Lee, Saet Byoul; Choi, Jaehyun; Murray, Joseph C; Zhou, Yan; Song, Paul H; Kim, Kyung-Ah; Weiner, Louis M

    2014-01-01

    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 EGFR 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 employing a siRNA-based synthetic lethal screening method. We found that knockdown of 69 genes in Met-amplified MKN45 cells sensitized the anti-tumor activity of SAIT301. Pathway analysis of these 69 genes implicated FGFR as a key regulator for anti-proliferative 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 ?3 is another potential target for combination treatment with SAIT301. Suppression of integrin ?3 decreased AKT phosphorylation in SAIT301-resistant cells and restores SAIT301 responsiveness in HCC1954 cells, which are resistant to SAIT301. Gene expression analysis using CCLE database shows cancer cells with high levels of FGFR and integrin ?3 are resistant to crizotinib treatment, suggesting FGFR and integrin ?3 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. Parallel in vivo and in vitro melanoma RNAi dropout screens reveal synthetic lethality between hypoxia and DNA damage response inhibition.

    PubMed

    Possik, Patricia A; Müller, Judith; Gerlach, Carmen; Kenski, Juliana C N; Huang, Xinyao; Shahrabi, Aida; Krijgsman, Oscar; Song, Ji-Ying; Smit, Marjon A; Gerritsen, Bram; Lieftink, Cor; Kemper, Kristel; Michaut, Magali; Beijersbergen, Roderick L; Wessels, Lodewyk; Schumacher, Ton N; Peeper, Daniel S

    2014-11-20

    To identify factors preferentially necessary for driving tumor expansion, we performed parallel in vitro and in vivo negative-selection short hairpin RNA (shRNA) screens. Melanoma cells harboring shRNAs targeting several DNA damage response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for the identification of pharmacologically tractable targets. PMID:25456132

  3. 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...

  4. Synthetic lethal screens as a means to understand and treat MYC-driven cancers

    PubMed Central

    Cermelli, Silvia; Jang, In Sock; Bernard, Brady; Grandori, Carla

    2014-01-01

    While therapeutics against MYC could potentially be employed against a wide range of human cancers, MYC targeted therapies have proven difficult to develop. The convergence of breakthroughs in human genomics and in gene silencing using RNA interference (RNAi), have recently allowed functional interrogation of the genome and systematic identification of synthetic lethal interactions with hyper-active MYC. Here, we focus on the pathways that have emerged through RNAi screens and present evidence that a subset of genes exhibiting synthetic lethality with MYC are significantly interconnected and linked to chromatin, and transcriptional processes, as well as to DNA repair and cell-cycle checkpoints. Other synthetic lethal interactions with MYC point to novel pathways and potentially broaden the repertoire of targeted therapies. The elucidation of MYC synthetic lethal interactions is still in its infancy and how these interactions may be influenced by tissue specific programs and by concurrent genetic change will require further investigation. Nevertheless, we predict that these studies may lead the way to novel therapeutic approaches and new insights into the role of MYC in cancer. PMID:24591535

  5. 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).

  6. A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis

    PubMed Central

    Kranz, Dominique; Boutros, Michael

    2014-01-01

    The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions. PMID:24442637

  7. 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.

  8. Synthetic lethality: General principles, utility and detection using genetic screens in human cells

    PubMed Central

    Nijman, Sebastian M.B.

    2011-01-01

    Synthetic lethality occurs when the simultaneous perturbation of two genes results in cellular or organismal death. Synthetic lethality also occurs between genes and small molecules, and can be used to elucidate the mechanism of action of drugs. This area has recently attracted attention because of the prospect of a new generation of anti-cancer drugs. Based on studies ranging from yeast to human cells, this review provides an overview of the general principles that underlie synthetic lethality and relates them to its utility for identifying gene function, drug action and cancer therapy. It also identifies the latest strategies for the large-scale mapping of synthetic lethalities in human cells which bring us closer to the generation of comprehensive human genetic interaction maps. PMID:21094158

  9. 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

  10. Alkylation sensitivity screens reveal a conserved cross-species functionome

    PubMed Central

    Svilar, David; Dyavaiah, Madhu; Brown, Ashley R.; Tang, Jiang-bo; Li, Jianfeng; McDonald, Peter R.; Shun, Tong Ying; Braganza, Andrea; Wang, Xiao-hong; Maniar, Salony; St Croix, Claudette M.; Lazo, John S.; Pollack, Ian F.; Begley, Thomas J.; Sobol, Robert W.

    2013-01-01

    To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases UNG and MYH as well as MPG to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in E. coli and S. cerevisiae. The conserved biological processes across all three species composes an Alkylation Functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance. PMID:23038810

  11. 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

  12. Identification of molecular vulnerabilities in human multiple myeloma cells by RNA interference lethality screening of the druggable genome.

    PubMed

    Tiedemann, Rodger E; Zhu, Yuan Xao; Schmidt, Jessica; Shi, Chang Xin; Sereduk, Chris; Yin, Hongwei; Mousses, Spyro; Stewart, A Keith

    2012-02-01

    Despite recent advances in targeted treatments for multiple myeloma, optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in multiple myeloma cells using siRNAs. We validated the top 160 lethal hits with four siRNAs per gene in three multiple myeloma cell lines and two non-myeloma cell lines, cataloging a total of 57 potent multiple myeloma survival genes. We identified the Bcl2 family member MCL1 and several 26S proteasome subunits among the most important and selective multiple myeloma survival genes. These results provided biologic validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation, and mitosis. Several of the multiple myeloma survival genes, especially MCL1, TNK2, CDK11, and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between multiple myeloma and non-multiple myeloma cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8, and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in multiple myeloma. PMID:22147262

  13. Identification of molecular vulnerabilities in human multiple myeloma cells by RNAi lethality screening of the druggable genome

    PubMed Central

    Tiedemann, Rodger E; Zhu, Yuan Xao; Schmidt, Jessica; Xin Shi, Chang; Sereduk, Chris; Yin, Hongwei; Mousses, Spyro; Stewart, A Keith

    2013-01-01

    Despite recent advances in targeted treatments for multiple myeloma (MM), optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in MM cells using small interfering RNAs (siRNA). We validated the top 160 lethal hits with 4 siRNAs per gene in three MM cell lines and two non-myeloma cell lines, cataloging a total of 57 potent MM survival genes. We identified the Bcl-2 family member MCL-1 and several 26S proteasome subunits amongst the most important and selective MM survival genes. These results provided biological validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation and mitosis. Several of the MM survival genes, especially MCL1, TNK2, CDK11 and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between MM and non-MM cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8 and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in MM. PMID:22147262

  14. Synthetic Lethality Screen Identifies RPS6KA2 as Modifier of Epidermal Growth Factor Receptor Activity in Pancreatic Cancer12

    PubMed Central

    Milosevic, Nada; Kühnemuth, Benjamin; Mühlberg, Leonie; Ripka, Stefanie; Griesmann, Heidi; Lölkes, Carolin; Buchholz, Malte; Aust, Daniela; Pilarsky, Christian; Krug, Sebastian; Gress, Thomas; Michl, Patrick

    2013-01-01

    Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR) inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA)-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2)/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy. PMID:24403857

  15. A synthetic lethal screen identifies the Vitamin D receptor as a novel gemcitabine sensitizer in pancreatic cancer cells.

    PubMed

    Bhattacharjee, V; Zhou, Y; Yen, T J

    2014-01-01

    Overcoming chemoresistance of pancreatic cancer (PCa) cells should significantly extend patient survival. The current treatment modalities rely on a variety of DNA damaging agents including gemcitabine, FOLFIRINOX, and Abraxane that activate cell cycle checkpoints, which allows cells to survive these drug treaments. Indeed, these treatment regimens have only extended patient survival by a few months. The complex microenvironment of PCa tumors has been shown to complicate drug delivery thus decreasing the sensitivity of PCa tumors to chemotherapy. In this study, a genome-wide siRNA library was used to conduct a synthetic lethal screen of Panc1 cells that was treated with gemcitabine. A sublethal dose (50 nM) of the drug was used to model situations of limiting drug availability to PCa tumors in vivo. Twenty-seven validated sensitizer genes were identified from the screen including the Vitamin D receptor (VDR). Gemcitabine sensitivity was shown to be VDR dependent in multiple PCa cell lines in clonogenic survival assays. Sensitization was not achieved through checkpoint override but rather through disrupting DNA repair. VDR knockdown disrupted the cells' ability to form phospho-?H2AX and Rad51 foci in response to gemcitabine treatment. Disruption of Rad51 foci formation, which compromises homologous recombination, was consistent with increased sensitivity of PCa cells to the PARP inhibitor Rucaparib. Thus inhibition of VDR in PCa cells provides a new way to enhance the efficacy of genotoxic drugs. PMID:25558828

  16. Synthetic lethal interactions in yeast reveal functional roles of J protein co-chaperones

    PubMed Central

    Gillies, Anne; Taylor, Rebecca; Gestwicki, Jason E.

    2012-01-01

    J proteins are a diverse family of co-chaperones that cooperate with heat shock protein 70 (Hsp70) to coordinate protein quality control, especially in response to cellular stress. Current models suggest that individual J proteins might play roles in recruiting Hsp70s to specific functions, such as maintaining cell wall integrity or promoting ribosome biogenesis. However, relatively few stresses have been used to test this model and, as a result, only a few specific activities have been identified. To expand our understanding of the J protein network, we used a synthetic lethal approach in which 11 Saccharomyces cerevisiae deletion strains were treated with 12 well-characterized chemical inhibitors. The results defined new roles for specific J proteins in major signaling pathways. For example, an important role for Swa2 in cell wall integrity was identified and activities of the under-explored Jjj1, Apj1, Jjj3 and Caj1 proteins were suggested. More generally, these findings support a model in which some J proteins, such as Ydj1 and Zuo1, play “generalist” roles, while others, such as Apj1 and Jjj2, are “specialists”, having roles in relatively few pathways. Together, these results provide new insight into the network of J proteins. PMID:22851130

  17. Synthetic lethal screening with small molecule inhibitors provides a pathway to rational combination therapies for melanoma

    PubMed Central

    Roller, Devin; Axelrod, Mark; Capaldo, Brian; Jensen, Karin; Mackey, Aaron; Weber, Michael J; Gioeli, Daniel

    2012-01-01

    Recent data demonstrate that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways suggesting why inhibition of a single component of a canonical pathway is insufficient for the treatment of cancer. The biological outcome of signaling through a network is inherently more robust and resistant to inhibition of a single network component. In this study, we performed a functional chemical genetic screen to identify novel interactions between signaling inhibitors that would not be predicted based on our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. The synergistic cytotoxicities identified did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. Among the most robust results was synergy between sorafenib, a multi-kinase inhibitor with activity against RAF, and diclofenac, a non-steroidal anti-inflammatory drug (NSAID). Drug substitution experiments using the NSAIDs celecoxib and ibuprofen or the MEK inhibitor PD325901 and the RAF inhibitor RAF265 suggest that inhibition of cyclooxygenase (COX) and MAP kinase signaling are targets for the synergistic cytotoxicity of sorafenib and diclofenac. Co-treatment with sorafenib and diclofenac interrupts a positive feedback signaling loop involving ERK, cPLA2, and COX. Genome-wide expression profiling demonstrates synergy-specific down-regulation of survival-related genes. This study has uncovered novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially depending on unexplored components of the cell genotype. PMID:22962324

  18. In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase.

    PubMed Central

    Geslain, R; Martin, F; Delagoutte, B; Cavarelli, J; Gangloff, J; Eriani, G

    2000-01-01

    Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination. PMID:10744027

  19. 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

  20. 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

  1. Discovery of a Role for Hsp82 in Histoplasma Virulence through a Quantitative Screen for Macrophage Lethality ?

    PubMed Central

    Edwards, Jessica A.; Zemska, Olga; Rappleye, Chad A.

    2011-01-01

    The application of forward genetics can reveal new factors required for the virulence of intracellular pathogens. To facilitate such virulence screens, we developed macrophage cell lines with which the number of intact host cells following infection with intracellular pathogens can be rapidly and easily ascertained through the expression of a constitutive lacZ transgene. Using known virulence mutants of Francisella novicida and Histoplasma capsulatum, we confirmed the applicability of these host cells for the quantitative assessment of bacterial and fungal virulence, respectively. To identify new genes required for Histoplasma virulence, we employed these transgenic macrophage cells to screen a collection of individual transfer DNA (T-DNA) insertion mutants. Among the mutants showing decreased virulence in macrophages, we identified an insertion in the locus encoding the Histoplasma Hsp82 homolog. The lesion caused by the T-DNA insertion localizes to the promoter region, resulting in significantly decreased HSP82 expression. Reduced HSP82 expression markedly attenuates the virulence of Histoplasma yeast in vivo. While the HSP82 hypomorph grows normally in vitro at 37°C and under acid and salinity stresses, its ability to recover from high-temperature stress is impaired. These results provide genetic proof of the role of stress chaperones in the virulence of a thermally dimorphic fungal pathogen. PMID:21606189

  2. Functional Genomics Reveals the Induction of Inflammatory Response and Metalloproteinase Gene Expression during Lethal Ebola Virus Infection?†

    PubMed Central

    Cilloniz, Cristian; Ebihara, Hideki; Ni, Chester; Neumann, Gabriele; Korth, Marcus J.; Kelly, Sara M.; Kawaoka, Yoshihiro; Feldmann, Heinz; Katze, Michael G.

    2011-01-01

    Ebola virus is the etiologic agent of a lethal hemorrhagic fever in humans and nonhuman primates with mortality rates of up to 90%. Previous studies with Zaire Ebola virus (ZEBOV), mouse-adapted virus (MA-ZEBOV), and mutant viruses (ZEBOV-NPma, ZEBOV-VP24ma, and ZEBOV-NP/VP24ma) allowed us to identify the mutations in viral protein 24 (VP24) and nucleoprotein (NP) responsible for acquisition of high virulence in mice. To elucidate specific molecular signatures associated with lethality, we compared global gene expression profiles in spleen samples from mice infected with these viruses and performed an extensive functional analysis. Our analysis showed that the lethal viruses (MA-ZEBOV and ZEBOV-NP/VP24ma) elicited a strong expression of genes 72 h after infection. In addition, we found that although the host transcriptional response to ZEBOV-VP24ma was nearly the same as that to ZEBOV-NP/VP24ma, the contribution of a mutation in the NP gene was required for a lethal phenotype. Further analysis indicated that one of the most relevant biological functions differentially regulated by the lethal viruses was the inflammatory response, as was the induction of specific metalloproteinases, which were present in our newly identify functional network that was associated with Ebola virus lethality. Our results suggest that this dysregulated proinflammatory response increased the severity of disease. Consequently, the newly discovered molecular signature could be used as the starting point for the development of new drugs and therapeutics. To our knowledge, this is the first study that clearly defines unique molecular signatures associated with Ebola virus lethality. PMID:21734050

  3. Glycan array screening reveals a candidate ligand for Siglec-8.

    PubMed

    Bochner, Bruce S; Alvarez, Richard A; Mehta, Padmaja; Bovin, Nicolai V; Blixt, Ola; White, John R; Schnaar, Ronald L

    2005-02-11

    Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is selectively expressed on human eosinophils, basophils, and mast cells, where it regulates their function and survival. Previous studies demonstrated sialic acid-dependent binding of Siglec-8 but failed to reveal significant substructure specificity or high affinity of that binding. To test a broader range of potential ligands, a Siglec-8-Ig chimeric protein was tested for binding to 172 different glycan structures immobilized as biotinylated glycosides on a 384-well streptavidin-coated plate. Of these, approximately 40 structures were sialylated. Among these, avid binding was detected to a single defined glycan, NeuAcalpha2-3(6-O-sulfo)Galbeta1-4[Fucalpha1-3]GlcNAc, also referred to in the literature as 6'-sulfo-sLex. Notably, neither unsulfated sLex (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc) nor an isomer with the sulfate on the 6-position of the GlcNAc residue (6-sulfo-sLex, NeuAcalpha2-3Galbeta1-4[Fucalpha1-3](6-O-sulfo)GlcNAc) supported detectable binding. Subsequent secondary screening was performed using surface plasmon resonance. Biotin glycosides immobilized on streptavidin biosensor chips were exposed to Siglec-8-Ig in solution. Whereas surfaces derivatized with sLex and 6-sulfo-sLex failed to support detectable Siglec-8 binding, 6'-sulfo-sLex supported significant binding with a Kd of 2.3 microm. In a separate test of binding specificity, aminopropyl glycosides were covalently immobilized at different concentrations on activated (N-hydroxysuccinimidyl) glass surfaces (Schott-Nexterion Slide H). Subsequent exposure to Siglec-8-Ig precomplexed with fluorescein isothiocyanate anti-human Fc resulted in fluorescent signals at immobilized concentrations of 6'-sulfo-sLex of <5 pmol/spot. In contrast, sLex and 6-sulfo-sLex did not support any Siglec-8 binding at the highest concentration tested (300 pmol/spot). We conclude that Siglec-8 binds preferentially to the sLex structure bearing an additional sulfate ester on the galactose 6-hydroxyl. PMID:15563466

  4. A novel system of fertility rescue in Drosophila hybrids reveals a link between hybrid lethality and female sterility.

    PubMed Central

    Barbash, Daniel A; Ashburner, Michael

    2003-01-01

    Hybrid daughters of crosses between Drosophila melanogaster females and males from the D. simulans species clade are fully viable at low temperature but have agametic ovaries and are thus sterile. We report here that mutations in the D. melanogaster gene Hybrid male rescue (Hmr), along with unidentified polymorphic factors, rescue this agametic phenotype in both D. melanogaster/D. simulans and D. melanogaster/D. mauritiana F(1) female hybrids. These hybrids produced small numbers of progeny in backcrosses, their low fecundity being caused by incomplete rescue of oogenesis as well as by zygotic lethality. F(1) hybrid males from these crosses remained fully sterile. Hmr(+) is the first Drosophila gene shown to cause hybrid female sterility. These results also suggest that, while there is some common genetic basis to hybrid lethality and female sterility in D. melanogaster, hybrid females are more sensitive to fertility defects than to lethality. PMID:12586709

  5. Colonization of America by Drosophila subobscura: Heterotic effect of chromosomal arrangements revealed by the persistence of lethal genes

    PubMed Central

    Mestres, F.; Balanyà, J.; Arenas, C.; Solé, E.; Serra, L.

    2001-01-01

    About 20 years ago Drosophila subobscura, a native Palearctic species, colonized both North and South America. In Palearctic populations lethal genes are not associated in general with particular chromosomal arrangements. In colonizing populations they are not randomly distributed and usually are associated to a different degree with chromosomal arrangements caused by the founder event. The persistence of two lethal genes in the colonizing populations, one completely associated with the O5 inversion and the other partially associated with the O3+4+7 arrangement, has been analyzed. In all populations studied (five North American and six South American) the observed frequency of the lethal gene completely associated with the O5 inversion is higher than expected, the difference being statistically significant in all South American and one North American populations. The observed frequency of the lethal gene partially associated with the O3+4+7 arrangement is also significantly higher than expected. Taking into account that the O5 inversion exhibits significant latitudinal clines both in North and South America, an overdominant model favoring the heterokaryotypes seems to be in operation. From this model, a polynomial expression has been developed that allows us to estimate the relative fitness and the coefficient of selection against all karyotypes not carrying the O5 inversion. The relative fitness of the O5 heterokaryotypes is higher in South American than in North American populations. Furthermore, the observed frequencies of the lethal genes studied are in general very close to those of the equilibrium. This case is an outstanding demonstration in nature of an heterotic effect of chromosomal segments associated with lethal genes on a large geographic scale. PMID:11470907

  6. 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 detail the rapid evolution of snake venoms. We found that the difference in venom properties resulted from major changes in expression levels of toxin gene families, differential gene-family expansion and loss, changes in which paralogs within gene families were expressed at high levels, and higher nonsynonymous substitution rates in the toxin genes relative to nontoxins. These massive alterations in the genetics of the venom phenotype emphasize the evolutionary lability and flexibility of this ecologically critical trait. PMID:23758969

  7. 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

  8. 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

  9. 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

  10. Lethal laryngopyocele.

    PubMed

    Byard, Roger W; Gilbert, John D

    2015-03-01

    A 44-year-old man collapsed after complaining of difficulty breathing. Layer dissection of the neck at autopsy revealed a large mixed internal and external laryngopyocele occluding the upper airway. It contained 30 mls of yellow-gray pus. Mechanisms of death in laryngoceles involve obstruction of the opening into the larynx resulting in accumulation of mucus or air within the sac causing airway occlusion. Once infection supervenes, deaths in laryngopyocoeles result either from accumulated pus causing airway occlusion from a mass effect (as in the current case) or the discharge of pus into the airway causing death from aspiration. Sudden death in laryngopyoceles is a very rare event that requires careful dissection at autopsy to demonstrate the characteristics of the underlying lesion and the possible mechanism of death. Laryngopyocele should be considered in the differential diagnosis of natural conditions causing acute, potentially lethal, upper airway obstruction. PMID:25556289

  11. Small Molecule Screen Reveals Regulation of Survival Motor Neuron Protein Abundance by Ras Proteins

    E-print Network

    Stockwell, Brent R.

    Small Molecule Screen Reveals Regulation of Survival Motor Neuron Protein Abundance by Ras Proteins States *S Supporting Information ABSTRACT: Small molecule modulators of protein activity have proven are relatively common, small molecules that can increase protein abundance are rare. Small molecule protein

  12. Research paper Screen of FDA-approved drug library reveals compounds that protect hair cells

    E-print Network

    Rubel, Edwin

    Research paper Screen of FDA-approved drug library reveals compounds that protect hair cells from Accepted 9 August 2012 Available online xxx a b s t r a c t Loss of mechanosensory hair cells in the inner cause hair cell death as a side effect. These include amino- glycoside antibiotics, such as neomycin

  13. RNAi Screen in Drosophila Cells Reveals the Involvement of the Tom Complex

    E-print Network

    Paris-Sud XI, Université de

    RNAi Screen in Drosophila Cells Reveals the Involvement of the Tom Complex in Chlamydia Infection developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells

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

    PubMed

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

    2015-01-16

    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 ?g 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. NCI: SBIR & STTR - Find Funding - Contracts - 290 siRNA Resource for Synthetic Lethal Screening of DNA Repair and Damage Signaling Networks

    Cancer.gov

    Human cancers appear to be highly and differentially vulnerable to targeted attack on individual gene expression within their DNA repair networks, especially those pathways that are closely associated with DNA replication. However such lethal combinations (i.e., synthetic lethals) are generally not obvious or easily predicted a priori.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24652767

  18. A chemical genetic screen reveals a resistance mechanism to PI3K inhibitors in cancer

    PubMed Central

    Muellner, Markus K; Uras, Iris Z; Gapp, Bianca V; Kerzendorfer, Claudia; Smida, Michal; Lechtermann, Hannelore; Craig-Mueller, Nils; Colinge, Jacques; Duernberger, Gerhard; Nijman, Sebastian MB

    2011-01-01

    Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. We establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to PI3K inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC overrode the dependency of cells on the PI3K/mTOR pathway for proliferation. These data reveal a novel mechanism of resistance to PI3K inhibitors with direct clinical implications. PMID:21946274

  19. A zebrafish model of lethal congenital contracture syndrome 1 reveals Gle1 function in spinal neural precursor survival and motor axon arborization.

    PubMed

    Jao, Li-En; Appel, Bruce; Wente, Susan R

    2012-04-01

    In humans, GLE1 is mutated in lethal congenital contracture syndrome 1 (LCCS1) leading to prenatal death of all affected fetuses. Although the molecular roles of Gle1 in nuclear mRNA export and translation have been documented, no animal models for this disease have been reported. To elucidate the function of Gle1 in vertebrate development, we used the zebrafish (Danio rerio) model system. gle1 mRNA is maternally deposited and widely expressed. Altering Gle1 using an insertional mutant or antisense morpholinos results in multiple defects, including immobility, small eyes, diminished pharyngeal arches, curved body axis, edema, underdeveloped intestine and cell death in the central nervous system. These phenotypes parallel those observed in LCCS1 human fetuses. Gle1 depletion also results in reduction of motoneurons and aberrant arborization of motor axons. Unexpectedly, the motoneuron deficiency results from apoptosis of neural precursors, not of differentiated motoneurons. Mosaic analyses further indicate that Gle1 activity is required extrinsically in the environment for normal motor axon arborization. Importantly, the zebrafish phenotypes caused by Gle1 deficiency are only rescued by expressing wild-type human GLE1 and not by the disease-linked Fin(Major) mutant form of GLE1. Together, our studies provide the first functional characterization of Gle1 in vertebrate development and reveal its essential role in actively dividing cells. We propose that defective GLE1 function in human LCCS1 results in both neurogenic and non-neurogenic defects linked to the apoptosis of proliferative organ precursors. PMID:22357925

  20. A zebrafish model of lethal congenital contracture syndrome 1 reveals Gle1 function in spinal neural precursor survival and motor axon arborization

    PubMed Central

    Jao, Li-En; Appel, Bruce; Wente, Susan R.

    2012-01-01

    In humans, GLE1 is mutated in lethal congenital contracture syndrome 1 (LCCS1) leading to prenatal death of all affected fetuses. Although the molecular roles of Gle1 in nuclear mRNA export and translation have been documented, no animal models for this disease have been reported. To elucidate the function of Gle1 in vertebrate development, we used the zebrafish (Danio rerio) model system. gle1 mRNA is maternally deposited and widely expressed. Altering Gle1 using an insertional mutant or antisense morpholinos results in multiple defects, including immobility, small eyes, diminished pharyngeal arches, curved body axis, edema, underdeveloped intestine and cell death in the central nervous system. These phenotypes parallel those observed in LCCS1 human fetuses. Gle1 depletion also results in reduction of motoneurons and aberrant arborization of motor axons. Unexpectedly, the motoneuron deficiency results from apoptosis of neural precursors, not of differentiated motoneurons. Mosaic analyses further indicate that Gle1 activity is required extrinsically in the environment for normal motor axon arborization. Importantly, the zebrafish phenotypes caused by Gle1 deficiency are only rescued by expressing wild-type human GLE1 and not by the disease-linked FinMajor mutant form of GLE1. Together, our studies provide the first functional characterization of Gle1 in vertebrate development and reveal its essential role in actively dividing cells. We propose that defective GLE1 function in human LCCS1 results in both neurogenic and non-neurogenic defects linked to the apoptosis of proliferative organ precursors. PMID:22357925

  1. Imaging-based chemical screening reveals activity-dependent neural differentiation of pluripotent stem cells.

    PubMed

    Sun, Yaping; Dong, Zhiqiang; Jin, Taihao; Ang, Kean-Hooi; Huang, Miller; Haston, Kelly M; Peng, Jisong; Zhong, Tao P; Finkbeiner, Steven; Weiss, William A; Arkin, Michelle R; Jan, Lily Y; Guo, Su

    2013-01-01

    Mammalian pluripotent stem cells (PSCs) represent an important venue for understanding basic principles regulating tissue-specific differentiation and discovering new tools that may facilitate clinical applications. Mechanisms that direct neural differentiation of PSCs involve growth factor signaling and transcription regulation. However, it is unknown whether and how electrical activity influences this process. Here we report a high throughput imaging-based screen, which uncovers that selamectin, an anti-helminthic therapeutic compound with reported activity on invertebrate glutamate-gated chloride channels, promotes neural differentiation of PSCs. We show that selamectin's pro-neurogenic activity is mediated by ?2-containing GABAA receptors in subsets of neural rosette progenitors, accompanied by increased proneural and lineage-specific transcription factor expression and cell cycle exit. In vivo, selamectin promotes neurogenesis in developing zebrafish. Our results establish a chemical screening platform that reveals activity-dependent neural differentiation from PSCs. Compounds identified in this and future screening might prove therapeutically beneficial for treating neurodevelopmental or neurodegenerative disorders. DOI:http://dx.doi.org/10.7554/eLife.00508.001. PMID:24040509

  2. Anthrax lethal factor inhibition

    PubMed Central

    Shoop, W. L.; Xiong, Y.; Wiltsie, J.; Woods, A.; Guo, J.; Pivnichny, J. V.; Felcetto, T.; Michael, B. F.; Bansal, A.; Cummings, R. T.; Cunningham, B. R.; Friedlander, A. M.; Douglas, C. M.; Patel, S. B.; Wisniewski, D.; Scapin, G.; Salowe, S. P.; Zaller, D. M.; Chapman, K. T.; Scolnick, E. M.; Schmatz, D. M.; Bartizal, K.; MacCoss, M.; Hermes, J. D.

    2005-01-01

    The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) ?50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit “point of no return” model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax. PMID:15911756

  3. High incidence of later-onset fabry disease revealed by newborn screening.

    PubMed

    Spada, Marco; Pagliardini, Severo; Yasuda, Makiko; Tukel, Turgut; Thiagarajan, Geetha; Sakuraba, Hitoshi; Ponzone, Alberto; Desnick, Robert J

    2006-07-01

    The classic phenotype of Fabry disease, X-linked alpha -galactosidase A (alpha -Gal A) deficiency, has an estimated incidence of approximately 1 in 50,000 males. The recent recognition of later-onset variants suggested that this treatable lysosomal disease is more frequent. To determine the disease incidence, we undertook newborn screening by assaying the alpha-Gal A activity in blood spots from 37,104 consecutive Italian male neonates. Enzyme-deficient infants were retested, and "doubly screened-positive" infants and their relatives were diagnostically confirmed by enzyme and mutation analyses. Twelve (0.03%) neonates had deficient alpha-Gal A activities and specific mutations, including four novel missense mutations (M51I, E66G, A73V, and R118C), three missense mutations (F113L, A143T, and N215S) identified previously in later-onset patients, and one splicing defect (IVS5(+1G-->T)) reported in a patient with the classic phenotype. Molecular modeling and in vitro overexpression of the missense mutations demonstrated structures and residual activities, which were rescued/enhanced by an alpha-Gal A-specific pharmacologic chaperone, consistent with mutations that cause the later-onset phenotype. Family studies revealed undiagnosed Fabry disease in affected individuals. In this population, the incidence of alpha-Gal A deficiency was 1 in approximately 3,100, with an 11 : 1 ratio of patients with the later-onset : classic phenotypes. If only known disease-causing mutations were included, the incidence would be 1 in approximately 4,600, with a 7 : 1 ratio of patients with the later-onset : classic phenotypes. These results suggest that the later-onset phenotype of Fabry disease is underdiagnosed among males with cardiac, cerebrovascular, and/or renal disease. Recognition of these patients would permit family screening and earlier therapeutic intervention. However, the higher incidence of the later-onset phenotype in patients raises ethical issues related to when screening should be performed--in the neonatal period or at early maturity, perhaps in conjunction with screening for other treatable adult-onset disorders. PMID:16773563

  4. Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology.

    PubMed

    Hulsman, Marc; Hulshof, Frits; Unadkat, Hemant; Papenburg, Bernke J; Stamatialis, Dimitrios F; Truckenmüller, Roman; van Blitterswijk, Clemens; de Boer, Jan; Reinders, Marcel J T

    2015-03-01

    Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces. PMID:25554402

  5. 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

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

    PubMed

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

    2014-03-01

    Pancreatic ?-cell death plays a role in both type 1 and type 2 diabetes, but clinical treatments that specifically target ?-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 ?-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-?, 10 ng/mL IL-1?, and 10 ng/mL IFN-?). 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 ?-cells has not been well studied. We analyzed the expression and characteristics of Na(+) currents in mature ?-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

  7. A Genomewide Screen for Suppressors of Alu-Mediated Rearrangements Reveals a Role for PIF1

    PubMed Central

    Chisholm, Karen M.; Aubert, Sarah D.; Freese, Krister P.; Zakian, Virginia A.; King, Mary-Claire; Welcsh, Piri L.

    2012-01-01

    Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition. PMID:22347400

  8. Functional genetic screen of human diversity reveals that a methionine salvage enzyme regulates inflammatory cell death.

    PubMed

    Ko, Dennis C; Gamazon, Eric R; Shukla, Kajal P; Pfuetzner, Richard A; Whittington, Dale; Holden, Tarah D; Brittnacher, Mitchell J; Fong, Christine; Radey, Matthew; Ogohara, Cassandra; Stark, Amy L; Akey, Joshua M; Dolan, M Eileen; Wurfel, Mark M; Miller, Samuel I

    2012-08-28

    Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1-mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5'-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology. PMID:22837397

  9. What to Do if Your Baby's Screening Reveals a Possible Hearing Problem

    MedlinePLUS

    ... the development of speech and language skills. Universal newborn hearing screening programs currently operate in every state ... hospitals and birthing centers throughout the country screen newborns for hearing loss. If you and your baby ...

  10. A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity.

    PubMed

    Chia, Na-Yu; Chan, Yun-Shen; Feng, Bo; Lu, Xinyi; Orlov, Yuriy L; Moreau, Dimitri; Kumar, Pankaj; Yang, Lin; Jiang, Jianming; Lau, Mei-Sheng; Huss, Mikael; Soh, Boon-Seng; Kraus, Petra; Li, Pin; Lufkin, Thomas; Lim, Bing; Clarke, Neil D; Bard, Frederic; Ng, Huck-Hui

    2010-11-11

    The derivation of human ES cells (hESCs) from human blastocysts represents one of the milestones in stem cell biology. The full potential of hESCs in research and clinical applications requires a detailed understanding of the genetic network that governs the unique properties of hESCs. Here, we report a genome-wide RNA interference screen to identify genes which regulate self-renewal and pluripotency properties in hESCs. Interestingly, functionally distinct complexes involved in transcriptional regulation and chromatin remodelling are among the factors identified in the screen. To understand the roles of these potential regulators of hESCs, we studied transcription factor PRDM14 to gain new insights into its functional roles in the regulation of pluripotency. We showed that PRDM14 regulates directly the expression of key pluripotency gene POU5F1 through its proximal enhancer. Genome-wide location profiling experiments revealed that PRDM14 colocalized extensively with other key transcription factors such as OCT4, NANOG and SOX2, indicating that PRDM14 is integrated into the core transcriptional regulatory network. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells. PMID:20953172

  11. 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

    Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin–dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be “purified” via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin–dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome’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

  12. 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

    Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome'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

  13. A Genetic Screen and Transcript Profiling Reveal a Shared Regulatory Program for Drosophila Linker Histone H1 and Chromatin Remodeler CHD1

    PubMed Central

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

    2015-01-01

    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. PMID:25628309

  14. A Targeted In Vivo RNAi Screen Reveals Deubiquitinases as New Regulators of Notch Signaling

    PubMed Central

    Zhang, Junzheng; Liu, Min; Su, Ying; Du, Juan; Zhu, Alan Jian

    2012-01-01

    Notch signaling is highly conserved in all metazoan animals and plays critical roles in cell fate specification, cell proliferation, apoptosis, and stem cell maintenance. Although core components of the Notch signaling cascade have been identified, many gaps in the understanding of the Notch signaling pathway remain to be filled. One form of posttranslational regulation, which is controlled by the ubiquitin-proteasome system, is known to modulate Notch signaling. The ubiquitination pathway is a highly coordinated process in which the ubiquitin moiety is either conjugated to or removed from target proteins by opposing E3 ubiquitin ligases and deubiquitinases (DUBs). Several E3 ubiquitin ligases have been implicated in ubiquitin conjugation to the receptors and the ligands of the Notch signaling cascade. In contrast, little is known about a direct role of DUBs in Notch signaling in vivo. Here, we report an in vivo RNA interference screen in Drosophila melanogaster targeting all 45 DUBs that we annotated in the fly genome. We show that at least four DUBs function specifically in the formation of the fly wing margin and/or the specification of the scutellar sensory organ precursors, two processes that are strictly dependent on the balanced Notch signaling activity. Furthermore, we provide genetic evidence suggesting that these DUBs are necessary to positively modulate Notch signaling activity. Our study reveals a conserved molecular mechanism by which protein deubiquitination process contributes to the complex posttranslational regulation of Notch signaling in vivo. PMID:23275879

  15. A single-cell imaging screen reveals multiple effects of secreted small molecules on bacteria

    PubMed Central

    Salje, Jeanne

    2014-01-01

    Bacteria cells exist in close proximity to other cells of both the same and different species. Bacteria secrete a large number of different chemical species, and the local concentrations of these compounds at the surfaces of nearby cells may reach very high levels. It is fascinating to imagine how individual cells might sense and respond to the complex mix of signals at their surface. However, it is difficult to measure exactly what the local environmental composition looks like, or what the effects of individual compounds on nearby cells are. Here, an electron microscopy imaging screen was designed that would detect morphological changes induced by secreted small molecules. This differs from conventional approaches by detecting structural changes in individual cells rather than gene expression or growth rate changes at the population level. For example, one of the changes detected here was an increase in outer membrane vesicle production, which does not necessarily correspond to a change in gene expression. This initial study focussed on Pseudomonas aeruginosa, Escherichia coli, and Burkholderia dolosa, and revealed an intriguing range of effects of secreted small molecules on cells both within and between species. PMID:24910069

  16. Zebrafish Chemical Screening Reveals the Impairment of Dopaminergic Neuronal Survival by Cardiac Glycosides

    PubMed Central

    Sun, Yaping; Dong, Zhiqiang; Khodabakhsh, Hadie; Chatterjee, Sandip; Guo, Su

    2012-01-01

    Parkinson's disease is a neurodegenerative disorder characterized by the prominent degeneration of dopaminergic (DA) neurons among other cell types. Here we report a first chemical screen of over 5,000 compounds in zebrafish, aimed at identifying small molecule modulators of DA neuron development or survival. We find that Neriifolin, a member of the cardiac glycoside family of compounds, impairs survival but not differentiation of both zebrafish and mammalian DA neurons. Cardiac glycosides are inhibitors of Na+/K+ ATPase activity and widely used for treating heart disorders. Our data suggest that Neriifolin impairs DA neuronal survival by targeting the neuronal enriched Na+/K+ ATPase ?3 subunit (ATP1A3). Modulation of ionic homeostasis, knockdown of p53, or treatment with antioxidants protects DA neurons from Neriifolin-induced death. These results reveal a previously unknown effect of cardiac glycosides on DA neuronal survival and suggest that it is mediated through ATP1A3 inhibition, oxidative stress, and p53. They also elucidate potential approaches for counteracting the neurotoxicity of this valuable class of medications. PMID:22563390

  17. Comparative drug pair screening across multiple glioblastoma cell lines reveals novel drug-drug interactions

    PubMed Central

    Schmidt, Linnéa; Kling, Teresia; Monsefi, Naser; Olsson, Maja; Hansson, Caroline; Baskaran, Sathishkumar; Lundgren, Bo; Martens, Ulf; Häggblad, Maria; Westermark, Bengt; Forsberg Nilsson, Karin; Uhrbom, Lene; Karlsson-Lindahl, Linda; Gerlee, Philip; Nelander, Sven

    2013-01-01

    Background Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults, and despite state-of-the-art treatment, survival remains poor and novel therapeutics are sorely needed. The aim of the present study was to identify new synergistic drug pairs for GBM. In addition, we aimed to explore differences in drug-drug interactions across multiple GBM-derived cell cultures and predict such differences by use of transcriptional biomarkers. Methods We performed a screen in which we quantified drug-drug interactions for 465 drug pairs in each of the 5 GBM cell lines U87MG, U343MG, U373MG, A172, and T98G. Selected interactions were further tested using isobole-based analysis and validated in 5 glioma-initiating cell cultures. Furthermore, drug interactions were predicted using microarray-based transcriptional profiling in combination with statistical modeling. Results Of the 5 × 465 drug pairs, we could define a subset of drug pairs with strong interaction in both standard cell lines and glioma-initiating cell cultures. In particular, a subset of pairs involving the pharmaceutical compounds rimcazole, sertraline, pterostilbene, and gefitinib showed a strong interaction in a majority of the cell cultures tested. Statistical modeling of microarray and interaction data using sparse canonical correlation analysis revealed several predictive biomarkers, which we propose could be of importance in regulating drug pair responses. Conclusion We identify novel candidate drug pairs for GBM and suggest possibilities to prospectively use transcriptional biomarkers to predict drug interactions in individual cases. PMID:24101737

  18. Manipulating individual decisions and environmental conditions reveal individual quality in decision-making and non-lethal costs of predation risk.

    PubMed

    Thomson, Robert L; Tomás, Gustavo; Forsman, Jukka T; Mönkkönen, Mikko

    2012-01-01

    Habitat selection is a crucial decision for any organism. Selecting a high quality site will positively impact survival and reproductive output. Predation risk is an important component of habitat quality that is known to impact reproductive success and individual condition. However, separating the breeding consequences of decision-making of wild animals from individual quality is difficult. Individuals face reproductive decisions that often vary with quality such that low quality individuals invest less. This reduced reproductive performance could appear a cost of increased risk but may simply reflect lower quality. Thus, teasing apart the effects of individual quality and the effect of predation risk is vital to understand the physiological and reproductive costs of predation risk alone on breeding animals. In this study we alter the actual territory location decisions of pied flycatchers by moving active nests relative to breeding sparrowhawks, the main predators of adult flycatchers. We experimentally measure the non-lethal effects of predation on adults and offspring while controlling for effects of parental quality, individual territory choice and initiation of breeding. We found that chicks from high predation risk nests (<50 m of hawk) were significantly smaller than chicks from low risk nests (>200 m from hawk). However, in contrast to correlative results, females in manipulated high risk nests did not suffer decreased body condition or increased stress response (HSP60 and HSP70). Our results suggest that territory location decisions relative to breeding avian predators cause spatial gradients in individual quality. Small adjustments in territory location decisions have crucial consequences and our results confirm non-lethal costs of predation risk that were expressed in terms of smaller offspring produced. However, females did not show costs in physiological condition which suggests that part of the costs incurred by adults exposed to predation risk are quality determined. PMID:23272226

  19. 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

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

    E-print Network

    Sabatini, David M.

    Cell microarrays are a promising tool for performing large-scale functional genomic screening in mammalian cells at reasonable cost, but owing to technical limitations they have been restricted for use with a narrow range ...

  1. Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds

    E-print Network

    Paris-Sud XI, Université de

    ), Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 2 Screening Technology & Pharmacology Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 3 Active Compound Space Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea, 4 Image Mining Group, Institut Pasteur Korea

  2. A Zebrafish Compound Screen Reveals Modulation of Neutrophil Reverse Migration as an Anti-Inflammatory Mechanism

    PubMed Central

    Robertson, Anne L.; Holmes, Geoffrey R.; Bojarczuk, Aleksandra N.; Burgon, Joseph; Loynes, Catherine A.; Chimen, Myriam; Sawtell, Amy K.; Hamza, Bashar; Willson, Joseph; Walmsley, Sarah R.; Anderson, Sean R.; Coles, Mark C.; Farrow, Stuart N.; Solari, Roberto; Jones, Simon; Prince, Lynne R.; Irimia, Daniel; Rainger, G. Ed; Kadirkamanathan, Visakan; Whyte, Moira K. B.; Renshaw, Stephen A.

    2014-01-01

    Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy. PMID:24574340

  3. Phenotypic Screening Reveals Topoisomerase I as a Breast Cancer Stem Cell Therapeutic Target

    PubMed Central

    Zhang, Fang; Rothermund, Kristi; Gangadharan, Sajithlal B.; Pommier, Yves; Prochownik, Edward V.; Lazo, John S.

    2012-01-01

    Cancer stem cells (CSCs) are a subpopulation generally thought to be responsible for cancer initiation and progression. Because CSCs are often rare in the total tumor cell population and differentiate rapidly when grown in culture, it has been challenging to uncover compounds that selectively target CSCs. We previously described CSC-emulating cells derived from breast cancer cell lines that maintained a stable undifferentiated state. We optimized a phenotypic assay with these cells and screened 1,280-bioactive compounds, identifying five that preferentially inhibited CSC-like cell proliferation. Using a compound-guided target identification approach, we found high topoisomerase I (Topo I) expression levels in breast CSC-like cells and primary breast CSCs. Structurally unrelated small molecules targeting Topo I preferentially inhibited CSC-like cells. These results illustrate the substantial power of this CSC phenotypic screening platform and promote Topo I as a potential molecular therapeutic target for therapies aimed at expunging CSCs. PMID:22948175

  4. 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. PMID:23124746

  5. Systematic combination screening reveals synergism between rapamycin and sunitinib against human lung cancer.

    PubMed

    Li, Xian; Tong, Lin-Jiang; Ding, Jian; Meng, Ling-Hua

    2014-01-01

    Mammalian target of rapamycin (mTOR) acts as a hub integrating signals from nutrient availability and growth factors and plays central roles in regulating protein synthesis and cell growth, which has been validated as a promising target for cancer therapy. Rapamycin and its analogues have emerged as the first generation of mTOR inhibitors, but their efficacy is modest in clinical settings. Combinatorial use of rapamycin with other drugs is a promising strategy to improve its anticancer activity. Here we developed an unbiased systematic binary screening platform aiming to discover new remedy for rapamycin-based cancer therapy. We found that sunitinib emerged as one of the clinically available anticancer drugs screened that displayed significant synergy with rapamycin in NSCLC cells. Combination of rapamycin with sunitinib resulted in enhanced cell cycle arrest in G1 phase, which was accompanied with enhanced suppression of mTOR signaling and disruption of the negative feedback loop that activate AKT upon mTORC1 inhibition. Furthermore, sunitinib and rapamycin displayed synergistic activity against tube formation by human microvessel endothelial cells as well as outgrowth of endothelial tubes and microvessels both in vitro and in vivo, which is associated with down-regulation of VEGF secretion and HIF1? expression. Our study demonstrated that new combinatorial regimen could be identified via systematic drug combination screening and established a mechanistic rationale for a combination approach using rapalogs and sunitinib in the treatment of human NSCLC. PMID:24018642

  6. Synthetic protection short interfering RNA screen reveals glyburide as a novel radioprotector.

    PubMed

    Jiang, Jianfei; McDonald, Peter R; Dixon, Tracy M; Franicola, Darcy; Zhang, Xichen; Nie, Suhua; Epperly, Laura D; Huang, Zhentai; Kagan, Valerian E; Lazo, John S; Epperly, Michael W; Greenberger, Joel S

    2009-10-01

    To assist in screening existing drugs for use as potential radioprotectors, we used a human unbiased 16,560 short interfering RNA (siRNA) library targeting the druggable genome. We performed a synthetic protection screen that was designed to identify genes that, when silenced, protected human glioblastoma T98G cells from gamma-radiation-induced cell death. We identified 116 candidate protective genes, then identified 10 small molecule inhibitors of 13 of these candidate gene products and tested their radioprotective effects. Glyburide, a clinically used second-generation hypoglycemic drug, effectively decreased radiation-induced cell death in several cell lines including T98G, glioblastoma U-87 MG, and normal lung epithelial BEAS-2B and in primary cultures of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective in vivo (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, P = 0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors. PMID:19772462

  7. A high-throughput phenotypic screen of cytotoxic T lymphocyte lytic granule exocytosis reveals candidate immunosuppressants.

    PubMed

    Zhao, Ziyan; Haynes, Mark K; Ursu, Oleg; Edwards, Bruce S; Sklar, Larry A; Zweifach, Adam

    2015-03-01

    We screened the National Institutes of Health's Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364,202 compounds in a 1536-well plate format, we identified 2404 initial hits: 161 were confirmed on retesting, and dose-response measurements were performed. Seventy-five of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of extracellular signal-regulated kinase (ERK) by upstream mitogen-activated protein kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For 8 compounds, we were unable to determine an MMOA. The activity of 1 of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOAs. PMID:25381253

  8. A Whole Cell Pathway Screen Reveals Seven Novel Chemosensitizers to Combat Chloroquine Resistant Malaria

    PubMed Central

    Ch'ng, Jun-Hong; Mok, Sachel; Bozdech, Zbynek; Lear, Martin James; Boudhar, Aicha; Russell, Bruce; Nosten, Francois; Tan, Kevin Shyong-Wei

    2013-01-01

    Due to the widespread prevalence of resistant parasites, chloroquine (CQ) was removed from front-line antimalarial chemotherapy in the 1990s despite its initial promise of disease eradication. Since then, resistance-conferring mutations have been identified in transporters such as the PfCRT, that allow for the efflux of CQ from its primary site of action, the parasite digestive vacuole. Chemosensitizing/chemoreversing compounds interfere with the function of these transporters thereby sensitizing parasites to CQ once again. However, compounds identified thus far have disappointing in vivo efficacy and screening for alternative candidates is required to revive this strategy. In this study, we propose a simple and direct means to rapidly screen for such compounds using a fluorescent-tagged CQ molecule. When this screen was applied to a small library, seven novel chemosensitizers (octoclothepin, methiothepin, metergoline, loperamide, chlorprothixene, L-703,606 and mibefradil) were quickly elucidated, including two which showed greater potency than the classical chemosensitizers verapamil and desipramine. PMID:23615863

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

    PubMed Central

    Servien?, Elena; Lukša, Juliana; Orentait?, 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

  10. Revealing molecular mechanisms by integrating high-dimensional functional screens with protein interaction data.

    PubMed

    Simeone, Angela; Marsico, Giovanni; Collinet, Claudio; Galvez, Thierry; Kalaidzidis, Yannis; Zerial, Marino; Beyer, Andreas

    2014-09-01

    Functional genomics screens using multi-parametric assays are powerful approaches for identifying genes involved in particular cellular processes. However, they suffer from problems like noise, and often provide little insight into molecular mechanisms. A bottleneck for addressing these issues is the lack of computational methods for the systematic integration of multi-parametric phenotypic datasets with molecular interactions. Here, we present Integrative Multi Profile Analysis of Cellular Traits (IMPACT). The main goal of IMPACT is to identify the most consistent phenotypic profile among interacting genes. This approach utilizes two types of external information: sets of related genes (IMPACT-sets) and network information (IMPACT-modules). Based on the notion that interacting genes are more likely to be involved in similar functions than non-interacting genes, this data is used as a prior to inform the filtering of phenotypic profiles that are similar among interacting genes. IMPACT-sets selects the most frequent profile among a set of related genes. IMPACT-modules identifies sub-networks containing genes with similar phenotype profiles. The statistical significance of these selections is subsequently quantified via permutations of the data. IMPACT (1) handles multiple profiles per gene, (2) rescues genes with weak phenotypes and (3) accounts for multiple biases e.g. caused by the network topology. Application to a genome-wide RNAi screen on endocytosis showed that IMPACT improved the recovery of known endocytosis-related genes, decreased off-target effects, and detected consistent phenotypes. Those findings were confirmed by rescreening 468 genes. Additionally we validated an unexpected influence of the IGF-receptor on EGF-endocytosis. IMPACT facilitates the selection of high-quality phenotypic profiles using different types of independent information, thereby supporting the molecular interpretation of functional screens. PMID:25188415

  11. Revealing Molecular Mechanisms by Integrating High-Dimensional Functional Screens with Protein Interaction Data

    PubMed Central

    Collinet, Claudio; Galvez, Thierry; Kalaidzidis, Yannis; Zerial, Marino; Beyer, Andreas

    2014-01-01

    Functional genomics screens using multi-parametric assays are powerful approaches for identifying genes involved in particular cellular processes. However, they suffer from problems like noise, and often provide little insight into molecular mechanisms. A bottleneck for addressing these issues is the lack of computational methods for the systematic integration of multi-parametric phenotypic datasets with molecular interactions. Here, we present Integrative Multi Profile Analysis of Cellular Traits (IMPACT). The main goal of IMPACT is to identify the most consistent phenotypic profile among interacting genes. This approach utilizes two types of external information: sets of related genes (IMPACT-sets) and network information (IMPACT-modules). Based on the notion that interacting genes are more likely to be involved in similar functions than non-interacting genes, this data is used as a prior to inform the filtering of phenotypic profiles that are similar among interacting genes. IMPACT-sets selects the most frequent profile among a set of related genes. IMPACT-modules identifies sub-networks containing genes with similar phenotype profiles. The statistical significance of these selections is subsequently quantified via permutations of the data. IMPACT (1) handles multiple profiles per gene, (2) rescues genes with weak phenotypes and (3) accounts for multiple biases e.g. caused by the network topology. Application to a genome-wide RNAi screen on endocytosis showed that IMPACT improved the recovery of known endocytosis-related genes, decreased off-target effects, and detected consistent phenotypes. Those findings were confirmed by rescreening 468 genes. Additionally we validated an unexpected influence of the IGF-receptor on EGF-endocytosis. IMPACT facilitates the selection of high-quality phenotypic profiles using different types of independent information, thereby supporting the molecular interpretation of functional screens. PMID:25188415

  12. 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. PMID:17179072

  13. Lethal factor VII deficiency due to novel mutations in the F7 promoter: functional analysis reveals disruption of HNF4 binding site.

    PubMed

    Giansily-Blaizot, Muriel; Lopez, Estelle; Viart, Victoria; Chafa, Ouerdia; Tapon-Bretaudière, Jacqueline; Claustres, Mireille; Taulan, Magali

    2012-08-01

    Hereditary factor VII (FVII) deficiency is a rare autosomal recessive disorder. Deleterious mutations that prevent the synthesis of any amount of functional FVII have been associated with life-threatening haemorrhage in neonates. Here we report two infants, of Maghrebian origin, who suffered a fatal spontaneous cerebral haemorrhage. Investigation of the molecular basis for their severe FVII deficiency revealed novel mutations in a homozygous state within the F7 gene promoter: a single nucleotide substitution (c.-65G>C) and a 2bp deletion (c.-60_-59delTT). To determine whether these promoter variants were responsible for the FVII deficiency, computer-assisted sequence analyses were performed. The data predicted a disrupted binding of both HNF4 and COUP-TF transcription factors with each variant. Concordantly, experimental results revealed an altered HNF4-induced transactivation in the promoter mutated variants. The execution of functional tests is critical to ensuring a complete understanding of the effect of any promoter mutant on FVII deficiency. Only then can an accurate molecular diagnosis be made and further genetic counselling and prenatal diagnosis be offered. PMID:22628013

  14. 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

  15. High incidence of lead poisoning revealed by erythrocyte protoporphyrin (EPP) screening in Arabian children.

    PubMed

    Shaltout, A A; Ghawaby, M M; Hassan, M F; Hunt, M C; Fernando, N; Devarajan, L V; Kollberg, H; Guthrie, R

    1985-12-01

    The measurement of erythrocyte protoporphyrin (EPP) has been used in screening infants for undue exposure to lead. The infants were from a high risk area, Al Jahra in Kuwait, and were selected from those attending the emergency department of Al Jahra District Hospital. Dried spots of blood on Guthrie filter paper were mailed to the Central Laboratory of the Children's Hospital in Buffalo, New York. Elevated EPP values (greater than 50 micrograms/dl) were present in 63 (66%) out of 96 tests. Forty-four of the infants with elevated values were investigated further: 16 had blood lead levels above 30 micrograms/dl (1.45 mmol/l) and 12 had evidence of iron deficiency anaemia. Five infants with blood lead levels above 80 micrograms/dl (3.9 mmol/l) needed urgent chelation and in addition an 8-month-old infant with the highest EPP value (478 micrograms/dl) died of acute lead encephalopathy. We conclude that lead poisoning is a serious problem in Al Jahra, Kuwait, and that a comprehensive programme for screening and health education are urgently needed to avoid the irreversible effects of lead toxicity. PMID:2418770

  16. 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

  17. A screening approach reveals the influence of mineral coating morphology on human mesenchymal stem cell differentiation

    PubMed Central

    Choi, Siyoung; Murphy, William L.

    2013-01-01

    “Biomimetic” inorganic coating on biomaterials has been an active area of research with the intention of providing bioactive surfaces that can regulate cell behavior. Previous studies have demonstrated that human mesenchymal stem cell (hMSC) behavior is differentially regulated by physical and chemical properties of inorganic mineral coatings, indicating that modulation of mineral properties has potential importance in regulating hMSC behavior. However, the lack of an efficient experimental context in which to study stem cell behavior on inorganic substrates has made it difficult to systematically study the effects of specific mineral coating parameters on hMSC behavior. In this study, we developed an efficient experimental platform to screen for the effects of mineral coating morphology on hMSC expansion and differentiation. hMSC expansion on mineral coatings was regulated by micro-scale morphology of mineral coatings, with greater expansion on small granule-like coatings when compared to plate-like or net-like coatings. In contrast, hMSC osteogenic differentiation was inversely correlated with cell expansion on mineral coating, indicating that mineral coating morphology was a key parameter regulating hMSC differentiation. The effect of mineral coating morphology on hMSC behavior suggests the utility of this inorganic screening platform to identify optimal coatings for medical devices and bone tissue engineering applications. PMID:23420758

  18. Mass spectrometry screening reveals widespread diversity in trichome specialized metabolites of tomato chromosomal substitution lines

    PubMed Central

    Schilmiller, Anthony; Shi, Feng; Kim, Jeongwoon; Charbonneau, Amanda L; Holmes, Daniel; Daniel Jones, A; Last, Robert L

    2010-01-01

    Glandular secreting trichomes of cultivated tomato (Solanum lycopersicum) and close relatives produce a variety of structurally diverse volatile and non-volatile specialized (‘secondary’) metabolites, including terpenes, flavonoids and acyl sugars. A genetic screen is described here to profile leaf trichome and surface metabolite extracts of nearly isogenic chromosomal substitution lines covering the tomato genome. These lines contain specific regions of the Solanum pennellii LA0716 genome in an otherwise ‘wild-type’ M82 tomato genetic background. Regions that have an impact on the total amount of extractable mono- and sesquiterpenes (IL2-2) or only sesquiterpenes (IL10-3) or specifically influence accumulation of the monoterpene ?-thujene (IL1-3 and IL1-4) were identified using GC-MS. A rapid LC-TOF-MS method was developed and used to identify changes in non-volatile metabolites through non-targeted analysis. Metabolite profiles generated using this approach led to the discovery of introgression lines producing different acyl chain substitutions on acyl sugar metabolites (IL1-3/1-4 and IL8-1/8-1-1), as well as two regions that influence the quantity of acyl sugars (IL5-3 and IL11-3). Chromosomal region 1-1/1-1-3 was found to influence the types of glycoalkaloids that are detected in leaf surface extracts. These results show that direct chemical screening is a powerful way to characterize genetic diversity in trichome specialized metabolism. PMID:20113441

  19. Screening.

    PubMed

    Colombo, Massimo

    2007-09-01

    Screening is the only practical approach for improving the management of hepatocellular carcinoma (HCC) patients, as early detection increases the application of curative treatments. A conference of experts from Japan, USA, and Europe (Barcelona 2005) advised surveillance every six months for patients with chronic liver disease at increased risk of HCC with abdominal US. Whether this approach benefits HCC patients in terms of survival is still uncertain, since available data are retrospective and biased by lead-time factors in the calculation of patient survival. Only one randomized controlled study in China showed the benefit of surveillance for HCC; however, in a population-based setting. Today, clinic-based, randomized studies are unfeasible for ethical reasons. In a cohort of 447 Italian patients with compensated cirrhosis, we compared the survival of HCC patients identified along three consecutive quinquennia of surveillance. HCC developed in 112 patients (3.4% per year) and was the prime cause of death. Forty-six patients (41%) had a single tumor with mean sizes of 3.7, 3.0, and 2.2 cm in the three quinquennia (first vs third, P = 0.0147; second vs third, P = 0.02) and 38(44%) underwent radical therapies. Mortality rates in HCC patients fell from 45% in the first quinquennium to 10% in the third (P = 0.0009), in parallel with a reduction in mortality of treated patients (34, 28, and 5%) (first vs third, P = 0.0024). Cirrhotic patients developing HCC during the last five years of surveillance survived longer than previously, as a consequence of improved management of the tumor and complications of cirrhosis. It remains controversial whether HCC screening is cost-effective, i.e. whether the cost of detection, confirmatory studies, and treatment are outbalanced by the number of life-years gained. In a retrospective study of Italian patients with cirrhosis, there was an incremental cost-efficacy ratio of surveillance vs no surveillance to be $USD 112 993 per liver-year saved. The cost of surveillance was increased by surgery applied to 15 patients with HCC detected during surveillance. PMID:17877477

  20. An ER-Mitochondria Tethering Complex Revealed by a Synthetic Biology Screen

    PubMed Central

    Kornmann, Benoît; Currie, Erin; Collins, Sean R.; Schuldiner, Maya; Nunnari, Jodi; Weissman, Jonathan S.; Walter, Peter

    2010-01-01

    Communication between organelles is an important feature of all eukaryotic cells. To uncover components involved in mitochondria/endoplasmic reticulum (ER) junctions, we screened for mutants that could be complemented by a synthetic protein designed to artificially tether the two organelles. We identified the Mmm1/Mdm10/Mdm12/Mdm34 complex as a molecular tether between ER and mitochondria. The tethering complex was composed of proteins resident of both ER and mitochondria. With the use of genome-wide mapping of genetic interactions, we showed that the components of the tethering complex were functionally connected to phospholipid biosynthesis and calcium-signaling genes. In mutant cells, phospholipid biosynthesis was impaired. The tethering complex localized to discrete foci, suggesting that discrete sites of close apposition between ER and mitochondria facilitate interorganelle calcium and phospholipid exchange. PMID:19556461

  1. Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis

    PubMed Central

    Sivan, Gilad; Martin, Scott E.; Myers, Timothy G.; Buehler, Eugen; Szymczyk, Krysia H.; Ormanoglu, Pinar; Moss, Bernard

    2013-01-01

    Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells. PMID:23401514

  2. Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration

    PubMed Central

    Samara, Chrysanthi; Rohde, Christopher B.; Gilleland, Cody L.; Norton, Stephanie; Haggarty, Stephen J.; Yanik, Mehmet Fatih

    2010-01-01

    Discovery of molecular mechanisms and chemical compounds that enhance neuronal regeneration can lead to development of therapeutics to combat nervous system injuries and neurodegenerative diseases. By combining high-throughput microfluidics and femtosecond laser microsurgery, we demonstrate for the first time large-scale in vivo screens for identification of compounds that affect neurite regeneration. We performed thousands of microsurgeries at single-axon precision in the nematode Caenorhabditis elegans at a rate of 20 seconds per animal. Following surgeries, we exposed the animals to a hand-curated library of approximately one hundred small molecules and identified chemicals that significantly alter neurite regeneration. In particular, we found that the PKC kinase inhibitor staurosporine strongly modulates regeneration in a concentration- and neuronal type-specific manner. Two structurally unrelated PKC inhibitors produce similar effects. We further show that regeneration is significantly enhanced by the PKC activator prostratin. PMID:20937901

  3. 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

  4. Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis.

    PubMed

    Sivan, Gilad; Martin, Scott E; Myers, Timothy G; Buehler, Eugen; Szymczyk, Krysia H; Ormanoglu, Pinar; Moss, Bernard

    2013-02-26

    Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells. PMID:23401514

  5. Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation

    PubMed Central

    Ambegaokar, Surendra S.; Jackson, George R.

    2011-01-01

    A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ?1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-? and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3? is strongly upregulated due to TDP-43 expression, and reduced GSK-3? dosage is also a common suppressor of A?42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation. PMID:21949350

  6. Virtual screening using combinatorial cyclic Peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    PubMed

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human ? thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened. PMID:25668361

  7. Alanine-scanning mutagenesis of WH2 domains of VopF reveals residues important for conferring lethality in a Saccharomyces cerevisiae model.

    PubMed

    Tripathi, Ranjana; Kaithwas, Vikas; Dureja, Chetna; Raychaudhuri, Saumya

    2013-08-01

    VopF, the type III effector molecule, has been implicated in the pathogenesis of non-O1, non-O139 strains of Vibrio cholerae. It is a protein of 530 amino acids, comprises of one formin homology 1-like (FH1-like) domain and three WASP homology 2 (WH2) domains. Previous works have demonstrated that WH2 domains are crucial for VopF function as a modulator of cellular actin homeostasis. Furthermore, domain deletion analysis also suggests that VopF variant constituted with only WH2 domain 3 is more efficient in restricting the growth of budding yeast than its congeners containing either only domain 1 or domain 2. Interestingly, a good degree of sequence diversity is present within each WH2 domain of VopF. In order to ascertain the importance of different amino acids in each WH2 domain, a systemic alanine scanning mutagenesis was employed. Using a yeast model system, the alanine derivatives of each amino acid of WH2 domain 1 and 3 of VopF were examined for growth restricting activity. Taken together, our mutagenesis results reveal the identification of critical residues of WH2 domain 1 and 3 of VopF. PMID:23680644

  8. Functional genomic screen reveals genes involved in lipid-droplet formation and utilization

    PubMed Central

    Guo, Yi; Walther, Tobias C.; Rao, Meghana; Stuurman, Nico; Goshima, Gohta; Terayama, Koji; Wong, Jinny S.; Vale, Ronald D.; Walter, Peter; Farese, Robert V.

    2009-01-01

    Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets1,2 enclosed in a monolayer of phospholipids and associated proteins3,4. These dynamic organelles5 serve as the principal reservoirs for storing cellular energy and for the building blocks for membrane lipids. Excessive lipid accumulation in cells is a central feature of obesity, diabetes and atherosclerosis, yet remarkably little is known about lipid-droplet cell biology. Here we show, by means of a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells that about 1.5% of all genes function in lipid-droplet formation and regulation. The phenotypes of the gene knockdowns sorted into five distinct phenotypic classes. Genes encoding enzymes of phospholipid biosynthesis proved to be determinants of lipid-droplet size and number, suggesting that the phospholipid composition of the monolayer profoundly affects droplet morphology and lipid utilization. A subset of the Arf1–COPI vesicular transport proteins also regulated droplet morphology and lipid utilization, thereby identifying a previously unrecognized function for this machinery. These phenotypes are conserved in mammalian cells, suggesting that insights from these studies are likely to be central to our understanding of human diseases involving excessive lipid storage. PMID:18408709

  9. Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

    PubMed Central

    Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T

    2014-01-01

    The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. PMID:24952591

  10. Chemoecological Screening Reveals High Bioactivity in Diverse Culturable Portuguese Marine Cyanobacteria

    PubMed Central

    Leão, Pedro N.; Ramos, Vitor; Gonçalves, Patrício B.; Viana, Flávia; Lage, Olga M.; Gerwick, William H.; Vasconcelos, Vitor M.

    2013-01-01

    Marine cyanobacteria, notably those from tropical regions, are a rich source of bioactive secondary metabolites. Tropical marine cyanobacteria often grow to high densities in the environment, allowing direct isolation of many secondary metabolites from field-collected material. However, in temperate environments culturing is usually required to produce enough biomass for investigations of their chemical constituents. In this work, we cultured a selection of novel and diverse cyanobacteria isolated from the Portuguese coast, and tested their organic extracts in a series of ecologically-relevant bioassays. The majority of the extracts showed activity in at least one of the bioassays, all of which were run in very small scale. Phylogenetically related isolates exhibited different activity profiles, highlighting the value of microdiversity for bioprospection studies. Furthermore, LC-MS analyses of selected active extracts suggested the presence of previously unidentified secondary metabolites. Overall, the screening strategy employed here, in which previously untapped cyanobacterial diversity was combined with multiple bioassays, proved to be a successful strategy and allowed the selection of several strains for further investigations based on their bioactivity profiles. PMID:23609580

  11. An Overexpression Screen of Toxoplasma gondii Rab-GTPases Reveals Distinct Transport Routes to the Micronemes

    PubMed Central

    Kremer, Katrin; Kamin, Dirk; Rittweger, Eva; Wilkes, Jonathan; Flammer, Halley; Mahler, Sabine; Heng, Joanne; Tonkin, Christopher J.; Langsley, Gordon; Hell, Stefan W.; Carruthers, Vernon B.; Ferguson, David J. P.; Meissner, Markus

    2013-01-01

    The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles. PMID:23505371

  12. A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity

    PubMed Central

    Arnold, Carrie N.; Pirie, Elaine; Dosenovic, Pia; McInerney, Gerald M.; Xia, Yu; Wang, Nathaniel; Li, Xiaohong; Siggs, Owen M.; Karlsson Hedestam, Gunilla B.; Beutler, Bruce

    2012-01-01

    Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble, cellophane, and Worker ascribed to mutations in Nfkbid, Zeb1, and Ruvbl2, respectively. We show that I?BNS, the nuclear I?B-like protein encoded by Nfkbid, is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system. PMID:22761313

  13. Proteomic screen reveals Fbw7 as a modulator of the NF-?B pathway

    PubMed Central

    Arabi, Azadeh; Ullah, Karim; Branca, Rui M.M.; Johansson, Johan; Bandarra, Daniel; Haneklaus, Moritz; Fu, Jing; Ariës, Ingrid; Nilsson, Peter; Den Boer, Monique L.; Pokrovskaja, Katja; Grandér, Dan; Xiao, Gutian; Rocha, Sonia; Lehtiö, Janne; Sangfelt, Olle

    2012-01-01

    Fbw7 is a ubiquitin-ligase that targets several oncoproteins for proteolysis, but the full range of Fbw7 substrates is not known. Here we show that by performing quantitative proteomics combined with degron motif searches, we effectively screened for a more complete set of Fbw7 targets. We identify 89 putative Fbw7 substrates, including several disease-associated proteins. The transcription factor NF-?B2 (p100/p52) is one of the candidate Fbw7 substrates. We show that Fbw7 interacts with p100 via a conserved degron and that it promotes degradation of p100 in a GSK3? phosphorylation-dependent manner. Fbw7 inactivation increases p100 levels, which in the presence of NF-?B pathway stimuli, leads to increased p52 levels and activity. Accordingly, the apoptotic threshold can be increased by loss of Fbw7 in a p100-dependent manner. In conclusion, Fbw7-mediated destruction of p100 is a regulatory component restricting the response to NF-?B2 pathway stimulation. PMID:22864569

  14. 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

  15. A high throughput RNAi screen reveals determinants of HIV-1 activity in host kinases

    PubMed Central

    Jiang, Wei-Min; Zhang, Xin-Yun; Zhang, Yun-Zhi; Liu, Li; Lu, Hong-Zhou

    2014-01-01

    Drug resistance remains a great challenge in HIV/AIDS treatment despite the recent advances in novel therapeutics. It may be a good strategy to develop drugs targeting the essential host factors to decrease the risk of drug resistance. Previous studies suggested that so many host kinases play roles in HIV life cycles. More importantly, many kinase genes are drugable targets, therefore, it is vital to figure out host kinases responsible for HIV-1 infection and replication to provide novel therapeutic regimens and to deepen our understanding to HIV-host interaction. In present work, a high throughput RNAi screen with a shRNA library against 474 kinases was applied to HEK293T cells stably expressed a HIV-1 LTR (long terminal repeat)-driven reporter plasmid. Four genes, AK1, EphB2, PRKACB and CDK5R2, were found to specifically suppress the HIV-1 LTR activity following effective knockdown. Furthermore, overexpression of AK1 and PRKACB upregulated the HIV-1 LTR activity. Therefore, AK1 and PRKACB are in positive control of HIV-1 activity. DNA microarray analysis demonstrated that overlapped genes between AK1-silenced and PRKACB-silenced cells were mainly enriched in several amino acid biosynthesis pathways, TGF-? signaling and p53 signaling pathways. These alterations may repress the viral infection by the downregulation of ERK1/2, p38MAPK and NF?B signaling pathways. Collectively, our work uncovers several host kinases involving the HIV-1 infection and may provide potential therapeutic targets for AIDS treatment in future. PMID:24966931

  16. Chemistry & Biology Synthetic Lethal Screening Identifies Compounds

    E-print Network

    Stockwell, Brent R.

    -Dependent, Nonapoptotic Cell Death in Oncogenic-RAS-Harboring Cancer Cells Wan Seok Yang1 and Brent R. Stockwell1,2,* 1, such as a nonapoptotic, MEK-de- pendent, and iron-dependent oxidative cell death. Erastin, a previously reported compound VDACs to activate the observed pathway. RSL3 acti- vated a similar death mechanism but in a VDAC

  17. 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. PMID:25533206

  18. 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

  19. 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 ?-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

  20. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

    PubMed

    Babu, Mohan; Arnold, Roland; Bundalovic-Torma, Cedoljub; Gagarinova, Alla; Wong, Keith S; 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

    2014-02-01

    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 ?-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

  1. Functional Screening of a Metagenomic Library Reveals Operons Responsible for Enhanced Intestinal Colonization by Gut Commensal Microbes

    PubMed Central

    Yoon, Mi Young; Lee, Kang-Mu; Yoon, Yujin; Go, Junhyeok; Park, Yongjin; Cho, Yong-Joon; Tannock, Gerald W.

    2013-01-01

    Evidence suggests that gut microbes colonize the mammalian intestine through propagation as an adhesive microbial community. A bacterial artificial chromosome (BAC) library of murine bowel microbiota DNA in the surrogate host Escherichia coli DH10B was screened for enhanced adherence capability. Two out of 5,472 DH10B clones, 10G6 and 25G1, exhibited enhanced capabilities to adhere to inanimate surfaces in functional screens. DNA segments inserted into the 10G6 and 25G1 clones were 52 and 41 kb and included 47 and 41 protein-coding open reading frames (ORFs), respectively. DNA sequence alignments, tetranucleotide frequency, and codon usage analysis strongly suggest that these two DNA fragments are derived from species belonging to the genus Bacteroides. Consistent with this finding, a large portion of the predicted gene products were highly homologous to those of Bacteroides spp. Transposon mutagenesis and subsequent experiments that involved heterologous expression identified two operons associated with enhanced adherence. E. coli strains transformed with the 10a or 25b operon adhered to the surface of intestinal epithelium and colonized the mouse intestine more vigorously than did the control strain. This study has revealed the genetic determinants of unknown commensals (probably resembling Bacteroides species) that enhance the ability of the bacteria to colonize the murine bowel. PMID:23584783

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

    PubMed

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

    2014-01-01

    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

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

    PubMed Central

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

    2013-01-01

    Summary The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two inter-related branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborated pathway centered on the three gonad-specific Argonaute proteins Piwi, Aubergine, and Argonaute3. 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 new key factors of piRNA-mediated transposon silencing, including the novel piRNA biogenesis factors, CG2183 (GASZ) and Deadlock. Last, our data uncovers a previously unanticipated set of factors preferentially required for repression of different transposons types. PMID:23665227

  4. Thiolactone modulators of quorum sensing revealed through library design and screening

    PubMed Central

    McInnis, Christine E.; Blackwell, Helen E.

    2011-01-01

    Quorum sensing (QS) is a process by which bacteria use small molecules or peptidic signals to assess their local population densities. At sufficiently high density, bacteria can alter gene expression levels to regulate group behaviors involved in a range of important and diverse phenotypes, including virulence factor production, biofilm formation, root nodulation, and bioluminescence. Gram-negative bacteria most commonly use N-acylated L-homoserine lactones (AHLs) as their QS signals. The AHL lactone ring is hydrolyzed relatively rapidly at biological pH, and the ring-opened product is QS inactive. We seek to identify AHL analogues with heightened hydrolytic stability, and thereby potentially heightened activity, for use as non-native modulators of bacterial QS. As part of this effort, we probed the utility of thiolactone analogues in the current study as QS agonists and antagonists in Gram-negative bacteria. A focused library of thiolactone analogs was designed and rapidly synthesized in solution. We examined the activity of the library as agonists and antagonists of LuxR-type QS receptors in Pseudomonas aeruginosa (LasR), Vibrio fischeri (LuxR), and Agrobacterium tumefaciens (TraR) using bacterial reporter strains. The thiolactone library contained several highly active compounds, including some of the most active LuxR inhibitors and the most active synthetic TraR agonist reported to date. Analysis of a representative thiolactone analog revealed that its hydrolysis half-life was almost double that of its parent AHL in bacterial growth medium. PMID:21798746

  5. Annotating novel genes by integrating synthetic lethals and genomic information

    Microsoft Academic Search

    Daniel Schöner; Markus Kalisch; Christian Leisner; Lukas Meier; Marc Sohrmann; Mahamadou Faty; Yves Barral; Matthias Peter; Wilhelm Gruissem; Peter Bühlmann

    2008-01-01

    BACKGROUND: Large scale screening for synthetic lethality serves as a common tool in yeast genetics to systematically search for genes that play a role in specific biological processes. Often the amounts of data resulting from a single large scale screen far exceed the capacities of experimental characterization of every identified target. Thus, there is need for computational tools that select

  6. Identification of ?-hematin inhibitors in a high-throughput screening effort reveals scaffolds with in vitro antimalarial activity.

    PubMed

    Sandlin, Rebecca D; Fong, Kim Y; Wicht, Kathryn J; Carrell, Holly M; Egan, Timothy J; Wright, David W

    2014-12-01

    The emergence of drug resistant strains of Plasmodium spp. creates a critical need for the development of novel antimalarials. Formation of hemozoin, a crystalline heme detoxification process vital to parasite survival serves as an important drug target. The quinoline antimalarials including chloroquine and amodiaquine owe their antimalarial activity to inhibition of hemozoin formation. Though in vivo formation of hemozoin occurs within the presence of neutral lipids, the lipophilic detergent NP-40 was previously shown to serve as a surrogate in the ?-hematin (synthetic hemozoin) formation process. Consequently, an NP-40 mediated ?-hematin formation assay was developed for use in high-throughput screening. Here, the assay was utilized to screen 144,330 compounds for the identification of inhibitors of crystallization, resulting in 530 hits. To establish the effectiveness of these target-based ?-hematin inhibitors against Plasmodium falciparum, each hit was further tested in cultures of parasitized red blood cells. This effort revealed that 171 of the ?-hematin inhibitors are also active against the parasite. Dose-response data identified 73 of these ?-hematin inhibitors have IC50 values ?5 ?M, including 25 compounds with nanomolar activity against P. falciparum. A scaffold-based analysis of this data identified 14 primary scaffolds that represent 46% of the 530 total hits. Representative compounds from each of the classes were further assessed for hemozoin inhibitory activity in P. falciparum infected human erythrocytes. Each of the hit compounds tested were found to be positive inhibitors, while a negative control did not perturb this biological pathway in culture. PMID:25516843

  7. 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 specifically impact alternative splicing. PMID:24643257

  8. Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion

    PubMed Central

    Schiebler, Mark; Brown, Karen; Hegyi, Krisztina; Newton, Sandra M; Renna, Maurizio; Hepburn, Lucy; Klapholz, Catherine; Coulter, Sarah; Obregón-Henao, Andres; Henao Tamayo, Marcela; Basaraba, Randall; Kampmann, Beate; Henry, Katherine M; Burgon, Joseph; Renshaw, Stephen A; Fleming, Angeleen; Kay, Robert R; Anderson, Karen E; Hawkins, Phillip T; Ordway, Diane J; Rubinsztein, David C; Floto, Rodrigo Andres

    2015-01-01

    Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection. PMID:25535254

  9. Chemical genetic screen in fission yeast reveals roles for vacuolar acidification, mitochondrial fission, and cellular GMP levels in lifespan extension.

    PubMed

    Stephan, Jessica; Franke, Jacqueline; Ehrenhofer-Murray, Ann E

    2013-08-01

    The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age-related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti-aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V-ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J? displayed anti-aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G-protein-coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti-aging activities for several phytochemicals and open up opportunities for the development of novel anti-aging therapies. PMID:23521895

  10. A suppressor/enhancer screen in Drosophila reveals a role for wnt-mediated lipid metabolism in primordial germ cell migration.

    PubMed

    McElwain, Mark A; Ko, Dennis C; Gordon, Michael D; Fyrst, Henrik; Saba, Julie D; Nusse, Roel

    2011-01-01

    Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-?B protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/?-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC) to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase) redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P), the product of Dcerk, allowing proper PGC migration. PMID:22069480

  11. A Suppressor/Enhancer Screen in Drosophila Reveals a Role for Wnt-Mediated Lipid Metabolism in Primordial Germ Cell Migration

    PubMed Central

    McElwain, Mark A.; Ko, Dennis C.; Gordon, Michael D.; Fyrst, Henrik; Saba, Julie D.; Nusse, Roel

    2011-01-01

    Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-?B protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/?-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC) to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase) redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P), the product of Dcerk, allowing proper PGC migration. PMID:22069480

  12. Pathway-Based Analysis of Genome-Wide siRNA Screens Reveals the Regulatory Landscape of App Processing.

    PubMed

    Camargo, Luiz Miguel; Zhang, Xiaohua Douglas; Loerch, Patrick; Caceres, Ramon Miguel; Marine, Shane D; Uva, Paolo; Ferrer, Marc; Rinaldis, Emanuele de; Stone, David J; Majercak, John; Ray, William J; Yi-An, Chen; Shearman, Mark S; Mizuguchi, Kenji

    2015-01-01

    The progressive aggregation of Amyloid-? (A?) in the brain is a major trait of Alzheimer's Disease (AD). A? is produced as a result of proteolytic processing of the ?-amyloid precursor protein (APP). Processing of APP is mediated by multiple enzymes, resulting in the production of distinct peptide products: the non-amyloidogenic peptide sAPP? and the amyloidogenic peptides sAPP?, A?40, and A?42. Using a pathway-based approach, we analyzed a large-scale siRNA screen that measured the production of different APP proteolytic products. Our analysis identified many of the biological processes/pathways that are known to regulate APP processing and have been implicated in AD pathogenesis, as well as revealing novel regulatory mechanisms. Furthermore, we also demonstrate that some of these processes differentially regulate APP processing, with some mechanisms favouring production of certain peptide species over others. For example, synaptic transmission having a bias towards regulating A?40 production over A?42 as well as processes involved in insulin and pancreatic biology having a bias for sAPP? production over sAPP?. In addition, some of the pathways identified as regulators of APP processing contain genes (CLU, BIN1, CR1, PICALM, TREM2, SORL1, MEF2C, DSG2, EPH1A) recently implicated with AD through genome wide association studies (GWAS) and associated meta-analysis. In addition, we provide supporting evidence and a deeper mechanistic understanding of the role of diabetes in AD. The identification of these processes/pathways, their differential impact on APP processing, and their relationships to each other, provide a comprehensive systems biology view of the "regulatory landscape" of APP. PMID:25723573

  13. Pathway-Based Analysis of Genome-Wide siRNA Screens Reveals the Regulatory Landscape of App Processing

    PubMed Central

    Camargo, Luiz Miguel; Zhang, Xiaohua Douglas; Loerch, Patrick; Caceres, Ramon Miguel; Marine, Shane D.; Uva, Paolo; Ferrer, Marc; de Rinaldis, Emanuele; Stone, David J.; Majercak, John; Ray, William J.; Yi-An, Chen; Shearman, Mark S.; Mizuguchi, Kenji

    2015-01-01

    The progressive aggregation of Amyloid-? (A?) in the brain is a major trait of Alzheimer's Disease (AD). A? is produced as a result of proteolytic processing of the ?-amyloid precursor protein (APP). Processing of APP is mediated by multiple enzymes, resulting in the production of distinct peptide products: the non-amyloidogenic peptide sAPP? and the amyloidogenic peptides sAPP?, A?40, and A?42. Using a pathway-based approach, we analyzed a large-scale siRNA screen that measured the production of different APP proteolytic products. Our analysis identified many of the biological processes/pathways that are known to regulate APP processing and have been implicated in AD pathogenesis, as well as revealing novel regulatory mechanisms. Furthermore, we also demonstrate that some of these processes differentially regulate APP processing, with some mechanisms favouring production of certain peptide species over others. For example, synaptic transmission having a bias towards regulating A?40 production over A?42 as well as processes involved in insulin and pancreatic biology having a bias for sAPP? production over sAPP?. In addition, some of the pathways identified as regulators of APP processing contain genes (CLU, BIN1, CR1, PICALM, TREM2, SORL1, MEF2C, DSG2, EPH1A) recently implicated with AD through genome wide association studies (GWAS) and associated meta-analysis. In addition, we provide supporting evidence and a deeper mechanistic understanding of the role of diabetes in AD. The identification of these processes/pathways, their differential impact on APP processing, and their relationships to each other, provide a comprehensive systems biology view of the “regulatory landscape” of APP. PMID:25723573

  14. A shRNA functional screen reveals Nme6 and Nme7 are crucial for embryonic stem cell renewal.

    PubMed

    Wang, Chia-Hui; Ma, Nianhan; Lin, Yu-Tsen; Wu, Cheng-Chung; Hsiao, Michael; Lu, Frank Leigh; Yu, Ching-Chia; Chen, Shao-Yin; Lu, Jean

    2012-10-01

    In contrast to the somatic cells, embryonic stem cells (ESCs) are characterized by its immortalization ability, pluripotency, and oncogenicity. Revealing the underlying mechanism of ESC characteristics is important for the application of ESCs in clinical medicine. We performed systematic functional screen in mouse ESCs with 4,801 shRNAs that target 929 kinases and phosphatases. One hundred and thirty-two candidate genes that regulate both ESC expansion and stem cell marker expression were identified. Twenty-seven out of the 132 genes were regarded as most important since knockdown of each gene induces morphological changes from undifferentiated to differentiated state. Among the 27 genes, we chose nonmetastatic cell 6 (Nme6, also named as Nm23-H6) and nonmetastatic cell 7 (Nme7, also designated as Nm23-H7) to study first. Nme6 and Nme7 both belong to the members of nucleoside diphosphate kinase family. We demonstrate that Nme6 and Nme7 are important for the regulation of Oct4, Nanog, Klf4, c-Myc, telomerase, Dnmt3B, Sox2, and ERas expression. Either knockdown of Nme6 or Nme7 reduces the formation of embryoid body (EB) and teratoma. The overexpression of either Nme6 or Nme7 can rescue the stem cell marker expression and the EB formation in the absence of leukemia inhibiting factor. This implies the importance of Nme6 and Nme7 in ESC renewal. This finding not only pinpoints Nme6 or Nme7 can regulate several critical regulators in ESC renewal but also increases our understanding of the ESC renewal and oncogenesis. PMID:22899353

  15. Unusual multisystemic involvement and a novel BAG3 mutation revealed by NGS screening in a large cohort of myofibrillar myopathies

    PubMed Central

    2014-01-01

    Background Myofibrillar myopathies (MFM) are a group of phenotypically and genetically heterogeneous neuromuscular disorders, which are characterized by protein aggregations in muscle fibres and can be associated with multisystemic involvement. Methods We screened a large cohort of 38 index patients with MFM for mutations in the nine thus far known causative genes using Sanger and next generation sequencing (NGS). We studied the clinical and histopathological characteristics in 38 index patients and five additional relatives (n?=?43) and particularly focused on the associated multisystemic symptoms. Results We identified 14 heterozygous mutations (diagnostic yield of 37%), among them the novel p.Pro209Gln mutation in the BAG3 gene, which was associated with onset in adulthood, a mild phenotype and an axonal sensorimotor polyneuropathy, in the absence of giant axons at the nerve biopsy. We revealed several novel clinical phenotypes and unusual multisystemic presentations with previously described mutations: hearing impairment with a FLNC mutation, dysphonia with a mutation in DES and the first patient with a FLNC mutation presenting respiratory insufficiency as the initial symptom. Moreover, we described for the first time respiratory insufficiency occurring in a patient with the p.Gly154Ser mutation in CRYAB. Interestingly, we detected a polyneuropathy in 28% of the MFM patients, including a BAG3 and a MYOT case, and hearing impairment in 13%, including one patient with a FLNC mutation and two with mutations in the DES gene. In four index patients with a mutation in one of the MFM genes, typical histological findings were only identified at the ultrastructural level (29%). Conclusions We conclude that extraskeletal symptoms frequently occur in MFM, particularly cardiac and respiratory involvement, polyneuropathy and/or deafness. BAG3 mutations should be considered even in cases with a mild phenotype or an adult onset. We identified a genetic defect in one of the known genes in less than half of the MFM patients, indicating that more causative genes are still to be found. Next generation sequencing techniques should be helpful in achieving this aim. PMID:25208129

  16. Proteomic screening of variola virus reveals a unique NF-?B 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-?B1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-?B1/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-?B signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses. PMID:19451633

  17. Synthetic Lethal Interaction between Oncogenic KRAS Dependency and STK33 Suppression in Human Cancer Cells

    E-print Network

    Scholl, Claudia

    An alternative to therapeutic targeting of oncogenes is to perform “synthetic lethalityscreens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference ...

  18. Identification of genes important for cutaneous function revealed by a large scale reverse genetic screen in the mouse.

    PubMed

    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

    2014-10-01

    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

  19. 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

  20. A Genome-Wide siRNA Screen Reveals Positive and Negative Regulators of the NOD2 and NF-?B Signaling Pathways

    PubMed Central

    Warner, Neil; Burberry, Aaron; Franchi, Luigi; Kim, Yun-Gi; McDonald, Christine; Sartor, Maureen A.; Núñez, Gabriel

    2013-01-01

    The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways that lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn’s disease and Blau syndrome. We used a genome-wide, small interfering RNA (siRNA) screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn’s disease risk were identified in the screen, supporting a role for NOD2 and nuclear factor ?B (NF-?B) pathways in the pathogenesis of Crohn’s disease. A comparison of hits from this screen with other “omics” data sets revealed interconnected networks of genes implicated in NF-?B signaling. Secondary assays, including the measurement of interleukin-8 secretion, served to validate many of the regulators. Knockdown of putative regulators in HEK293 cells followed by stimulation with tumor necrosis factor ? revealed that most of the genes identified were general regulators of NF-?B signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-?B–mediated inflammation. PMID:23322906

  1. A genome-wide siRNA screen reveals positive and negative regulators of the NOD2 and NF-?B signaling pathways.

    PubMed

    Warner, Neil; Burberry, Aaron; Franchi, Luigi; Kim, Yun-Gi; McDonald, Christine; Sartor, Maureen A; Núñez, Gabriel

    2013-01-15

    The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways, including activation of nuclear factor ?B (NF-?B) signaling, which lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn's disease and Blau syndrome. We used a genome-wide small interfering RNA screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn's disease risk were identified in the screen. A comparison of candidates from this screen with other "omics" data sets revealed interconnected networks of genes implicated in NF-?B signaling, thus supporting a role for NOD2 and NF-?B pathways in the pathogenesis of Crohn's disease. Many of these regulators were validated in secondary assays, such as measurement of interleukin-8 secretion, which is partially dependent on NF-?B. Knockdown of putative regulators in human embryonic kidney 293 cells followed by stimulation with tumor necrosis factor-? revealed that most of the genes identified were general regulators of NF-?B signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-?B-mediated inflammation. PMID:23322906

  2. Less lethal technology: medical issues

    Microsoft Academic Search

    Gary M. Vilke; Theodore C. Chan

    2007-01-01

    Purpose – Less lethal weapons have become a critical tool for law enforcement when confronting dangerous, combative individuals in the field. The purpose of this paper is to review the medical aspects and implications of three different types of less lethal weapons. Design\\/methodology\\/approach – The paper conducted a comprehensive medical literature review on blunt projectiles, irritant sprays including oleoresin capsicum

  3. High throughput flow cytometry screening reveals a novel role for JAM-A as a cancer stem cell maintenance factor

    PubMed Central

    Lathia, Justin D.; Li, Meizhang; Sinyuk, Maksim; Alvarado, Alvaro G.; Flavahan, William A.; Stoltz, Kevin; Rosager, Ann Mari; Hale, James; Hitomi, Masahiro; Gallagher, Joseph; Wu, Qiulian; Martin, Jody; Vidal, Jason G.; Nakano, Ichiro; Dahlrot, Rikke H.; Hansen, Steinbjørn; McLendon, Roger E.; Sloan, Andrew E.; Bao, Shideng; Hjelmeland, Anita B.; Carson, Christian T.; Naik, Ulhas P.; Kristensen, Bjarne; Rich, Jeremy N.

    2014-01-01

    Summary Stem cells reside in niches that regulate the balance between self-renewal and differentiation. The identity of a stem cell is linked with the ability to interact with its niche through adhesion mechanisms. To identify targets that disrupt cancer stem cell (CSC) adhesion, we performed a flow cytometry screen on patient derived glioblastoma (GBM) cells and identified junctional adhesion molecule-A (JAM-A) as a CSC adhesion mechanism essential for self-renewal and tumor growth. JAM-A was dispensable for normal neural stem/progenitor cell (NPC) function and JAM-A expression was reduced in normal brain versus GBM. Targeting JAM-A compromises the self-renewal of CSCs. JAM-A expression negatively correlated to GBM patient prognosis. Our results demonstrate that novel GBM targeting strategies can be identified through screening adhesion receptors and JAM-A represents a novel mechanism for niche driven CSC maintenance. PMID:24373972

  4. Genomewide Screen for Negative Regulators of Sirtuin Activity in Saccharomyces cerevisiae Reveals 40 Loci and Links to Metabolism

    Microsoft Academic Search

    Ryan M. Raisner; Hiten D. Madhani

    2008-01-01

    Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Silent information regulator 2 (Sir2) promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genomewide screen for factors that negatively regulate Sir activity at a reporter geneplacedimmediatelyoutsideasilencedregion.Afterlinkageanalysis,assessmentofSirdependency,and knockout tag verification, 40

  5. 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

  6. Lethal Myocardial Ischemic Injury

    PubMed Central

    Jennings, Robert B.; Reimer, Keith A.

    1981-01-01

    The biologic changes occurring in severely ischemic myocytes in vivo as the affected cells pass through the phase of reversible to the phase of lethal or irreversible injury are reviewed with special emphasis on the effect of ischemia on the production and utilization of highenergy phosphate, the destruction of the adenine nucleotide pool, and the appearance of signs of damage to the plasma membrane of the sarcolemma. Evidence is presented that indicates that the events occurring in severe ischemia in vivo are essentially identical to those found in total ischemia in vitro except that the biologic changes of ischemia develop more slowly in total ischemia in vitro than in severe ischemia in vivo. The slower time course of injury, together with the uniformity of injury provided by total ischemia in vitro, may allow for more precise identification of potential lethal cellular events in ischemic injury. The production of highenergy phosphates (HEP) from anaerobic glycolysis have been estimated in both in vivo and in vitro ischemia by the measurement of lactate accumulation, and total HEP utilization has been estimated from the depletion of stores of preformed HEP. The results show that between 80% and 90% of the HEP utilized by ischemic dog left ventricle is produced by anaerobic glycolysis. The onset of irreversibility is associated with marked depletion of the HEP and adenine nucleotide pools of the tissue and the cessation of energy production via glycolysis. The cessation of anaerobic glycolysis may be caused by the low sarcoplasmic, adenosine triphosphate (ATP) concentration of the dying myocyte. In addition to the foregoing changes, irreversibly injured tissue exhibits both ultrastructural and functional evidence of disruption of the plasmalemma of the sarcolemma. The possible relationships, causal and otherwise, between severe HEP depletion and membrane damage are discussed. Both HEP depletion (ATP < 3-8% of control) and membrane damage are considered to be objective signs of the presence of irreversible myocardial ischemic injury. However, at the present time, there is no proof that these changes are causally related either to each other or to cell death in severe in vivo ischemia. ImagesFigure 3Figure 4 PMID:7008621

  7. 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

  8. Fragment screening reveals salicylic hydroxamic acid as an inhibitor of Trypanosoma brucei GPI GlcNAc-PI de-N-acetylase.

    PubMed

    Urbaniak, Michael D; Capes, Amy S; Crossman, Arthur; O'Neill, Sandra; Thompson, Stephen; Gilbert, Ian H; Ferguson, Michael A J

    2014-03-31

    The zinc-metalloenzyme GlcNAc-PI de-N-acetylase is essential for the biosynthesis of mature GPI anchors and has been genetically validated in the bloodstream form of Trypanosoma brucei, which causes African sleeping sickness. We screened a focused library of zinc-binding fragments and identified salicylic hydroxamic acid as a GlcNAc-PI de-N-acetylase inhibitor with high ligand efficiency. This is the first small molecule inhibitor reported for the trypanosome GPI pathway. Investigating the structure activity relationship revealed that hydroxamic acid and 2-OH are essential for potency, and that substitution is tolerated at the 4- and 5-positions. PMID:24589444

  9. Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin.

    PubMed

    Vlasits, Anna L; Simon, Julian A; Raible, David W; Rubel, Edwin W; Owens, Kelly N

    2012-12-01

    Loss of mechanosensory hair cells in the inner ear accounts for many hearing loss and balance disorders. Several beneficial pharmaceutical drugs cause hair cell death as a side effect. These include aminoglycoside antibiotics, such as neomycin, kanamycin and gentamicin, and several cancer chemotherapy drugs, such as cisplatin. Discovering new compounds that protect mammalian hair cells from toxic insults is experimentally difficult because of the inaccessibility of the inner ear. We used the zebrafish lateral line sensory system as an in vivo screening platform to survey a library of FDA-approved pharmaceuticals for compounds that protect hair cells from neomycin, gentamicin, kanamycin and cisplatin. Ten compounds were identified that provide protection from at least two of the four toxins. The resulting compounds fall into several drug classes, including serotonin and dopamine-modulating drugs, adrenergic receptor ligands, and estrogen receptor modulators. The protective compounds show different effects against the different toxins, supporting the idea that each toxin causes hair cell death by distinct, but partially overlapping, mechanisms. Furthermore, some compounds from the same drug classes had different protective properties, suggesting that they might not prevent hair cell death by their known target mechanisms. Some protective compounds blocked gentamicin uptake into hair cells, suggesting that they may block mechanotransduction or other routes of entry. The protective compounds identified in our screen will provide a starting point for studies in mammals as well as further research discovering the cellular signaling pathways that trigger hair cell death. PMID:22967486

  10. An unbiased in vivo screen reveals multiple transcription factors that control HPV E6-regulated hTERT in keratinocytes

    PubMed Central

    Xu, Mei; Katzenellenbogen, Rachel A.; Grandori, Carla; Galloway, Denise A.

    2013-01-01

    Activation of telomerase by human papillomavirus 16 (HPV16) E6 is a critical step for cell immortalization and transformation in human foreskin keratinocytes (HFKs). Multiple transcription factors have been identified as being involved in E6-induced hTERT expression. Here, we adapted an unbiased in vivo screen using a LacO-LacI system in human cells to discover hTERT promoter-interacting regulators. This approach allowed us to identify a novel hTERT repressor, Maz, which bound the hTERT promoter. E6 expression reduced Maz binding and correspondingly increased Sp1 binding at the hTERT promoter. Knockdown of Maz further increased histone acetylation, as well as hTERT expression in the presence of E6. Overall, these data indicate the utility of a novel screen for promoter-interacting and transcription-regulating proteins. These data also highlight multiple factors that normally regulate hTERT repression in HFKs, and therefore are targeted by E6 for hTERT expression. PMID:24074563

  11. Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin

    PubMed Central

    Vlasits, Anna L.; Simon, Julian A.; Raible, David W.; Rubel, Edwin W; Owens, Kelly N.

    2012-01-01

    Loss of mechanosensory hair cells in the inner ear accounts for many hearing loss and balance disorders. Several beneficial pharmaceutical drugs cause hair cell death as a side effect. These include aminoglycoside antibiotics, such as neomycin, kanamycin and gentamicin, and several cancer chemotherapy drugs, such as cisplatin. Discovering new compounds that protect mammalian hair cells from toxic insults is experimentally difficult because of the inaccessibility of the inner ear. We used the zebrafish lateral line sensory system as an in vivo screening platform to survey a library of FDA-approved pharmaceuticals for compounds that protect hair cells from neomycin, gentamicin, kanamycin and cisplatin. Ten compounds were identified that provide protection from at least two of the four toxins. The resulting compounds fall into several drug classes, including serotonin and dopamine-modulating drugs, adrenergic receptor ligands, and estrogen receptor modulators. The protective compounds show different effects against the different toxins, supporting the idea that each toxin causes hair cell death by distinct, but partially overlapping, mechanisms. Furthermore, some compounds from the same drug classes had different protective properties, suggesting that they might not prevent hair cell death by their known target mechanisms. Some protective compounds blocked gentamicin uptake into hair cells, suggesting that they may block mechanotransduction or other routes of entry. The protective compounds identified in our screen will provide a starting point for studies in mammals as well as further research discovering the cellular signaling pathways that trigger hair cell death. PMID:22967486

  12. Screening analysis of ubiquitin ligases reveals G2E3 as a potential target for chemosensitizing cancer cells

    PubMed Central

    Schmidt, Franziska; Kunze, Meike; Loock, Ann-Christine; Dobbelstein, Matthias

    2015-01-01

    Cisplatin is widely used against various tumors, but resistance is commonly encountered. By inducing DNA crosslinks, cisplatin triggers DNA damage response (DDR) and cell death. However, the molecular determinants of how cells respond to cisplatin are incompletely understood. Since ubiquitination plays a major role in DDR, we performed a high-content siRNA screen targeting 327 human ubiquitin ligases and 92 deubiquitinating enzymes in U2OS cells, interrogating the response to cisplatin. We quantified ?H2AX by immunofluorescence and image analysis as a read-out for DNA damage. Among known mediators of DDR, the screen identified the ubiquitin ligase G2E3 as a new player in the response to cisplatin. G2E3 depletion led to decreased ?H2AX levels and decreased phosphorylation of the checkpoint kinase 1 (Chk1) upon cisplatin. Moreover, loss of G2E3 triggered apoptosis and decreased proliferation of cancer cells. Treating cells with the nucleoside analogue gemcitabine led to increased accumulation of single-stranded DNA upon G2E3 depletion, pointing to a defect in replication. Furthermore, we show that endogenous G2E3 levels in cancer cells were down-regulated upon chemotherapeutic treatment. Taken together, our results suggest that G2E3 is a molecular determinant of the DDR and cell survival, and that its loss sensitizes tumor cells towards DNA-damaging treatment. PMID:25593194

  13. A directed RNAi screen based on larval growth arrest reveals new modifiers of C. elegans insulin signaling.

    PubMed

    Billing, Ola; Natarajan, Balasubramanian; Mohammed, Ateequrrahman; Naredi, Peter; Kao, Gautam

    2012-01-01

    Genes regulating Caenorhabditis elegans insulin/IGF signaling (IIS) have largely been identified on the basis of their involvement in dauer development or longevity. A third IIS phenotype is the first larval stage (L1) diapause, which is also influenced by asna-1, a regulator of DAF-28/insulin secretion. We reasoned that new regulators of IIS strength might be identified in screens based on the L1 diapause and the asna-1 phenotype. Eighty- six genes were selected for analysis by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest. ykt-6, mrps-2, mrps-10 and mrpl-43 were identified as genes which, when inactivated, caused larval arrest without any associated feeding defects. Several tests indicated that IIS strength was weaker and that insulin secretion was defective in these animals. This study highlights the role of the Golgi network and the mitochondria in insulin secretion and provides a new list of genes that modulate IIS in C. elegans. PMID:22511947

  14. A Directed RNAi Screen Based on Larval Growth Arrest Reveals New Modifiers of C. elegans Insulin Signaling

    PubMed Central

    Mohammed, Ateequrrahman; Naredi, Peter; Kao, Gautam

    2012-01-01

    Genes regulating Caenorhabditis elegans insulin/IGF signaling (IIS) have largely been identified on the basis of their involvement in dauer development or longevity. A third IIS phenotype is the first larval stage (L1) diapause, which is also influenced by asna-1, a regulator of DAF-28/insulin secretion. We reasoned that new regulators of IIS strength might be identified in screens based on the L1 diapause and the asna-1 phenotype. Eighty- six genes were selected for analysis by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest. ykt-6, mrps-2, mrps-10 and mrpl-43 were identified as genes which, when inactivated, caused larval arrest without any associated feeding defects. Several tests indicated that IIS strength was weaker and that insulin secretion was defective in these animals. This study highlights the role of the Golgi network and the mitochondria in insulin secretion and provides a new list of genes that modulate IIS in C. elegans. PMID:22511947

  15. 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 and be used as alternatives to traditional two-dimensional screens for better comparability to the in vivo state. PMID:24194875

  16. Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production.

    PubMed

    Özayd?n, Bilge; Burd, Helcio; Lee, Taek Soon; Keasling, Jay D

    2013-01-01

    Beside their essential cellular functions, isoprenoids have value as pharmaceuticals, nutriceuticals, pesticides, and fuel alternatives. Engineering microorganisms for production of isoprenoids is relatively easy, sustainable, and cost effective in comparison to chemical synthesis or extraction from natural producers. We introduced genes encoding carotenoid biosynthetic enzymes into the haploid yeast deletion collection to identify gene deletions that improved isoprenoid production. Deletions that showed significant improvement in carotenoid production were further screened for production of bisabolene, an isoprenoid alternative to petroleum-derived diesel. Combining those deletions with other mevalonate pathway modifications increased production of bisabolene from 40mg/L to 800mg/L in shake-flask cultures. In a fermentation process, this engineered strain produced 5.2g/L of bisabolene. PMID:22918085

  17. A genetic screen for mutations affecting gonad formation in Drosophila reveals a role for the slit/robo pathway

    PubMed Central

    Weyers, Jill J.; Milutinovich, Allison B.; Takeda, Yasuko; Jemc, Jennifer C.; Doren, Mark Van

    2013-01-01

    Organogenesis is a complex process requiring multiple cell types to associate with one another through correct cell contacts and in the correct location to achieve proper organ morphology and function. To better understand the mechanisms underlying gonad formation, we performed a mutagenesis screen in Drosophila and identified twenty-four genes required for gonadogenesis. These genes affect all different aspects of gonad formation and provide a framework for understanding the molecular mechanisms that control these processes. We find that gonad formation is regulated by multiple, independent pathways; some of these regulate the key cell adhesion molecule DE-cadherin, while others act through distinct mechanisms. In addition, we discover that the Slit/Roundabout pathway, best known for its role in regulating axonal guidance, is essential for proper gonad formation. Our findings shed light on the complexities of gonadogenesis and the genetic regulation required for proper organ formation. PMID:21377458

  18. A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

    PubMed

    Candido, Thiago De Souza; Gonçalves, Rodrigo Duarte; Felício, Ana Paula; Freitas, Fernanda Zanolli; Cupertino, Fernanda Barbosa; De Carvalho, Ana Carolina Gomes Vieira; Bertolini, Maria Célia

    2014-12-15

    Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development. PMID:25253091

  19. Genomewide screen for negative regulators of sirtuin activity in Saccharomyces cerevisiae reveals 40 loci and links to metabolism.

    PubMed

    Raisner, Ryan M; Madhani, Hiten D

    2008-08-01

    Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Silent information regulator 2 (Sir2) promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genomewide screen for factors that negatively regulate Sir activity at a reporter gene placed immediately outside a silenced region. After linkage analysis, assessment of Sir dependency, and knockout tag verification, 40 loci were identified, including 20 that have not been previously described to regulate Sir. In addition to chromatin-associated factors known to prevent ectopic silencing (Bdf1, SAS-I complex, Rpd3L complex, Ku), we identified the Rtt109 DNA repair-associated histone H3 lysine 56 acetyltransferase as an anti-silencing factor. Our findings indicate that Rtt109 functions independently of its proposed effectors, the Rtt101 cullin, Mms1, and Mms22, and demonstrate unexpected interplay between H3K56 and H4K16 acetylation. The screen also identified subunits of mediator (Soh1, Srb2, and Srb5) and mRNA metabolism factors (Kem1, Ssd1), thus raising the possibility that weak silencing affects some aspect of mRNA structure. Finally, several factors connected to metabolism were identified. These include the PAS-domain metabolic sensor kinase Psk2, the mitochondrial homocysteine detoxification enzyme Lap3, and the Fe-S cluster protein maturase Isa2. We speculate that PAS kinase may integrate metabolic signals to control sirtuin activity. PMID:18689887

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

    PubMed Central

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

    2014-01-01

    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. PMID:25159123

  1. 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. PMID:25756177

  2. NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species

    Microsoft Academic Search

    Frank C. Schroeder; Andrew E. Taggi; Matthew Gronquist; Rabia U. Malik; Jacqualine B. Grant; Thomas Eisner; Jerrold Meinwald

    2008-01-01

    Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to

  3. Large-scale screening of transcription factor–promoter interactions in spruce reveals a transcriptional network involved in vascular development

    PubMed Central

    Lachance, Denis; Giguère, Isabelle; Séguin, Armand

    2014-01-01

    This research aimed to investigate the role of diverse transcription factors (TFs) and to delineate gene regulatory networks directly in conifers at a relatively high-throughput level. The approach integrated sequence analyses, transcript profiling, and development of a conifer-specific activation assay. Transcript accumulation profiles of 102 TFs and potential target genes were clustered to identify groups of coordinately expressed genes. Several different patterns of transcript accumulation were observed by profiling in nine different organs and tissues: 27 genes were preferential to secondary xylem both in stems and roots, and other genes were preferential to phelloderm and periderm or were more ubiquitous. A robust system has been established as a screening approach to define which TFs have the ability to regulate a given promoter in planta. Trans-activation or repression effects were observed in 30% of TF–candidate gene promoter combinations. As a proof of concept, phylogenetic analysis and expression and trans-activation data were used to demonstrate that two spruce NAC-domain proteins most likely play key roles in secondary vascular growth as observed in other plant species. This study tested many TFs from diverse families in a conifer tree species, which broadens the knowledge of promoter–TF interactions in wood development and enables comparisons of gene regulatory networks found in angiosperms and gymnosperms. PMID:24713992

  4. Large-scale screening of transcription factor-promoter interactions in spruce reveals a transcriptional network involved in vascular development.

    PubMed

    Duval, Isabelle; Lachance, Denis; Giguère, Isabelle; Bomal, Claude; Morency, Marie-Josée; Pelletier, Gervais; Boyle, Brian; MacKay, John J; Séguin, Armand

    2014-06-01

    This research aimed to investigate the role of diverse transcription factors (TFs) and to delineate gene regulatory networks directly in conifers at a relatively high-throughput level. The approach integrated sequence analyses, transcript profiling, and development of a conifer-specific activation assay. Transcript accumulation profiles of 102 TFs and potential target genes were clustered to identify groups of coordinately expressed genes. Several different patterns of transcript accumulation were observed by profiling in nine different organs and tissues: 27 genes were preferential to secondary xylem both in stems and roots, and other genes were preferential to phelloderm and periderm or were more ubiquitous. A robust system has been established as a screening approach to define which TFs have the ability to regulate a given promoter in planta. Trans-activation or repression effects were observed in 30% of TF-candidate gene promoter combinations. As a proof of concept, phylogenetic analysis and expression and trans-activation data were used to demonstrate that two spruce NAC-domain proteins most likely play key roles in secondary vascular growth as observed in other plant species. This study tested many TFs from diverse families in a conifer tree species, which broadens the knowledge of promoter-TF interactions in wood development and enables comparisons of gene regulatory networks found in angiosperms and gymnosperms. PMID:24713992

  5. A Genetic Screen Based on in Vivo RNA Imaging Reveals Centrosome-Independent Mechanisms for Localizing gurken Transcripts in Drosophila

    PubMed Central

    Hayashi, Rippei; Wainwright, S. Mark; Liddell, Sophie J.; Pinchin, Sheena M.; Horswell, Stuart; Ish-Horowicz, David

    2014-01-01

    We have screened chromosome arm 3L for ethyl methanesulfonate?induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis. PMID:24531791

  6. Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism

    PubMed Central

    Freimoser, Florian M; Hürlimann, Hans Caspar; Jakob, Claude A; Werner, Thomas P; Amrhein, Nikolaus

    2006-01-01

    Background Inorganic polyphosphate (poly P) occurs universally in all organisms from bacteria to man. It functions, for example, as a phosphate and energy store, and is involved in the activation and regulation of proteins. Despite its ubiquitous occurrence and important functions, it is unclear how poly P is synthesized or how poly P metabolism is regulated in higher eukaryotes. This work describes a systematic analysis of poly P levels in yeast knockout strains mutated in almost every non-essential gene. Results After three consecutive screens, 255 genes (almost 4% of the yeast genome) were found to be involved in the maintenance of normal poly P content. Many of these genes encoded proteins functioning in the cytoplasm, the vacuole or in transport and transcription. Besides reduced poly P content, many strains also exhibited reduced total phosphate content, showed altered ATP and glycogen levels and were disturbed in the secretion of acid phosphatase. Conclusion Cellular energy and phosphate homeostasis is suggested to result from the equilibrium between poly P, ATP and free phosphate within the cell. Poly P serves as a buffer for both ATP and free phosphate levels and is, therefore, the least essential and consequently most variable component in this network. However, strains with reduced poly P levels are not only affected in their ATP and phosphate content, but also in other components that depend on ATP or free phosphate content, such as glycogen or secreted phosphatase activity. PMID:17107617

  7. A BLOC-1 Mutation Screen Reveals that PLDN Is Mutated in Hermansky-Pudlak Syndrome Type 9

    PubMed Central

    Cullinane, Andrew R.; Curry, James A.; Carmona-Rivera, Carmelo; Summers, C. Gail; Ciccone, Carla; Cardillo, Nicholas D.; Dorward, Heidi; Hess, Richard A.; White, James G.; Adams, David; Huizing, Marjan; Gahl, William A.

    2011-01-01

    Hermansky-Pudlak Syndrome (HPS) is an autosomal-recessive condition characterized by oculocutaneous albinism and a bleeding diathesis due to absent platelet delta granules. HPS is a genetically heterogeneous disorder of intracellular vesicle biogenesis. We first screened all our patients with HPS-like symptoms for mutations in the genes responsible for HPS-1 through HPS-6 and found no functional mutations in 38 individuals. We then examined all eight genes encoding the biogenesis of lysosome-related organelles complex-1, or BLOC-1, proteins in these individuals. This identified a homozygous nonsense mutation in PLDN in a boy with characteristic features of HPS. PLDN is mutated in the HPS mouse model pallid and encodes the protein pallidin, which interacts with the early endosomal t-SNARE syntaxin-13. We could not detect any full-length pallidin in our patient's cells despite normal mRNA expression of the mutant transcript. We could detect an alternative transcript that would skip the exon that harbored the mutation, but we demonstrate that if this transcript is translated into protein, although it correctly localizes to early endosomes, it does not interact with syntaxin-13. In our patient's melanocytes, the melanogenic protein TYRP1 showed aberrant localization, an increase in plasma-membrane trafficking, and a failure to reach melanosomes, explaining the boy's severe albinism and establishing his diagnosis as HPS-9. PMID:21665000

  8. A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling.

    PubMed

    Dani, Neil; Nahm, Minyeop; Lee, Seungbok; Broadie, Kendal

    2012-01-01

    A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects. PMID:23144627

  9. Screening for Depression in the Postpartum using the Beck Depression Inventory-II: What Logistic Regression Reveals

    PubMed Central

    Conradt, Elisabeth; Manian, Nanmathi; Bornstein, Marc H.

    2013-01-01

    Objective To identify items on the BDI-II that best discriminate between clinically depressed and nondepressed postpartum women. Background Postpartum depression is a serious and widespread health burden, and the Beck Depression Inventory (BDI-II) is commonly used to detect depression in the postpartum. Yet certain depressive symptoms are “normative” sequelae of childbirth, calling into question the discriminative utility of the BDI-II. Methods We examined the prospective contribution of BDI-II items to identify items that have the strongest relation with clinical postpartum depression. Women with BDI-II scores >12 were invited to participate in a structured clinical interview. A logistic regression was conducted to determine which BDI-II items discriminated between women who were later diagnosed as Depressed (n = 75) and Nondepressed (n = 78). Results Of the 11 BDI-II items that differed between the two groups, eight represented cognitive/affective symptoms. Results from the logistic regression indicated that four BDI-II symptoms were significant predictors of Depression status: sadness, pessimism, loss of interest, and changes in appetite. Conclusion The BDI-II should be used in the postpartum with caution. Professionals who screen for postpartum depression should pay particular attention to cognitive/affective symptoms, as they appear more robust to normative physical and emotional changes that occur in the postpartum. PMID:23569330

  10. Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage

    PubMed Central

    Hill, Sarah J.; Rolland, Thomas; Adelmant, Guillaume; Xia, Xianfang; Owen, Matthew S.; Dricot, Amélie; Zack, Travis I.; Sahni, Nidhi; Jacob, Yves; Hao, Tong; McKinney, Kristine M.; Clark, Allison P.; Reyon, Deepak; Tsai, Shengdar Q.; Joung, J. Keith; Beroukhim, Rameen; Marto, Jarrod A.; Vidal, Marc; Gaudet, Suzanne; Hill, David E.

    2014-01-01

    BRCA1 is a breast and ovarian tumor suppressor. Given its numerous incompletely understood functions and the possibility that more exist, we performed complementary systematic screens in search of new BRCA1 protein-interacting partners. New BRCA1 functions and/or a better understanding of existing ones were sought. Among the new interacting proteins identified, genetic interactions were detected between BRCA1 and four of the interactors: TONSL, SETX, TCEANC, and TCEA2. Genetic interactions were also detected between BRCA1 and certain interactors of TONSL, including both members of the FACT complex. From these results, a new BRCA1 function in the response to transcription-associated DNA damage was detected. Specifically, new roles for BRCA1 in the restart of transcription after UV damage and in preventing or repairing damage caused by stabilized R loops were identified. These roles are likely carried out together with some of the newly identified interactors. This new function may be important in BRCA1 tumor suppression, since the expression of several interactors, including some of the above-noted transcription proteins, is repeatedly aberrant in both breast and ovarian cancers. PMID:25184681

  11. A Targeted Glycan-Related Gene Screen Reveals Heparan Sulfate Proteoglycan Sulfation Regulates WNT and BMP Trans-Synaptic Signaling

    PubMed Central

    Dani, Neil; Nahm, Minyeop; Lee, Seungbok; Broadie, Kendal

    2012-01-01

    A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects. PMID:23144627

  12. A genetic screen based on in vivo RNA imaging reveals centrosome-independent mechanisms for localizing gurken transcripts in Drosophila.

    PubMed

    Hayashi, Rippei; Wainwright, S Mark; Liddell, Sophie J; Pinchin, Sheena M; Horswell, Stuart; Ish-Horowicz, David

    2014-04-01

    We have screened chromosome arm 3L for ethyl methanesulfonate-induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis. PMID:24531791

  13. Genetic screening identifies cyanogenesis-deficient mutants of Lotus japonicus and reveals enzymatic specificity in hydroxynitrile glucoside metabolism.

    PubMed

    Takos, Adam; Lai, Daniela; Mikkelsen, Lisbeth; Abou Hachem, Maher; Shelton, Dale; Motawia, Mohammed Saddik; Olsen, Carl Erik; Wang, Trevor L; Martin, Cathie; Rook, Fred

    2010-05-01

    Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (alpha-hydroxynitrile glucosides) by specific beta-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the beta-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related beta-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants. PMID:20453117

  14. Drug Screening Using a Library of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Disease Specific Patterns of Cardiotoxicity

    PubMed Central

    Liang, Ping; Lan, Feng; Lee, Andrew S.; Gong, Tingyu; Sanchez-Freire, Veronica; Wang, Yongming; Diecke, Sebastian; Sallam, Karim; Knowles, Joshua W.; Wang, Paul J.; Nguyen, Patricia K.; Bers, Donald M.; Robbins, Robert C.; Wu, Joseph C.

    2013-01-01

    Background Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with pre-existing heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. Methods and Results Action potential duration (APD) and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome (LQT), familial hypertrophic cardiomyopathy (HCM), and familial dilated cardiomyopathy (DCM). Disease phenotypes were verified in LQT, HCM, and DCM iPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene (hERG) expressing human embryonic kidney (HEK293) cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in HEK293 cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by APD and quantification of drug-induced arrhythmias such as early after depolarizations (EADs) and delayed after depolarizations (DADs). Conclusions We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, LQT, HCM, and DCM patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than standard hERG test or healthy control hiPSC-CM/hESC-CM screening assays. PMID:23519760

  15. Unbiased Modifier Screen Reveals That Signal Strength Determines the Regulatory Role Murine TLR9 Plays in Autoantibody Production.

    PubMed

    Mills, Robyn E; Lam, Viola C; Tan, Allison; Cresalia, Nicole; Oksenberg, Nir; Zikherman, Julie; Anderson, Mark; Weiss, Arthur; Hermiston, Michelle L

    2015-04-15

    The autoimmune disease systemic lupus erythematosus has a complex environmental and multifactorial genetic basis. Genome-wide association studies have recently identified numerous disease-associated polymorphisms, but it remains unclear in which cells and during which step of pathogenesis specific polymorphisms interact to cause disease. Using a mouse model in which the same activating mutation (CD45E613R) causes distinct genetic background-dependent disease phenotypes, we performed a screen for genetic modifiers of autoreactivity between anti-nuclear Ab (ANA)-resistant CD45E613R.B6 and ANA-permissive CD45E613R.BALB/c mice. Within a novel autoreactivity-associated locus on chromosome 9, we identify a putative modifier, TLR9. Validating a role for TLR9 in modifying autoreactivity in the context of the CD45E613R mutation, manipulation of TLR9 gene dosage eliminates ANA in CD45E613R.BALB/c mice, but confoundingly permits ANA in CD45E613R.B6 mice. We demonstrate that sensitivity to ANA is modulated by strength of TLR9 signal, because stronger TLR9(B6) signals, but not weaker TLR9(BALB/c) signals, negatively regulate CD45E613R B cell development during competitive reconstitution at the central tolerance checkpoint. Our results identify a novel autoreactivity-associated locus and validate Tlr9 as a candidate gene within the locus. We further demonstrate a novel role for TLR9 signal strength in central tolerance, providing insight into the interplay of disease-associated polymorphisms at a discrete step of systemic lupus erythematosus pathogenesis. PMID:25769918

  16. Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

    PubMed Central

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

    2009-01-01

    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. PMID:19662168

  17. Escherichia coli genes that reduce the lethal effects of stress

    PubMed Central

    2010-01-01

    Background The continuing emergence of antimicrobial resistance requires the development of new compounds and/or enhancers of existing compounds. Genes that protect against the lethal effects of antibiotic stress are potential targets of enhancers. To distinguish such genes from those involved in drug uptake and efflux, a new susceptibility screen is required. Results Transposon (Tn5)-mediated mutagenesis was used to create a library of Escherichia coli mutants that was screened for hypersensitivity to the lethal action of quinolones and counter-screened to have wild-type bacteriostatic susceptibility. Mutants with this novel "hyperlethal" phenotype were found. The phenotype was transferable to other E. coli strains by P1-mediated transduction, and for a subset of the mutants the phenotype was complemented by the corresponding wild-type gene cloned into a plasmid. Thus, the inactivation of these genes was responsible for hyperlethality. Nucleotide sequence analysis identified 14 genes, mostly of unknown function, as potential factors protecting from lethal effects of stress. The 14 mutants were killed more readily than wild-type cells by mitomycin C and hydrogen peroxide; nine were also more readily killed by UV irradiation, and several exhibited increased susceptibility to killing by sodium dodecyl sulfate. No mutant was more readily killed by high temperature. Conclusions A new screening strategy identified a diverse set of E. coli genes involved in the response to lethal antimicrobial and environmental stress, with some genes being involved in the response to multiple stressors. The gene set, which differed from sets previously identified with bacteriostatic assays, provides an entry point for obtaining small-molecule enhancers that will affect multiple antimicrobial agents. PMID:20128927

  18. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient ?-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 ?-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, are necessary to induce the transcription of sulfate assimilation genes during early cadmium stress. PMID:22283708

  19. NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species.

    PubMed

    Schroeder, Frank C; Taggi, Andrew E; Gronquist, Matthew; Malik, Rabia U; Grant, Jacqualine B; Eisner, Thomas; Meinwald, Jerrold

    2008-09-23

    Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules. These include polyamines, common neurotransmitters, and amino acid derivatives as well as two additional members of a recently discovered family of natural products, the sulfated nucleosides. In the case of the well studied brown recluse spider, Loxosceles reclusa, sulfated guanosine derivatives were found to comprise the major small-molecule components of the venom. PMID:18794518

  20. NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species

    PubMed Central

    Schroeder, Frank C.; Taggi, Andrew E.; Gronquist, Matthew; Malik, Rabia U.; Grant, Jacqualine B.; Eisner, Thomas; Meinwald, Jerrold

    2008-01-01

    Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules. These include polyamines, common neurotransmitters, and amino acid derivatives as well as two additional members of a recently discovered family of natural products, the sulfated nucleosides. In the case of the well studied brown recluse spider, Loxosceles reclusa, sulfated guanosine derivatives were found to comprise the major small-molecule components of the venom. PMID:18794518

  1. Screening of Metagenomic and Genomic Libraries Reveals Three Classes of Bacterial Enzymes That Overcome the Toxicity of Acrylate

    PubMed Central

    Curson, Andrew R. J.; Burns, Oliver J.; Voget, Sonja; Daniel, Rolf; Todd, Jonathan D.; McInnis, Kathryn; Wexler, Margaret; Johnston, Andrew W. B.

    2014-01-01

    Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again, through the removal of the toxic product acrylyl-CoA. PMID:24848004

  2. 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 as good, general representatives of protostome genomes. Currently sequenced insect and nematode genomes are less suitable models for deducing bilaterian ancestral states than lophotrochozoan genomes, despite the array of powerful genetic and mechanistic manipulation techniques in these ecdysozoans. A distinct category of genes that includes those present in non-bilaterians and lophotrochozoans, but which are absent from ecdysozoans and deuterostomes, highlights the need for further lophotrochozoan data to gain a more complete understanding of the gene complement of the bilaterian ancestor. PMID:19781084

  3. Lethal Traffic Jam

    NSDL National Science Digital Library

    Eefjan Breukink (Utrecht University; Department of Chemical Biology and Organic Chemistry)

    2009-08-07

    This perspective discusses new discoveries on the effects of the antibiotics chloramphenicol and tetracycline. The antibiotics chloramphenicol and tetracycline were discovered in the late 1940s shortly after the introduction of penicillin. Elucidation of the structure of the ribosome revealed how they bind to this target structure and inhibit protein synthesis. In a recent report, van Stelten et al. demonstrate another mode of action for these antibiotics, involving the destruction of a complex that allows proteins to be translocated across (or into) the bacterial membrane.

  4. Heterochromatin position effects on circularized sex chromosomes cause filicidal embryonic lethality in Drosophila melanogaster.

    PubMed

    Ferree, Patrick M; Gomez, Karina; Rominger, Peter; Howard, Dagnie; Kornfeld, Hannah; Barbash, Daniel A

    2014-04-01

    Some circularized X-Y chromosomes in Drosophila melanogaster are mitotically unstable and induce early embryonic lethality, but the genetic basis is unknown. Our experiments suggest that a large region of X-linked satellite DNA causes anaphase bridges and lethality when placed into a new heterochromatic environment within certain circularized X-Y chromosomes. These results reveal that repetitive sequences can be incompatible with one another in cis. The lethal phenotype also bears a remarkable resemblance to a case of interspecific hybrid lethality. PMID:24478337

  5. Synthetic Antibodies with a Human Framework That Protect Mice from Lethal Sudan Ebolavirus Challenge

    PubMed Central

    2014-01-01

    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

  6. Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma

    PubMed Central

    Lock, Frances E.; Rebollo, Rita; Miceli-Royer, Katharine; Gagnier, Liane; Kuah, Sabrina; Babaian, Artem; Sistiaga-Poveda, Maialen; Lai, C. Benjamin; Nemirovsky, Oksana; Serrano, Isabel; Steidl, Christian; Karimi, Mohammad M.; Mager, Dixie L.

    2014-01-01

    Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients. PMID:25114248

  7. Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma.

    PubMed

    Lock, Frances E; Rebollo, Rita; Miceli-Royer, Katharine; Gagnier, Liane; Kuah, Sabrina; Babaian, Artem; Sistiaga-Poveda, Maialen; Lai, C Benjamin; Nemirovsky, Oksana; Serrano, Isabel; Steidl, Christian; Karimi, Mohammad M; Mager, Dixie L

    2014-08-26

    Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients. PMID:25114248

  8. A genome-wide screen for methyl methanesulfonate-sensitive mutants reveals genes required for S phase progression in the presence of DNA damage

    PubMed Central

    Chang, Michael; Bellaoui, Mohammed; Boone, Charles; Brown, Grant W.

    2002-01-01

    We performed a systematic screen of the set of ?5,000 viable Saccharomyces cerevisiae haploid gene deletion mutants and have identified 103 genes whose deletion causes sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). In total, 40 previously uncharacterized alkylation damage response genes were identified. Comparison with the set of genes known to be transcriptionally induced in response to MMS revealed surprisingly little overlap with those required for MMS resistance, indicating that transcriptional regulation plays little, if any, role in the response to MMS damage. Clustering of the MMS response genes on the basis of their cross-sensitivities to hydroxyurea, UV radiation, and ionizing radiation revealed a DNA damage core of genes required for responses to a broad range of DNA-damaging agents. Of particular significance, we identified a subset of genes that show a specific MMS response, displaying defects in S phase progression only in the presence of MMS. These genes may promote replication fork stability or processivity during encounters between replication forks and DNA damage. PMID:12482937

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

    SciTech Connect

    Coulbault, Laurent [Laboratoire de Biochimie A, Centre Hospitalier et Universitaire de Caen, Avenue Cote de Nacre, 14033 Caen cedex (France); Herlicoviez, Danielle [Service d'Anatomie Pathologique, Centre Hospitalier et Universitaire de Caen, Avenue Cote de Nacre, 14033 Caen cedex (France); Chapon, Francoise [Service d'Anatomie Pathologique, Centre Hospitalier et Universitaire de Caen, Avenue Cote de Nacre, 14033 Caen cedex (France); Read, Marie-Helene [Departement de Genetique et Reproduction, Centre Hospitalier et Universitaire de Caen, Avenue Cote de Nacre, 14033 Caen cedex (France); Penniello, Marie-Jose [Departement de Pediatrie, Centre Hospitalier et Universitaire de Caen, Avenue Cote de Nacre, 14033 Caen cedex (France); Reynier, Pascal [INSERM U694, Laboratoire de Biochimie et Biologie Moleculaire, Centre Hospitalier et Universitaire, 49033 Angers cedex 1 (France); Fayet, Guillemette [INSERM U582-Institut de Myologie, Centre Hospitalier et Universitaire de Pitie-Salpetriere, 75013 Paris (France); Lombes, Anne [INSERM U582-Institut de Myologie, Centre Hospitalier et Universitaire de Pitie-Salpetriere, 75013 Paris (France); Jauzac, Philippe [Laboratoire de Biochimie A, Centre Hospitalier et Universitaire de Caen, Avenue Cote de Nacre, 14033 Caen cedex (France); Allouche, Stephane [Laboratoire de Biochimie A, Centre Hospitalier et Universitaire de Caen, Avenue Cote de Nacre, 14033 Caen cedex (France)]. 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.

  10. Uncovering Buffered Pleiotropy: A Genome-Scale Screen for mel-28 Genetic Interactors in Caenorhabditis elegans

    PubMed Central

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

    2013-01-01

    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. PMID:24281427

  11. Chemical Genomic Screening of a Saccharomyces cerevisiae Genomewide Mutant Collection Reveals Genes Required for Defense against Four Antimicrobial Peptides Derived from Proteins Found in Human Saliva

    PubMed Central

    Bhatt, Sanjay; Schoenly, Nathan E.; Lee, Anna Y.; Nislow, Corey; Bobek, Libuse A.

    2013-01-01

    To compare the effects of four antimicrobial peptides (MUC7 12-mer, histatin 12-mer, cathelicidin KR20, and a peptide containing lactoferricin amino acids 1 to 11) on the yeast Saccharomyces cerevisiae, we employed a genomewide fitness screen of combined collections of mutants with homozygous deletions of nonessential genes and heterozygous deletions of essential genes. When an arbitrary fitness score cutoffs of 1 (indicating a fitness defect, or hypersensitivity) and ?1 (indicating a fitness gain, or resistance) was used, 425 of the 5,902 mutants tested exhibited altered fitness when treated with at least one peptide. Functional analysis of the 425 strains revealed enrichment among the identified deletions in gene groups associated with the Gene Ontology (GO) terms “ribosomal subunit,” “ribosome biogenesis,” “protein glycosylation,” “vacuolar transport,” “Golgi vesicle transport,” “negative regulation of transcription,” and others. Fitness profiles of all four tested peptides were highly similar, particularly among mutant strains exhibiting the greatest fitness defects. The latter group included deletions in several genes involved in induction of the RIM101 signaling pathway, including several components of the ESCRT sorting machinery. The RIM101 signaling regulates response of yeasts to alkaline and neutral pH and high salts, and our data indicate that this pathway also plays a prominent role in regulating protective measures against all four tested peptides. In summary, the results of the chemical genomic screens of S. cerevisiae mutant collection suggest that the four antimicrobial peptides, despite their differences in structure and physical properties, share many interactions with S. cerevisiae cells and consequently a high degree of similarity between their modes of action. PMID:23208710

  12. MicroRNA screen of human embryonic stem cell differentiation reveals miR-105 as an enhancer of megakaryopoiesis from adult CD34+ cells.

    PubMed

    Kamat, Viraj; Paluru, Prasuna; Myint, Melissa; French, Deborah L; Gadue, Paul; Diamond, Scott L

    2014-05-01

    MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96-well plate electroporation, we screened 466 human miRNA mimics by four-color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4-5 days post-transfection, flow cytometry of erythroid (CD235(+)CD41(-)), megakaryocyte (CD41(+)CD42(+)), and myeloid (CD18(+)CD235(-)) lineages revealed miR-105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC-derived CMPs, miR-105 caused a sixfold enhancement in megakaryocyte production. miR-513a, miR-571, and miR-195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR-105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34(+) cells derived from peripheral blood. In addition, adult CD34(+) cells express endogenous miR-105 during megakaryocyte differentiation. siRNA knockdown of the hematopoietic transcription factor c-Myb caused a similar enhancement of megakaryocyte production as miR-105. Finally, a luciferase/c-Myb-3'UTR construct and Western blot analysis demonstrated that the hematopoietic transcription factor c-Myb mRNA was a target of miR-105. We report a novel hESC-based miR screening platform and demonstrate that miR-105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis. PMID:24446170

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

    PubMed Central

    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.

  14. Identification of potential synthetic lethal genes to p53 using a computational biology approach

    PubMed Central

    2013-01-01

    Background Identification of genes that are synthetic lethal to p53 is an important strategy for anticancer therapy as p53 mutations have been reported to occur in more than half of all human cancer cases. Although genome-wide RNAi screening is an effective approach to finding synthetic lethal genes, it is costly and labor-intensive. Methods To illustrate this approach, we identified potentially druggable genes synthetically lethal for p53 using three microarray datasets for gene expression profiles of the NCI-60 cancer cell lines, one next-generation sequencing (RNA-Seq) dataset from the Cancer Genome Atlas (TCGA) project, and one gene expression data from the Cancer Cell Line Encyclopedia (CCLE) project. We selected the genes which encoded kinases and had significantly higher expression in the tumors with functional p53 mutations (somatic mutations) than in the tumors without functional p53 mutations as the candidates of druggable synthetic lethal genes for p53. We identified important regulatory networks and functional categories pertinent to these genes, and performed an extensive survey of literature to find experimental evidence that support the synthetic lethality relationships between the genes identified and p53. We also examined the drug sensitivity difference between NCI-60 cell lines with functional p53 mutations and NCI-60 cell lines without functional p53 mutations for the compounds that target the kinases encoded by the genes identified. Results Our results indicated that some of the candidate genes we identified had been experimentally verified to be synthetic lethal for p53 and promising targets for anticancer therapy while some other genes were putative targets for development of cancer therapeutic agents. Conclusions Our study indicated that pre-screening of potential synthetic lethal genes using gene expression profiles is a promising approach for improving the efficiency of synthetic lethal RNAi screening. PMID:24025726

  15. Introduction to Lethal School Violence

    Microsoft Academic Search

    Jeffrey A. Daniels; Mary C. Bradley

    \\u000a In this chapter, we offer an introduction to the topic of the book, lethal school violence (LSV). We begin with an introduction\\u000a to and definition of LSV, and then highlight five different situations that often result in fatalities (i.e., suicide, rampage\\u000a shootings, gang-related deaths, domestic murder\\/suicide that occurs on campus, and barricaded captive events). We then turn\\u000a our attention to

  16. Electroshock weapons can be lethal!

    Microsoft Academic Search

    Marjorie Lundquist

    2008-01-01

    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

  17. 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 are urgently needed to avoid underestimating tropical diversity, and the use of mtDNA markers can be instrumental in identifying and prioritizing taxa for species discovery. PMID:22815761

  18. Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

    PubMed Central

    McMahon, Matthew D.

    2014-01-01

    Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we conducted in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate), and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. In this study, we introduce novel thioesterases with improved specificity for saturated, branched, and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent. PMID:24271180

  19. A signal peptide secretion screen in Fucus distichus embryos reveals expression of glucanase, EGF domain-containing, and LRR receptor kinase-like polypeptides during asymmetric cell growth.

    PubMed

    Belanger, Kenneth D; Wyman, Aaron J; Sudol, Michelle N; Singla-Pareek, Sneh L; Quatrano, Ralph S

    2003-10-01

    Zygotes of the brown alga Fucus distichus (L.) Powell develop polarity prior to the first embryonic cell division and retain a pattern of asymmetric growth during early embryogenesis. In order to identify F. distichus polypeptides secreted during asymmetric cell growth, we used a functional assay in Saccharomyces cerevisiae to screen a cDNA library generated from asymmetrically growing Fucus embryos for sequences encoding polypeptides that function as signal peptides for secretion. We isolated and sequenced 222 plasmids containing Fucus cDNAs encoding signal peptide activity. The cDNA inserts from these plasmids were translated in silico into 244 potential polypeptide sequences, 169 of which are predicted to contain signal peptides. BlastP analysis of the Fucus sequences revealed similarity between many Fucus proteins and cell surface proteins that function in development in other eukaryotes, including epidermal growth factor (EGF)-like repeat-containing proteins, plant leucine-rich repeat (LRR)-receptor kinases, and algal beta-1, 3-exoglucanase. However, most of the isolated Fucus polypeptides lack similarity to known proteins. The isolation of cDNAs encoding secreted Fucus proteins provides an important step toward characterizing cell surface proteins important for asymmetric organization and growth in fucoid embryos. PMID:12836024

  20. 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

  1. Harnessing synthetic lethal interactions in anticancer drug discovery

    PubMed Central

    Chan, Denise A.; Giaccia, Amato J.

    2013-01-01

    Unique features of tumours that can be exploited by targeted therapies are a key focus of current cancer research. One such approach is known as synthetic lethality screening, which involves searching for genetic interactions of two mutations whereby the presence of either mutation alone has no effect on cell viability but the combination of the two mutations results in cell death. The presence of one of these mutations in cancer cells but not in normal cells can therefore create opportunities to selectively kill cancer cells by mimicking the effect of the second genetic mutation with targeted therapy. Here, we summarize strategies that can be used to identify synthetic lethal interactions for anticancer drug discovery, describe examples of such interactions that are currently being investigated in preclinical and clinical studies of targeted anticancer therapies, and discuss the challenges of realizing the full potential of such therapies. PMID:21532565

  2. Early events of lethal action by tobramycin in Pseudomonas aeruginosa

    SciTech Connect

    Raulston, J.E.

    1988-01-01

    The immediate activities of the aminoglycoside antibiotic, tobramycin, were investigated in Pseudomonas aeruginosa PAO1. The influence of carbon growth substate and the antibiotic exposure environment in the magnitude of activity were examined. Lethality by 8 {mu}g/ml tobramycin occurred rapidly (1 to 3 minutes). The release of specific cellular components into the supernatant was associated with lethality. This material was initially detected as an increase in UV-absorbance. Magnesium in the reaction mixture provided protection against lethality and leakage, but did not reverse lethal damage after a 3 minute tobramycin treatment. Also, uptake of {sup 3}H-tobramycin was reduced in the presence of magnesium. Cells grown with glucose as a carbon source were more susceptible than organic acid grown cells as was the rapidity and amount of cell damage. Analyses of the leakage material revealed a 2-fold increase of protein in the supernatant after a 1-3 minute treatment which paralleled lethality. A prominent 29 kDa protein was observed by SDS-PAGE in the released material, which has been identified as the periplasmic enzyme, {beta}-lactamase. The immediate activities of tobramycin did not involve (i) release of overall cell protein, (ii) massive loss of total pool amino acids, (iii) cell lysis, (iv) inhibition of proline uptake, (v) release of lipopolysaccharide, or (vi) leakage of ATP. Electron microscopy showed no apparent damage after a 3 minute exposure. 40% inhibition of protein synthesis had occurred by 3 minutes of exposure, while release of UV-absorbing material and lethality were detectable after only 1 minute. Resistant cystic fibrosis isolates of P. aeruginosa did not leak under the same experimental conditions, but one of two susceptible strains examined did show increased UV-absorbance following treatment.

  3. Genome-wide screen reveals WNT11, a non-canonical WNT gene, as a direct target of ETS transcription factor ERG.

    PubMed

    Mochmann, L H; Bock, J; Ortiz-Tánchez, J; Schlee, C; Bohne, A; Neumann, K; Hofmann, W K; Thiel, E; Baldus, C D

    2011-04-28

    E26 transforming sequence-related gene (ERG) is a transcription factor involved in normal hematopoiesis and is dysregulated in leukemia. ERG mRNA overexpression was associated with poor prognosis in a subset of patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Herein, a genome-wide screen of ERG target genes was conducted by chromatin immunoprecipitation-on-chip (ChIP-chip) in Jurkat cells. In this screen, 342 significant annotated genes were derived from this global approach. Notably, ERG-enriched targets included WNT signaling genes: WNT11, WNT2, WNT9A, CCND1 and FZD7. Furthermore, chromatin immunoprecipitation (ChIP) of normal and primary leukemia bone marrow material also confirmed WNT11 as a target of ERG in six of seven patient samples. A larger sampling of patient diagnostic material revealed that ERG and WNT11 mRNA were co-expressed in 80% of AML (n=30) and 40% in T-ALL (n=30) bone marrow samples. Small interfering RNA (siRNA)-mediated knockdown of ERG confirmed downregulation of WNT11 transcripts. Conversely, in a tet-on ERG-inducible assay, WNT11 transcripts were co-stimulated. A WNT pathway agonist, 6-bromoindirubin-3-oxime (BIO), was used to determine the effect of cell growth on the ERG-inducible cells. The addition of BIO resulted in an ERG-dependent proliferative growth advantage over ERG-uninduced cells. Finally, ERG induction prompted morphological transformation whereby round unpolarized K562 cells developed elongated protrusions and became polarized. This morphological transformation could effectively be inhibited with BIO and with siRNA knockdown of WNT11. In conclusion, ERG transcriptional networks in leukemia converge on WNT signaling targets. Specifically, WNT11 emerged as a direct target of ERG. Potent ERG induction promoted morphological transformation through WNT11 signals. The findings in this study unravel new ERG-directed molecular signals that may contribute to the resistance of current therapies in acute leukemia patients with poor prognosis characterized by high ERG mRNA expression. PMID:21242973

  4. 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 ‘lethalreveals 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

  5. 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 (...

  6. 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:24911315

  7. Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q

    PubMed Central

    Dekel-Naftali, Michal; Aviram-Goldring, Ayala; Litmanovitch, Talia; Shamash, Jana; Reznik-Wolf, Haike; Laevsky, Ilana; Amit, Michal; Itskovitz-Eldor, Joseph; Yung, Yuval; Hourvitz, Ariel; Schiff, Eyal; Rienstein, Shlomit

    2012-01-01

    Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells. PMID:22713809

  8. 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. PMID:25036619

  9. 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).

  10. 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. PMID:24344743

  11. Screening for HBV, HCV and HIV genomes in blood donations: shortcomings of pooling revealed by a multicentre study simulating real-time testing

    Microsoft Academic Search

    Jean-Jacques Lefrère; Jean-François Cantaloube; Christine Defer; Bernard Mercier; Pascale Loiseau; Dominique Vignon; Jean-Michel Pawlotsky; Philippe Biagini; Joelle Lerable; Philippe Rouger; Françoise Roudot-Thoraval; Claude Férec

    1999-01-01

    This study was undertaken in order to determine whether screening of viremic blood donations by testing of pooled donor samples could constitute a technically feasible transfusional safety measure. A pilot study of real-time simulation, on a day-to-day basis, of screening of three viral genomes (hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV)) was conducted by

  12. Newborn Screening

    MedlinePLUS

    ... participate in the community. Home > Baby > Newborn screening Newborn screening Newborn screening checks for serious but rare ... been added to your dashboard . All babies get newborn screening Your baby gets newborn screening tests before ...

  13. PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila

    PubMed Central

    Johnson, Matthew L.; Nagengast, Alexis A.; Salz, Helen K.

    2010-01-01

    Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance. PMID:20221253

  14. 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…

  15. A Barcode Screen for Epigenetic Regulators Reveals a Role for the NuB4\\/HAT-B Histone Acetyltransferase Complex in Histone Turnover

    Microsoft Academic Search

    Kitty F. Verzijlbergen; Tibor van Welsem; Daoud Sie; Tineke L. Lenstra; Daniel J. Turner; Frank C. P. Holstege; Ron M. Kerkhoven; Fred van Leeuwen

    2011-01-01

    Dynamic modification of histone proteins plays a key role in regulating gene expression. However, histones themselves can also be dynamic, which potentially affects the stability of histone modifications. To determine the molecular mechanisms of histone turnover, we developed a parallel screening method for epigenetic regulators by analyzing chromatin states on DNA barcodes. Histone turnover was quantified by employing a genetic

  16. 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...

  17. 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.

  18. 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

  19. 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

  20. 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-1?, TNF-?, IFN-?, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-?-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

  1. 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.

  2. Two-Color Cell Array Screen Reveals Interdependent Roles for Histone Chaperones and a Chromatin Boundary Regulator in Histone Gene Repression

    Microsoft Academic Search

    Jeffrey Fillingham; Pinay Kainth; Jean-Philippe Lambert; Harm van Bakel; Kyle Tsui; Lourdes Peña-Castillo; Corey Nislow; Daniel Figeys; Timothy R. Hughes; Jack Greenblatt; Brenda J. Andrews

    2009-01-01

    SUMMARY We describe a fluorescent reporter system that exploits the functional genomic tools available in budding yeast to systematically assess conse- quences of genetic perturbations on gene expres- sion. We used our Reporter-Synthetic Genetic Array (R-SGA) method to screen for regulators of core histone gene expression. We discovered that the histone chaperone Rtt106 functions in a pathway with two other

  3. Genome-wide screen reveals WNT11, a non-canonical WNT gene, as a direct target of ETS transcription factor ERG

    Microsoft Academic Search

    L H Mochmann; J Bock; J Ortiz-Tánchez; C Schlee; A Bohne; K Neumann; W K Hofmann; E Thiel; C D Baldus

    2011-01-01

    E26 transforming sequence-related gene (ERG) is a transcription factor involved in normal hematopoiesis and is dysregulated in leukemia. ERG mRNA overexpression was associated with poor prognosis in a subset of patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Herein, a genome-wide screen of ERG target genes was conducted by chromatin immunoprecipitation-on-chip (ChIP-chip) in Jurkat cells. In

  4. Synthetic lethal genes to mutant p53.

    PubMed

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

    2015-04-20

    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. PMID:25881697

  5. Reaming experiments for the lethality test system

    SciTech Connect

    Hooten, D.; Stanley, P.

    1988-01-01

    Various reaming techniques were tried for use on the barrel of the Lethality Test System railgun. This report covers the successes and failures of the reamers and the techniques that were tried. 5 figs.

  6. 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 Central

    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

  7. Synthetic Lethality of Cohesins with PARPs and Replication Fork Mediators

    E-print Network

    Jessica L. Mclellan; Nigel J. O’neil; Irene Barrett; Elizabeth Ferree; Derek M. Van Pel; Payal Sipahimalani; Jennifer Bryan; Ann M. Rose; Philip Hieter

    Synthetic lethality has been proposed as a way to leverage the genetic differences found in tumor cells to affect their selective killing. Cohesins, which tether sister chromatids together until anaphase onset, are mutated in a variety of tumor types. The elucidation of synthetic lethal interactions with cohesin mutants therefore identifies potential therapeutic targets. We used a cross-species approach to identify robust negative genetic interactions with cohesin mutants. Utilizing essential and non-essential mutant synthetic genetic arrays in Saccharomyces cerevisiae, we screened genome-wide for genetic interactions with hypomorphic mutations in cohesin genes. A somatic cell proliferation assay in Caenorhabditis elegans demonstrated that the majority of interactions were conserved. Analysis of the interactions found that cohesin mutants require the function of genes that mediate replication fork progression. Conservation of these interactions between replication fork mediators and cohesin in both yeast and C. elegans prompted us to test whether other replication fork mediators not found in the yeast were required for viability in cohesin mutants. PARP1 has roles in the DNA damage response but also in the restart of stalled replication forks. We found that a hypomorphic allele of the C. elegans SMC1 orthologue, him-1(e879), genetically interacted with mutations in the orthologues of PAR metabolism genes resulting in a reduced brood size and somatic cell defects. We then demonstrated that this interaction is conserved in human cells by showing that PARP inhibitors reduce the viability of cultured human cells depleted for cohesin components. This work

  8. 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

  9. A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3?,4?-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

    PubMed Central

    Lee, Yan Quan; Goh, Amanda S. P.; Ch'ng, Jun Hong; Nosten, François H.; Preiser, Peter Rainer; Pervaiz, Shazib; Yadav, Sanjiv Kumar

    2014-01-01

    Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca2+. This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3?,4?-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs. PMID:24217693

  10. Artistic screening

    Microsoft Academic Search

    Victor Ostromoukhov; Roger D. Hersch

    1995-01-01

    Artistic screening is a new image reproduction technique incorpo- rating freely created artistic screen elements for generating halftones. Fixed predefined dot contours associated with given intensity lev- els determine the screen dot shape's growing behavior. Screen dot contours associated with each intensity level are obtained by inter- polation between the fixed predefined dot contours. A user-defined mapping transforms screen elements

  11. Hybrid lethal systems in the Drosophila melanogaster species complex

    Microsoft Academic Search

    Kyoichi Sawamura; Takao K. Watanabe; Masa-Toshi Yamamoto

    1993-01-01

    Lethal phases of the hybrids betweenDrosophila melanogaster and its sibling species,D. simulans are classified into three types: (1) embryonic lethality in hybrids carryingD. simulans cytoplasm andD. melanogaster X chromosome, (2) larval lethality in hybrids not carryingD. simulans X, and (3) temperature-sensitive pupal lethality in hybrids carryingD. simulans X. The same lethal phases are also observed when either of the two

  12. Sensitivity of seven PCRs for early detection of koi herpesvirus in experimentally infected carp, Cyprinus carpio L., by lethal and non-lethal sampling methods.

    PubMed

    Monaghan, S J; Thompson, K D; Adams, A; Bergmann, S M

    2015-03-01

    Koi herpesvirus (KHV) causes an economically important, highly infectious disease in common carp and koi, Cyprinus carpio L. Since the occurrence of mass mortalities worldwide, highly specific and sensitive molecular diagnostic methods have been developed for KHV detection. The sensitivity and reliability of these assays have essentially focused at the detection of low viral DNA copy numbers during latent or persistent infections. However, the efficacy of these assays has not been investigated with regard to low-level viraemia during acute infection stages. This study was conducted to compare the sensitivity of seven different polymerase chain reaction (PCR) assays to detect KHV during the first hours and days post-infection (hpi; dpi), using lethal and non-lethal sampling methods. The results highlight the limitations of the assays for detecting virus during the first 4 dpi despite rapid mortality in experimentally infected carp. False-negative results were associated with time post-infection and the tissue sampled. Non-lethal sampling appears effective for KHV screening, with efficient detection in mucus samples obtained from external swabs during this early infection period (<5 dpi), while biopsies from gills and kidney were negative using the same PCR assays. Non-lethal sampling may improve the reliability of KHV detection in subclinical, acutely infected carp. PMID:24547985

  13. 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 any possible deleterious effects on host cellular biology. PMID:25144302

  14. 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 Central

    Mudhasani, Rajini; Kota, Krishna P.; Retterer, Cary; Tran, Julie P.; Whitehouse, Chris A.; Bavari, Sina

    2014-01-01

    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 any possible deleterious effects on host cellular biology. PMID:25144302

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

    SciTech Connect

    Saebel, Crystal E.; Carbone, Ryan; Dabous, John R.; Lo, Suet Y. [Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6 (Canada)] [Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6 (Canada); Siemann, Stefan, E-mail: ssiemann@laurentian.ca [Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6 (Canada)] [Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6 (Canada)

    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.

  16. A screen for modifiers of decapentaplegic mutant phenotypes identifies lilliputian, the only member of the Fragile-X/Burkitt's Lymphoma family of transcription factors in Drosophila melanogaster.

    PubMed

    Su, M A; Wisotzkey, R G; Newfeld, S J

    2001-02-01

    The decapentaplegic (dpp) gene directs numerous developmental events in Drosophila melanogaster. dpp encodes a member of the Transforming Growth Factor-beta family of secreted signaling molecules. At this time, mechanisms of dpp signaling have not yet been fully described. Therefore we conducted a genetic screen for new dpp signaling pathway components. The screen exploited a transvection-dependent dpp phenotype: heldout wings. The screen generated 30 mutations that appear to disrupt transvection at dpp. One of the mutations is a translocation with a recessive lethal breakpoint in cytological region 23C1-2. Genetic analyses identified a number of mutations allelic to this breakpoint. The 23C1-2 complementation group includes several mutations in the newly discovered gene lilliputian (lilli). lilli mutations that disrupt the transvection-dependent dpp phenotype are also dominant maternal enhancers of recessive embryonic lethal alleles of dpp and screw. lilli zygotic mutant embryos exhibit a partially ventralized phenotype similar to dpp embryonic lethal mutations. Phylogenetic analyses revealed that lilli encodes the only Drosophila member of a family of transcription factors that includes the human genes causing Fragile-X mental retardation (FMR2) and Burkitt's Lymphoma (LAF4). Taken together, the genetic and phylogenetic data suggest that lilli may be an activator of dpp expression in embryonic dorsal-ventral patterning and wing development. PMID:11156991

  17. Temperature-Sensitive Mutations in Drosophila Melanogaster, III. Dominant Lethals and Semilethals on Chromosome 2

    Microsoft Academic Search

    David T. Suzuki; Douglas Procunier

    1969-01-01

    Dominant temperature-sensitive lethal and semilethal (DTS-L) mutations induced by ethyl methanesulfonate have been recovered in chromosome 2 of Drosophila melanogaster. The 19 (0.39% of the chromosomes screened) characterized had greater than 65 per cent viability at 22 degrees ± 2 degrees C and less than 5 per cent at 29 degrees ± 0.5 degrees C. Fifteen of the DTS-L's map

  18. A high-throughput fatty acid profiling screen reveals novel variations in fatty acid biosynthesis in Chlamydomonas reinhardtii and related algae.

    PubMed

    Pflaster, Erin L; Schwabe, Michael J; Becker, Joyanne; Wilkinson, Melissa S; Parmer, Ashley; Clemente, Thomas E; Cahoon, Edgar B; Riekhof, Wayne R

    2014-11-01

    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 ?-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ?-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make ?6-unsaturated polyunsaturated fatty acids (PUFA) rather than the ?5 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

  19. Identification and classification of genes required for tolerance to high-sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiae.

    PubMed

    Ando, Akira; Tanaka, Fumiko; Murata, Yoshinori; Takagi, Hiroshi; Shima, Jun

    2006-03-01

    Yeasts used in bread making are exposed to high concentrations of sucrose during sweet dough fermentation. Despite its importance, tolerance to high-sucrose stress is poorly understood at the gene level. To clarify the genes required for tolerance to high-sucrose stress, genome-wide screening was undertaken using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 273 deletions that yielded high sucrose sensitivity, approximately 20 of which were previously uncharacterized. These 273 deleted genes were classified based on their cellular function and localization of their gene products. Cross-sensitivity of the high-sucrose-sensitive mutants to high concentrations of NaCl and sorbitol was studied. Among the 273 sucrose-sensitive deletion mutants, 269 showed cross-sensitivities to sorbitol or NaCl, and four (i.e. ade5,7, ade6, ade8, and pde2) were specifically sensitive to high sucrose. The general stress response pathways via high-osmolarity glycerol and stress response element pathways and the function of the invertase in the ade mutants were similar to those in the wild-type strain. In the presence of high-sucrose stress, intracellular contents of ATP in ade mutants were at least twofold lower than that of the wild-type cells, suggesting that depletion of ATP is a factor in sensitivity to high-sucrose stress. The genes identified in this study might be important for tolerance to high-sucrose stress, and therefore should be target genes in future research into molecular modification for breeding of yeast tolerant to high-sucrose stress. PMID:16487347

  20. 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

  1. Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

    PubMed

    Agarwal, A; MacKenzie, R J; Eide, C A; Davare, M A; Watanabe-Smith, K; Tognon, C E; Mongoue-Tchokote, S; Park, B; Braziel, R M; Tyner, J W; Druker, B J

    2014-08-11

    To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (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 (IL2R?) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2R? abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2R? in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2R? completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2R? interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2R? 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 IL2R?, indicating IL2R? and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2R? in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, 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.Oncogene advance online publication, 11 August 2014; doi:10.1038/onc.2014.243. PMID:25109334

  2. A genome-wide small interfering RNA (siRNA) screen reveals nuclear factor-?B (NF-?B)-independent regulators of NOD2-induced interleukin-8 (IL-8) secretion.

    PubMed

    Warner, Neil; Burberry, Aaron; Pliakas, Maria; McDonald, Christine; Núñez, Gabriel

    2014-10-10

    NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNF? counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8. PMID:25170077

  3. 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. PMID:23233475

  4. 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…

  5. 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

  6. A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching

    PubMed Central

    Hopkins, Kaycie C.; McLane, Laura M.; Maqbool, Tariq; Panda, Debasis; Gordesky-Gold, Beth; Cherry, Sara

    2013-01-01

    Bunyaviruses are an emerging group of medically important viruses, many of which are transmitted from insects to mammals. To identify host factors that impact infection, we performed a genome-wide RNAi screen in Drosophila and identified 131 genes that impacted infection of the mosquito-transmitted bunyavirus Rift Valley fever virus (RVFV). Dcp2, the catalytic component of the mRNA decapping machinery, and two decapping activators, DDX6 and LSM7, were antiviral against disparate bunyaviruses in both insect cells and adult flies. Bunyaviruses 5? cap their mRNAs by “cap-snatching” the 5? ends of poorly defined host mRNAs. We found that RVFV cap-snatches the 5? ends of Dcp2 targeted mRNAs, including cell cycle-related genes. Loss of Dcp2 allows increased viral transcription without impacting viral mRNA stability, while ectopic expression of Dcp2 impedes viral transcription. Furthermore, arresting cells in late S/early G2 led to increased Dcp2 mRNA targets and increased RVFV replication. Therefore, RVFV competes for the Dcp2-accessible mRNA pool, which is dynamically regulated and can present a bottleneck for viral replication. PMID:23824541

  7. 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

  8. A BLOC-1 Mutation Screen Reveals a Novel BLOC1S3 Mutation in Hermansky-Pudlak Syndrome Type 8 (HPS-8)

    PubMed Central

    Cullinane, Andrew R; Curry, James A; Golas, Gretchen; Pan, James; Carmona-Rivera, Carmelo; Hess, Richard A; White, James G; Huizing, Marjan; Gahl, William A

    2012-01-01

    Summary Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1–6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all 8 genes encoding the Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1) proteins in these individuals. Here we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6 year-old Iranian boy. This mutation caused nonsense mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient’s melanocytes showed aberrant localization of TYRP1, with increased plasma-membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only 2 patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified. PMID:22709368

  9. Genome-wide RNAi screen reveals the E3 SUMO-protein ligase gene SIZ1 as a novel determinant of furfural tolerance in Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    Background Furfural is a major growth inhibitor in lignocellulosic hydrolysates and improving furfural tolerance of microorganisms is critical for rapid and efficient fermentation of lignocellulosic biomass. In this study, we used the RNAi-Assisted Genome Evolution (RAGE) method to select for furfural resistant mutants of Saccharomyces cerevisiae, and identified a new determinant of furfural tolerance. Results By using a genome-wide RNAi (RNA-interference) screen in S. cerevisiae for genes involved in furfural tolerance, we identified SIZ1, a gene encoding an E3 SUMO-protein ligase. Disruption of SIZ1 gene function by knockdown or deletion conferred significantly higher furfural tolerance compared to other previously reported metabolic engineering strategies in S. cerevisiae. This improved furfural tolerance of siz1? cells is accompanied by rapid furfural reduction to furfuryl alcohol and leads to higher ethanol productivity in the presence of furfural. In addition, the siz1? mutant also exhibited tolerance towards oxidative stress, suggesting that oxidative stress tolerance related proteins may be under the SUMO regulation of SIZ1p and responsible for furfural tolerance. Conclusions Using a genome-wide approach, we identified a novel determinant for furfural tolerance, providing valuable insights into the design of recombinant microbes for efficient lignocellulose fermentation. PMID:24904688

  10. A touch screen-automated cognitive test battery reveals impaired attention, memory abnormalities, and increased response inhibition in the TgCRND8 mouse model of Alzheimer's disease.

    PubMed

    Romberg, Carola; Horner, Alexa E; Bussey, Timothy J; Saksida, Lisa M

    2013-03-01

    Transgenic mouse models of Alzheimer's disease (AD) with abundant ?-amyloid develop memory impairments. However, multiple nonmnemonic cognitive domains such as attention and executive control are also compromised early in AD individuals, but have not been routinely assessed in animal models. Here, we assessed the cognitive abilities of TgCRND8 mice-a widely used model of ?-amyloid pathology-with a touch screen-based automated test battery. The test battery comprises highly translatable tests of multiple cognitive constructs impaired in human AD, such as memory, attention, and response control, as well as appropriate control tasks. We found that familial AD mutations affect not only memory, but also cause significant alterations of sustained attention and behavioral flexibility. Because changes in attention and response inhibition may affect performance on tests of other cognitive abilities including memory, our findings have important consequences for the assessment of disease mechanisms and therapeutics in animal models of AD. A more comprehensive phenotyping with specialized, multicomponent cognitive test batteries for mice might significantly advance translation from preclinical mouse studies to the clinic. PMID:22959727

  11. A touch screen-automated cognitive test battery reveals impaired attention, memory abnormalities, and increased response inhibition in the TgCRND8 mouse model of Alzheimer's disease

    PubMed Central

    Romberg, Carola; Horner, Alexa E.; Bussey, Timothy J.; Saksida, Lisa M.

    2013-01-01

    Transgenic mouse models of Alzheimer's disease (AD) with abundant ?-amyloid develop memory impairments. However, multiple nonmnemonic cognitive domains such as attention and executive control are also compromised early in AD individuals, but have not been routinely assessed in animal models. Here, we assessed the cognitive abilities of TgCRND8 mice—a widely used model of ?-amyloid pathology—with a touch screen-based automated test battery. The test battery comprises highly translatable tests of multiple cognitive constructs impaired in human AD, such as memory, attention, and response control, as well as appropriate control tasks. We found that familial AD mutations affect not only memory, but also cause significant alterations of sustained attention and behavioral flexibility. Because changes in attention and response inhibition may affect performance on tests of other cognitive abilities including memory, our findings have important consequences for the assessment of disease mechanisms and therapeutics in animal models of AD. A more comprehensive phenotyping with specialized, multicomponent cognitive test batteries for mice might significantly advance translation from preclinical mouse studies to the clinic. PMID:22959727

  12. CSNK1E/CTNNB1 Are Synthetic Lethal To TP53 in Colorectal Cancer and Are Markers for Prognosis

    PubMed Central

    Tiong, Khong-Loon; Chang, Kuo-Ching; Yeh, Kun-Tu; Liu, Ting-Yuan; Wu, Jia-Hong; Hsieh, Ping-Heng; Lin, Shu-Hui; Lai, Wei-Yun; Hsu, Yu-Chin; Chen, Jeou-Yuan; Chang, Jan-Gowth; Shieh, Grace S.

    2014-01-01

    Two genes are called synthetic lethal (SL) if their simultaneous mutations lead to cell death, but each individual mutation does not. Targeting SL partners of mutated cancer genes can kill cancer cells specifically, but leave normal cells intact. We present an integrated approach to uncovering SL pairs in colorectal cancer (CRC). Screening verified SL pairs using microarray gene expression data of cancerous and normal tissues, we first identified potential functionally relevant (simultaneously differentially expressed) gene pairs. From the top-ranked pairs, ~ 20 genes were chosen for immunohistochemistry (IHC) staining in 171 CRC patients. To find novel SL pairs, all 169 combined pairs from the individual IHC were synergistically correlated to five clinicopathological features, e.g. overall survival. Of the 11 predicted SL pairs, MSH2-POLB and CSNK1E-MYC were consistent with literature, and we validated the top two pairs, CSNK1E-TP53 and CTNNB1-TP53 using RNAi knockdown and small molecule inhibitors of CSNK1E in isogenic HCT-116 and RKO cells. Furthermore, synthetic lethality of CSNK1E and TP53 was verified in mouse model. Importantly, multivariate analysis revealed that CSNK1E-P53, CTNNB1-P53, MSH2-RB1, and BRCA1-WNT5A were independent prognosis markers from stage, with CSNK1E-P53 applicable to early-stage and the remaining three throughout all stages. Our findings suggest that CSNK1E is a promising target for TP53-mutant CRC patients which constitute ~ 40% to 50% of patients, while to date safety regarding inhibition of TP53 is controversial. Thus the integrated approach is useful in finding novel SL pairs for cancer therapeutics, and it is readily accessible and applicable to other cancers. PMID:24947187

  13. 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

  14. A genome-wide miRNA screen revealed miR-603 as a MGMT-regulating miRNA in glioblastomas

    PubMed Central

    Ng, Kimberly; Steed, Tyler; Nguyen, Thien; Futalan, Diahnn; Akers, Johnny C.; Sarkaria, Jann; Jiang, Tao; Chowdhury, Dipanjan; Carter, Bob S.; Chen, Clark C.

    2014-01-01

    MGMT expression is a critical determinant for therapeutic resistance to DNA alkylating agents. We previously demonstrated that MGMT expression is post-transcriptionally regulated by miR-181d and other miRNAs. Here, we performed a genome-wide screen to identify MGMT regulating miRNAs. Candidate miRNAs were further tested for inverse correlation with MGMT expression in clinical specimens. We identified 15 candidate miRNAs and characterized the top candidate, miR-603. Transfection of miR-603 suppressed MGMT mRNA/protein expression in vitro and in vivo; this effect was reversed by transfection with antimiR-603. miR-603 affinity-precipitated with MGMT mRNA and suppressed luciferase activity in an MGMT-3'UTR-luciferase assay, suggesting direct interaction between miR-603 and MGMT 3'UTR. miR-603 transfection enhanced the temozolomide (TMZ) sensitivity of MGMT-expressing glioblastoma cell lines. Importantly, miR-603 mediated MGMT suppression and TMZ resistance were reversed by expression of an MGMT cDNA. In a collection of 74 clinical glioblastoma specimens, both miR-603 and miR-181d levels inversely correlated with MGMT expression. Moreover, a combined index of the two miRNAs better reflected MGMT expression than each individually. These results suggest that MGMT is co-regulated by independent miRNAs. Characterization of these miRNAs should contribute toward strategies for enhancing the efficacy of DNA alkylating agents. PMID:24994119

  15. Non-lethal technologies—an overview

    Microsoft Academic Search

    Nick LEWER; Neil DAVISON

    hilst the focus for this issue of Disarmament Forum is on chemical and biological weapons, sight should not be lost of the spectrum of non-lethal technologies that are being deployed or under development. These technologies will have an increasing impact on war fighting, peace support operations, civil policing and prison control. It is our purpose here to briefly review the

  16. Crystal structure of the anthrax lethal factor

    Microsoft Academic Search

    Andrew D. Pannifer; Thiang Yian Wong; Robert Schwarzenbacher; Martin Renatus; Carlo Petosa; Jadwiga Bienkowska; D. Borden Lacy; R. John Collier; Stephen H. Leppla; Philip Hanna; Robert C. Liddington

    2001-01-01

    Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its

  17. 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

  18. Chemical mutagenesis testing in Drosophila. I. Comparison of positive and negative control data for sex-linked recessive lethal mutations and reciprocal translocations in three laboratories

    SciTech Connect

    Woodruff, R.C.; Mason, J.M.; Valencia, R.; Zimmering, S.

    1984-01-01

    As part of the validation phase of the Drosophila melanogaster segment of the National Toxicology Program, a comparison has been made of positive and negative controls for sex-linked recessive lethal mutations and reciprocal translocations from three laboratories. This comparison involves approximately 700,000 spontaneous recessive lethal mutation tests, 70,000 spontaneous translocation tests, and screens for genetic damage induced by N-nitrosodimethylamine and ..beta..-propiolactone. Spontaneous frequencies for lethal mutations and translocations were homogeneous in the laboratories regardless of solvent or broods sampled. Inhomogeneity was observed in induced frequencies among laboratories, but the variation was no greater than that found within a laboratory.

  19. 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

  20. A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter.

    PubMed

    Laser, H; Bongards, C; Schüller, J; Heck, S; Johnsson, N; Lehming, N

    2000-12-01

    The split-ubiquitin assay detects protein interactions in vivo. To identify proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we converted the split-ubiquitin assay into a generally applicable screen for binding partners of specific proteins in vivo. A library of genomic Saccharomyces cerevisiae DNA fragments fused to the N-terminal half of ubiquitin was constructed and transformed into yeast strains carrying either Gal4p or Tup1p as a bait. Both proteins were C-terminally extended by the C-terminal half of ubiquitin followed by a modified Ura3p with an arginine in position 1, a destabilizing residue in the N-end rule pathway. The bait fusion protein alone is stable and enzymatically active. However, upon interaction with its prey, a native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the ubiquitin-specific proteases and rapidly degraded by the N-end rule pathway. In both screens, Nhp6B was identified as a protein in close proximity to Gal4p as well as to Tup1p. Direct interaction between either protein and Nhp6B was confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a member of the HMG1 family of DNA-binding proteins, can influence transcriptional activation as well as repression at a specific locus in the chromosome of the yeast S. cerevisiae. PMID:11095729

  1. Lethal thyroid storm after uncontrolled intake of liothyronine in order to lose weight.

    PubMed

    Hartung, Benno; Schott, Matthias; Daldrup, Thomas; Ritz-Timme, Stefanie

    2010-11-01

    Thyroid hormones are sometimes used for purposes for which they are not approved. Reasons for off-label use can be overweight, prevailing depressive mood, or various somatic symptoms. Information about the intake of thyroid hormones in order to lose weight can be easily obtained from inappropriate/nonmedical websites. The objective of this case report is to describe the first case of a lethal abuse of liothyronine. The case was a 29-year-old male (BMI 32) without relevant illnesses. An autopsy was performed and followed by histological, toxicological, and clinical chemistry examinations. The autopsy revealed no relevant pathology. Histology showed multiple areas of focal cell necrosis in the myocardium and signs of acute heart failure including severe edema of the lungs; the follicles of the thyroid gland were markedly plump. Postmortem laboratory results indicated lethal liothyronine intoxication. Despite prevailing opinion, uncontrolled intake of liothyronine can cause lethal thyroid storm in a euthyroid patient without manifested cardiac illnesses. PMID:20145940

  2. Candidate Gene Screen in the Red Flour Beetle Tribolium Reveals Six3 as Ancient Regulator of Anterior Median Head and Central Complex Development

    PubMed Central

    Hein, Hendrikje Jeannette; Bucher, Gregor

    2011-01-01

    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. PMID:22216011

  3. In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2.

    PubMed

    Oh, Sang-Keun; Young, Carolyn; Lee, Minkyoung; Oliva, Ricardo; Bozkurt, Tolga O; Cano, Liliana M; Win, Joe; Bos, Jorunn I B; Liu, Hsin-Yin; van Damme, Mireille; Morgan, William; Choi, Doil; Van der Vossen, Edwin A G; Vleeshouwers, Vivianne G A A; Kamoun, Sophien

    2009-09-01

    The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD36(45-1), suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34-amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2. PMID:19794118

  4. A cancer screen in zebrafish identifies many ribosomal proteins as haploinsufficient tumor suppressors

    E-print Network

    Lai, Kevin

    2006-01-01

    A collection of over 500 lines of zebrafish (Danio rerio), each heterozygous for a recessive embryonic lethal mutation caused by a retroviral insertion, was screened for lines that displayed early mortality and externally ...

  5. Henipaviruses-unanswered questions of lethal zoonoses.

    PubMed

    Field, Hume; Kung, Nina

    2011-12-01

    The highly lethal Hendra and Nipah viruses have been described for little more than a decade, yet within that time have been aetiologically associated with major livestock and human health impacts, albeit on a limited scale. Do these emerging pathogens pose a broader threat, or are they inconsequential 'viral chatter'. Given their lethality, and the evident multi-generational human-to-human transmission associated with Nipah virus in Bangladesh, it seems prudent to apply the precautionary principle. While much is known of their clinical, pathogenic and epidemiologic features in livestock species and humans, a number of fundamental questions regarding the relationship between the viruses, their natural fruit-bat host and the environment remain unanswered. In this paper, we pose and probe these questions in context, and offer perspectives based primarily on our experience with Hendra virus in Australia, augmented with Nipah virus parallels. PMID:22440924

  6. Lethal recurrent hemorrhages of a brainstem cavernoma

    Microsoft Academic Search

    Alexandru Vlad Ciurea; Cristian Nastase; Alexandru Tascu; Felix Mircea Brehar

    2007-01-01

    Hemorrhages of brainstem cavernomas may cause severe neurological deficits. Surgical strategies are frequently described,\\u000a and advanced neuromonitoring with intraoperative imaging can help neurosurgeons to achieve good results. However, patients\\u000a are often confronted with significant therapeutic risks by the primary doctor before talking to an experienced brainstem neurosurgeon.\\u000a On the other hand, lethal progression with repeated hemorrhages is rarely described, although

  7. 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, ...

  8. Toxicology screen

    MedlinePLUS

    A toxicology screen refers to various tests to determine the type and approximate amount of legal and illegal drugs ... Toxicology screening is most often done using a blood or urine sample. However, it may be done ...

  9. 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 were some isolates where either feeding decline was not associated with high virulence, or virulence did not automatically prompt large declines in feeding. These results are discussed in the context of choosing optimum fungal isolates for biopesticide development. PMID:23126549

  10. Lethal and Sub-lethal Effects of UVB on Juvenile Biomphalaria glabrata (Mollusca: Pulmonata)

    PubMed Central

    Ruelas, Debbie S.; Karentz, Deneb; Sullivan, John T.

    2007-01-01

    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 (290–320 nm) 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

  11. 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

  12. Fluorescence-based phenotypic selection allows forward genetic screens in haploid human cells.

    PubMed

    Duncan, Lidia M; Timms, Richard T; Zavodszky, Eszter; Cano, Florencia; Dougan, Gordon; Randow, Felix; Lehner, Paul J

    2012-01-01

    The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway. PMID:22745803

  13. 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 ?-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 ?-glucan for growth. Seven of the strains of L. ruminis were, however, able to utilize tetrasaccharides (3-O-?-cellotriosyl-d-glucose [LDP4] or 4-O-?-laminaribiosyl-d-cellobiose [CDP4]) present in ?-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-?-cellotriosyl-d-glucose released by ?-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

  14. 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

  15. Tolerance to effects of high doses of ethanol: 1. Lethal effects in mice.

    PubMed

    Tsibulsky, V L; Amit, Z

    1993-06-01

    Male Swiss Webster mice were injected with ethanol doses ranging from 6.5-10.5 g/kg (20% w/v, IP). Survival time distribution revealed three waves of deaths with peaks around 5 min, 300 min, and 33 h. There were two windows with very low density of probability of death between 30-130 min and between 22-25 h following lethal injections. This time structure of the probability density function did not significantly depend upon ethanol overdose, novelty of the experimental environment, or prior injections of saline and/or 3.5 g/kg ethanol. Injections of high doses of ethanol in BALB/c mice showed that this strain of mice was more sensitive to ethanol-induced lethality (LD50 = 6.6 g/kg) and over 99% of deaths occurred between 5-200 min following injections of the doses from 5.5-7.5 g/kg. Preexposure to ethanol increased tolerance to ethanol-induced lethality. LD50 increased from 8.1 g/kg (at 24 h following lethal injections in ethanol-naive Swiss Webster mice) to 8.5 and 9.0 g/kg in mice following four and eight injections of 3.5 g/kg ethanol, respectively. In BALB/c mice, eight prior injections of 3.5 g/kg ethanol increased LD50 also slightly but significantly to 7.15 g/kg. The results suggest that: a) Ethanol-induced lethality is not a unitary phenomenon and that deaths that occurred within distinct waves probably have different causes; b) mice strains have different susceptibility to different causes of ethanol-induced deaths; c) preexposure to 3.5 g/kg ethanol results in significant but small increase in tolerance to ethanol-induced lethality. PMID:8327553

  16. 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 than concentrations considered to be lethal. In contrast, carcass levels of DDT and DDD were significantly lower, and DDE was only slightly higher, in the birds that died in the second period of food restriction. It is concluded that stored DDT residues present a hazard to birds, which utilize stored fat during periods of stress due to reproduction, cold weather, disease, injury, limited food supply, or migration.

  17. Comparative Genome-Wide Screening Identifies a Conserved Doxorubicin Repair Network That Is Diploid Specific in Saccharomyces cerevisiae

    Microsoft Academic Search

    Tammy J. Westmoreland; Sajith M. Wickramasekara; Andrew Y. Guo; Alice L. Selim; Tiffany S. Winsor; Arno L. Greenleaf; Kimberly L. Blackwell; John A. Olson; Jeffrey R. Marks; Craig B. Bennett; Michael Polymenis

    2009-01-01

    The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the

  18. Lethal congenital contracture syndrome (LCCS) and other lethal arthrogryposes in Finland--an epidemiological study.

    PubMed

    Pakkasjärvi, Niklas; Ritvanen, Annukka; Herva, Riitta; Peltonen, Leena; Kestilä, Marjo; Ignatius, Jaakko

    2006-09-01

    Arthrogryposis multiplex congenita is a heterogeneous group of disorders characterized by multiple contractures with an estimated frequency of 1 in 3,000 births. With improving diagnostic methods, increasing numbers of fetuses with arthrogryposis are found. The pathogenetic mechanisms are relatively well known but the epidemiology and genetics of the prenatally lethal forms of arthrogryposis are less well known. In this study we collected all cases of a multiple contractures diagnosed in Finland during 1987-2002 including live born infants, stillbirths, and terminated pregnancies. Ninety-two cases of 214 suffered intrauterine demise (68 selective pregnancy terminations and 24 stillbirths) and 58 died in infancy. In 141 out of these cases the diagnosis could be included within lethal arthrogryposes, with a prevalence of 1 in 6,985 (1.43/10,000) births. Of these, 59 had spinal cord pathology at autopsy and thus were of neurogenic origin. Thirty-nine cases had lethal congenital contracture syndrome (LCCS) clinically characterized by total immobility of the fetus at all ultrasound examinations (12 weeks or later), multiple joint contractures in both upper and lower limbs, hydrops, and fetal death before the 32nd week of pregnancy. LCCS is noted as a unique Finnish disorder with a prevalence of 1 in 25,250 (0.40/10,000) births and is a major cause of lethal arthrogryposis in Finland. PMID:16892327

  19. 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.

  20. Lethal mutagenesis in a structured environment

    PubMed Central

    Steinmeyer, Shelby H.; Wilke, Claus O.

    2010-01-01

    We analyze how lethal mutagenesis operates in a compartmentalized host. We assume that different compartments receive different amounts of mutagen and that virions can migrate among compartments. We address two main questions: 1. To what extent can refugia, i.e., compartments that receive little mutagen, prevent extinction? 2. Does migration among compartments limit the effectiveness of refugia? We find that if there is little migration, extinction has to be achieved separately in all compartments. In this case, the total dose of mutagen administered to the host needs to be so high that the mutagen is effective even in the refugia. By contrast, if migration is extensive, then lethal mutagenesis is effective as long as the average growth in all compartments is reduced to below replacement levels. The effective-ness of migration is governed by the ratio of virion replication and death rates, R0. The smaller R0, the less migration is necessary to neutralize refugia and the less mutagen is necessary to achieve extinction at high migration rates. PMID:19627995

  1. Synthetic lethality for linking the mycophenolate mofetil mode of action with molecular disease and drug profiles.

    PubMed

    Söllner, Johannes; Mayer, Paul; Heinzel, Andreas; Fechete, Raul; Siehs, Christian; Oberbauer, Rainer; Mayer, Bernd

    2012-10-30

    Systematic study of the effect of mycophenolate mofetil (MMF) on the molecular level in the context of other drugs and molecular disease profiles became possible due to the availability of large scale molecular profiles on both disease characterization and drug mode of action. Such analysis is of particular value in elucidating alternative drug use for addressing clinically unmet needs, and the concept of synthetic lethality provides an alternative tool for such repositioning strategies. Resting on consolidation of transcriptomics data and literature mining, a MMF molecular footprint became available including a set of 170 genes specifically affected by the drug. Analysis of this profile on a molecular pathway level reveals a set of 14 pathways as affected. Next to assignment of molecular pathways and associated diseases synergistic drug combinations are proposed by utilizing the synthetic lethal interaction network. Of particular interest is the combination of MMF with adenosine deaminase inhibitors, sulfasalazine, and other selected drugs interfering with calcium-based regulatory pathways and metabolism. Indeed analysis of drugs in clinical trials positively identifies combinations with MMF in the context of synthetic lethality and affected pathways, particularly in diseases such as multiple sclerosis, vasculitis, GVHD and lupus nephritis. Importantly, the synthetic lethal interaction of the drug mode of action is an interesting basis for rational repositioning strategies by suggesting combinations which exhibit a synergistic rather than a mere additive effect, as for example is evident for the combination of tacrolimus and MMF. Inherent is also the assessment of possible adverse effects of drug combinations. PMID:23014771

  2. Genetic Screening

    NSDL National Science Digital Library

    Irwin Slesnick

    2004-01-01

    Many genetic disorders can be detected with tests of blood and chromosomes. Genetic screening is the large-scale use of these tests as part of the public health program. Different members of society, worldwide, have advocated genetic screening to achieve different goals. This chapter provides a critical analysis of this controversial issue.

  3. Newborn screening.

    PubMed

    Wilcken, Bridget; Wiley, Veronica

    2008-02-01

    The aim of newborn screening is to detect newborns with serious, treatable disorders so as to facilitate appropriate interventions to avoid or ameliorate adverse outcomes. Mass biochemical testing of newborn babies was pioneered in the 1960s with the introduction of screening for phenylketonuria, a rare inborn error of metabolism, tested by using a dried blood spot sample. The next disorder introduced into screening programs was congenital hypothyroidism and a few more much rarer disorders were gradually included. Two recent advances have greatly changed the pace: modification of tandem mass spectrometry and DNA extraction and analysis from newborn screening dried blood spot. These two technologies make the future possibilities of newborn screening seem almost unlimited. Newborn screening tests are usually carried out on a dried blood spot sample, for which there are special analytical considerations. Dried blood spot calibrators and controls, prepared on the same lot number of filter paper, are needed. Methods have a co-efficient of variation of about 10% due to the increased variability of a dried filter paper sample compared with other biochemical samples. The haematocrit is an additional variable not able to be measured. Also of importance is obtaining a balance between the sensitivity and specificity of each assay. Fixing cut-off points for action needs consideration of what is an acceptable percentage of the population to recall for further testing. Few assays are 100% discriminatory. Programs in Australasia currently screen for at least 30 disorders. Detection of these requires not only the assay of a primary marker but often determination of a ratio of that marker with another, or possibly an alternative assay, for example a DNA mutation. The most important disorders screened for are described briefly: phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, the galactosaemias, medium-chain acyl-CoA dehydrogenase deficiency, glutaryl-CoA dehydrogenase deficiency and congenital adrenal hyperplasia, together with several other disorders detectable by tandem mass spectrometry. Newborn screening deals with rare disorders and benefit cannot be shown easily without very large pilot studies. There have been randomised controlled trials of screening for cystic fibrosis, and now several studies are beginning to establish the benefit of tandem mass spectrometry screening for disorders of fatty acid and amino acid metabolism. Two things will influence the new directions for newborn screening: the development of effective treatments for hitherto untreatable disorders, and advancing technology, enabling new testing strategies to be developed. There are novel treatments on the horizon for many discrete disorders. Susceptibility testing has recently been considered for newborn screening application, but is more controversial. Newborn screening has entered a new and exciting phase, with an explosion of new treatments, new technologies, and, possibly in the future, new preventive strategies. PMID:18203033

  4. Hyperactivated Wnt Signaling Induces Synthetic Lethal Interaction with Rb Inactivation by Elevating TORC1 Activities

    PubMed Central

    Hsu, Fu-Ning; Zhang, Robin; Searle, Jennifer S.; Pei, Xun; Li, Xuan; Ryoo, Hyung Don; Ji, Jun-Yuan; Du, Wei

    2014-01-01

    Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor. PMID:24809668

  5. Screen Time

    NSDL National Science Digital Library

    2012-06-26

    This game asks you a series of questions about how much time you spend in front of a screen, not being active. It begins by pointing out that since we spend a lot of time in front of computer screens at work or school, additional time at home can really affect how healthy we are. It asks how much time you spend watching TV, playing computer games, and using the computer each day. It then adds up the total amount of screen time you spend every day, and calculates how many hours you spend a year in front of a screen. It also tells you if that's a healthy amount, and suggests ways to stay active while in front of screens.

  6. Genetic screening

    PubMed Central

    Andermann, Anne; Blancquaert, Ingeborg

    2010-01-01

    Abstract OBJECTIVE To provide a primer for primary care professionals who are increasingly called upon to discuss the growing number of genetic screening services available and to help patients make informed decisions about whether to participate in genetic screening, how to interpret results, and which interventions are most appropriate. QUALITY OF EVIDENCE As part of a larger research program, a wide literature relating to genetic screening was reviewed. PubMed and Internet searches were conducted using broad search terms. Effort was also made to identify the gray literature. MAIN MESSAGE Genetic screening is a type of public health program that is systematically offered to a specified population of asymptomatic individuals with the aim of providing those identified as high risk with prevention, early treatment, or reproductive options. Ensuring an added benefit from screening, as compared with standard clinical care, and preventing unintended harms, such as undue anxiety or stigmatization, depends on the design and implementation of screening programs, including the recruitment methods, education and counseling provided, timing of screening, predictive value of tests, interventions available, and presence of oversight mechanisms and safeguards. There is therefore growing apprehension that economic interests might lead to a market-driven approach to introducing and expanding screening before program effectiveness, acceptability, and feasibility have been demonstrated. As with any medical intervention, there is a moral imperative for genetic screening to do more good than harm, not only from the perspective of individuals and families, but also for the target population and society as a whole. CONCLUSION Primary care professionals have an important role to play in helping their patients navigate the rapidly changing terrain of genetic screening services by informing them about the benefits and risks of new genetic and genomic technologies and empowering them to make more informed choices. PMID:20393090

  7. Lethal fish hook attachment - An unusual occurrence.

    PubMed

    Byard, Roger W

    2013-02-01

    A 39-year-old fisherman is reported who was dragged into the water from a boat after he became entangled in fishing line. His death was attributed to salt water drowning. At autopsy the cause of death was confirmed and the mechanism of the lethal event elucidated. Specifically, a large fish hook attached to line was embedded in his right wrist. The hook had passed beneath flexor tendons and had firmly attached him to fishing line that was being dropped from the vessel. There were no other significant injuries or underlying diseases present. This case demonstrates another rare situation in the commercial fishing industry that may result in a victim being dragged from a boat and drowned. PMID:23357398

  8. Synthetic Lethality of Yeast slt Mutations with U2 Small Nuclear RNA Mutations Suggests Functional Interactions between U2 and U5 snRNPs That Are Important for Both Steps of Pre-mRNA Splicing

    Microsoft Academic Search

    DEMING XU; DEBORAH J. FIELD; SHOU-JIANG TANG; ARNAUD MORIS; BRIAN P. BOBECHKO; JAMES D. FRIESEN

    1998-01-01

    A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5* end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor

  9. Differentiation of lethal and non lethal prostate cancer: PSA and PSA isoforms and kinetics

    PubMed Central

    Ballentine Carter, H

    2012-01-01

    Prostate-specific antigen (PSA) testing for the early diagnosis of prostate cancer has led to a decrease in cancer mortality. However, the high prevalence of low-grade prostate cancer and its long natural history, competing causes of death in older men and treatment patterns of prostate cancer, have led to dramatic overtreatment of the disease. Improved markers of prostate cancer lethality are needed to reduce the overtreatment of prostate cancer that leads to a reduced quality of life without extending life for a high proportion of men. The PSA level prior to treatment is routinely used in multivariable models to predict prostate cancer aggressiveness. PSA isoforms and PSA kinetics have been associated with more aggressive phenotypes, but are not routinely employed as part of prediction tools prior to treatment. PSA kinetics is a valuable marker of lethality post treatment and routinely used in determining the need for salvage therapy. PMID:22343493

  10. Technical Report GITGVU0901 Responsibility and Lethality for Unmanned Systems

    E-print Network

    a responsibility advisor in autonomous systems capable lethal target engagement. The ramifications surrounding demonstrated a relevant military scenario. 1. INTRODUCTION advent autonomous lethal robotic systems is underway it is simple matter time before autonomous engagements targets present battlefield. We written extensively

  11. 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

  12. 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

  13. Airport Screening

    MedlinePLUS

    ... cannot become radioactive from this procedure. However, some photographic film may need to be hand-screened because ... professional organization whose mission is excellence in the science and practice of radiation safety. Formed in 1956, ...

  14. MRSA Screening

    MedlinePLUS

    ... in a molecular test, but the specimen is analyzed for the genetic markers to identify S. aureus and ... Skin Infections In the News: MRSA Prevention Strategies Analyzed, Screening Policies Challenged (2013) Elsewhere On The Web ...

  15. TORCH screen

    MedlinePLUS

    ... different infections in a newborn. TORCH stands for toxoplasmosis , rubella , cytomegalovirus, herpes simplex, and HIV, but it ... used to screen infants for infections such as toxoplasmosis, cytomegalovirus, herpes simplex, syphilis and others. These infections ...

  16. Developmental Screening

    MedlinePLUS

    Developmental Screening FACT SHEET What is child development? A child’s growth is more than just physical. Children grow, develop, and learn throughout their lives, starting at birth. A child’s development can be followed ...

  17. The effects of oil sands wastewater on fish resulting from exposure to sub-lethal concentrations

    SciTech Connect

    Birkholz, D.A. [Enviro-Test Labs., Edmonton, Alberta (Canada); Goudey, J.S.; Balch, G.C. [HydroQual Labs. Ltd., Calgary, Alberta (Canada); Nelson, L.R. [Alberta Department of Energy, Edmonton, Alberta (Canada). Oil Sands and Research Division; Gulley, J. [Suncor Inc., Fort McMurray, Alberta (Canada); MacKinnon, M. [Syncrude Canada Ltd., Edmonton, Alberta (Canada)

    1995-12-31

    Rainbow trout, Oncorhynchus mykiss, were exposed to sub-lethal concentrations of oil sands wastewater in flow through laboratory experiments as well as to artificial ponds containing sub-lethal concentrations of tailings pond water and fine tails in order to study the viability of the wet landscape remediation option. Large (200--300 g) fish were used for all the exposures in this preliminary study and the following data were collected: blood cell counts, sex hormone concentrations, sexual maturation, stress protein concentrations, PAH-metabolites in bile, condition factors, liver somatic indices, mixed function oxygenase induction, PAHs in muscle, external condition and the condition of internal organs. The data obtained from this study revealed no adverse effects upon fish during extended field exposures. Given similar exposure conditions in the release waters of a wet landscape reclamation, the data suggest that there may be no adverse effects upon fish, however, longer term studies, other indicator organisms and additional chronic tests should be conducted.

  18. Pathways to high-lethality suicide attempts in individuals with borderline personality disorder.

    PubMed

    Chesin, Megan S; Jeglic, Elizabeth L; Stanley, Barbara

    2010-01-01

    The purpose of this study was to develop and test a model of high-lethality suicide attempts (HLSA) in individuals with Borderline Personality Disorder (BPD). An increased number of prior suicide attempts, substance use immediately prior to the attempt, and objective planning were proposed to lead directly to a HLSA, while aggression and impulsivity were hypothesized to lead indirectly to a HLSA through their associations with prior attempts. Path analysis revealed a revised model in which impulsivity was found to be significantly associated with both the lethality of the most recent attempt and the number of prior attempts. These results are discussed in terms of trait and crescendo models of suicidal behavior and their implications for suicide risk assessment among individuals with BPD. PMID:21082450

  19. Synthetic lethality in ATM-deficient RAD50-mutant tumors underlie outlier response to cancer therapy

    PubMed Central

    Al-Ahmadie, Hikmat; Iyer, Gopa; Hohl, Marcel; Asthana, Saurabh; Inagaki, Akiko; Schultz, Nikolaus; Hanrahan, Aphrothiti J.; Scott, Sasinya N.; Brannon, A. Rose; McDermott, Gregory C.; Pirun, Mono; Ostrovnaya, Irina; Kim, Philip; Socci, Nicholas D.; Viale, Agnes; Schwartz, Gary K.; Reuter, Victor; Bochner, Bernard H.; Rosenberg, Jonathan E.; Bajorin, Dean F.; Berger, Michael F.; Petrini, John H.J.; Solit, David B.; Taylor, Barry S.

    2014-01-01

    Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small cell cancer to combined checkpoint kinase 1 (Chk1) inhibition and DNA damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of Chk1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy whereby checkpoint inhibition in combination with DNA damaging chemotherapy is synthetically lethal in tumor but not normal cells with somatic mutations that impair Mre11 complex function. PMID:24934408

  20. Sub-lethal effects of Roundup™ on tadpole anti-predator responses.

    PubMed

    Moore, Harrison; Chivers, Douglas P; Ferrari, Maud C O

    2015-01-01

    Roundup™ is a commonly used pesticide applied to agriculture and forest habitats. These areas are generally ideal for amphibians due to the presence of small, ephemeral water bodies. While Roundup™ has been shown to have lethal effects on many species of amphibians, effects on behaviour and sensory perception have yet to be considered. Here, we exposed wood frog tadpoles to a sub-lethal concentration of Roundup™ and showed that the ability of tadpoles to respond to injured conspecific cues, an important source of information regarding local predation risk, was impaired. Subsequent experiments revealed that impaired responses likely result from a chemical reaction between the Roundup™ and the cues and that tadpoles chronically exposed to Roundup™ had reduced basal movement rates compared with unexposed tadpoles. Our data demonstrate that environmentally-relevant concentrations of Roundup™ can drastically alter movement and anti-predator responses of tadpoles, with potential negative consequences for the population. PMID:25450945

  1. A Single Phosphorodiamidate Morpholino Oligomer Targeting VP24 Protects Rhesus Monkeys against Lethal Ebola Virus Infection

    PubMed Central

    Warren, Travis K.; Whitehouse, Chris A.; Wells, Jay; Welch, Lisa; Heald, Alison E.; Charleston, Jay S.; Sazani, Pete; Reid, St. Patrick; Iversen, Patrick L.

    2015-01-01

    ABSTRACT Ebola viruses (EBOV) cause severe disease in humans and nonhuman primates with high mortality rates and continue to emerge in new geographic locations, including several countries in West Africa, the site of a large ongoing outbreak. Phosphorodiamidate morpholino oligomers (PMOs) are synthetic antisense molecules that are able to target mRNAs in a sequence-specific fashion and suppress translation through steric hindrance. We previously showed that the use of PMOs targeting a combination of VP35 and VP24 protected rhesus monkeys from lethal EBOV infection. Surprisingly, the present study revealed that a PMOplus compound targeting VP24 alone was sufficient to confer protection from lethal EBOV infection but that a PMOplus targeting VP35 alone resulted in no protection. This study further substantiates recent data demonstrating that VP24 may be a key virulence factor encoded by EBOV and suggests that VP24 is a promising target for the development of effective anti-EBOV countermeasures. PMID:25670780

  2. 40 CFR 798.5275 - Sex-linked recessive lethal test in drosophila melanogaster.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...Sex-linked recessive lethal test in drosophila melanogaster. 798.5275 Section...Sex-linked recessive lethal test in drosophila melanogaster. (a) Purpose. ...recessive lethal (SLRL) test using Drosophila melanogaster detects the...

  3. 40 CFR 798.5275 - Sex-linked recessive lethal test in drosophila melanogaster.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...Sex-linked recessive lethal test in drosophila melanogaster. 798.5275 Section...Sex-linked recessive lethal test in drosophila melanogaster. (a) Purpose. ...recessive lethal (SLRL) test using Drosophila melanogaster detects the...

  4. 40 CFR 798.5275 - Sex-linked recessive lethal test in drosophila melanogaster.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...Sex-linked recessive lethal test in drosophila melanogaster. 798.5275 Section...Sex-linked recessive lethal test in drosophila melanogaster. (a) Purpose. ...recessive lethal (SLRL) test using Drosophila melanogaster detects the...

  5. 40 CFR 798.5275 - Sex-linked recessive lethal test in drosophila melanogaster.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...Sex-linked recessive lethal test in drosophila melanogaster. 798.5275 Section...Sex-linked recessive lethal test in drosophila melanogaster. (a) Purpose. ...recessive lethal (SLRL) test using Drosophila melanogaster detects the...

  6. 40 CFR 798.5275 - Sex-linked recessive lethal test in drosophila melanogaster.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...Sex-linked recessive lethal test in drosophila melanogaster. 798.5275 Section...Sex-linked recessive lethal test in drosophila melanogaster. (a) Purpose. ...recessive lethal (SLRL) test using Drosophila melanogaster detects the...

  7. Human cooperation by lethal group competition

    PubMed Central

    Egas, Martijn; Kats, Ralph; van der Sar, Xander; Reuben, Ernesto; Sabelis, Maurice W.

    2013-01-01

    Why humans are prone to cooperate puzzles biologists, psychologists and economists alike. Between-group conflict has been hypothesized to drive within-group cooperation. However, such conflicts did not have lasting effects in laboratory experiments, because they were about luxury goods, not needed for survival (“looting”). Here, we find within-group cooperation to last when between-group conflict is implemented as “all-out war” (eliminating the weakest groups). Human subjects invested in helping group members to avoid having the lowest collective pay-off, whereas they failed to cooperate in control treatments with random group elimination or with no subdivision in groups. When the game was repeated, experience was found to promote helping. Thus, not within-group interactions alone, not random group elimination, but pay-off-dependent group elimination was found to drive within-group cooperation in our experiment. We suggest that some forms of human cooperation are maintained by multi-level selection: reciprocity within groups and lethal competition among groups acting together. PMID:23459158

  8. 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. PMID:25232177

  9. Lethal body burdens of polar narcotics: Chlorophenols

    SciTech Connect

    Wezel, A.P. van; Punte, S.S. [Utrecht Univ. (Netherlands). Environmental Chemistry Group; Opperhuizen, A. [Ministry of Transport, Public Works and Water Management, The Hague (Netherlands). National Institute for Coastal and Marine Management

    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.

  10. Synthetic lethal interactions for the development of cancer therapeutics: biological and methodological advancements

    Microsoft Academic Search

    Shinji Mizuarai; Hidehito Kotani

    2010-01-01

    Synthetic lethal interaction is defined as a combination of two mutations that is lethal when present in the same cell; each\\u000a individual mutation is non-lethal. Synthetic lethal interactions attract attention in cancer research fields since the discovery\\u000a of synthetic lethal genes with either oncogenes or tumor suppressor genes (TSGs) provides novel cancer therapeutic targets.\\u000a Due to the selective lethal effect

  11. Electron Screening

    NASA Astrophysics Data System (ADS)

    Raiola, F.

    Understanding the electron screening effect in the laboratory is critical for a correct interpretation of low-energy nuclear reactions in stars. The theory of electron screening in a stellar plasma is still based on the physics model introduced in 1954 by Salpeter, although the mathematical treatments have been improved significatly in the meantime. Extensive studies of the electron screening effect in different nuclear reactions have shown always the same discrepancy, i.e. the experimental U_egg adiabatic U_e. In the recent years extensive studies of the elctron screening effect in deuterated metals (54 metals and 4 insulators) and other environments have been carried out in Bochum. Experimental results of anomalous enhancements have been interpreted in terms of the Debye plasma model, applied to quasi-free metallic electrons. Within this model, the deduced number of valence electrons per metallic atom also agrees with the corresponding number from the Hall coefficient. The expected temperature dependence of the screening potential has been verified together with the expected Zt scaling (with the metallic host) of the Debye radius.

  12. 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

  13. An Evolutionarily Conserved Synthetic Lethal Interaction Network Identifies FEN1 as a Broad-Spectrum Target for Anticancer Therapeutic Development

    PubMed Central

    van Pel, Derek M.; Barrett, Irene J.; Shimizu, Yoko; Sajesh, Babu V.; Guppy, Brent J.; Pfeifer, Tom; McManus, Kirk J.; Hieter, Philip

    2013-01-01

    Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease FEN1, was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through in vitro biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in CDC4, a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between FEN1 and MRE11A, another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model. PMID:23382697

  14. A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System

    PubMed Central

    Ghabrial, Amin S.; Levi, Boaz P.; Krasnow, Mark A.

    2011-01-01

    Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (?40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general “house-keeping” genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ?70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system. PMID:21750678

  15. 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. PMID:25230351

  16. Endogenous EPCR/aPC-PAR1 signaling prevents inflammation-induced vascular leakage and lethality

    PubMed Central

    Niessen, Frank; Furlan-Freguia, Christian; Fernández, José A.; Mosnier, Laurent O.; Castellino, Francis J.; Weiler, Hartmut; Rosen, Hugh; Griffin, John H.

    2009-01-01

    Protease activated receptor 1 (PAR1) signaling can play opposing roles in sepsis, either promoting dendritic cell (DC)–dependent coagulation and inflammation or reducing sepsis lethality due to activated protein C (aPC) therapy. To further define this PAR1 paradox, we focused on the vascular effects of PAR1 signaling. Pharmacological perturbations of the intravascular coagulant balance were combined with genetic mouse models to dissect the roles of endogenously generated thrombin and aPC during escalating systemic inflammation. Acute blockade of the aPC pathway with a potent inhibitory antibody revealed that thrombin-PAR1 signaling increases inflammation-induced vascular hyperpermeability. Conversely, aPC-PAR1 signaling and the endothelial cell PC receptor (EPCR) prevented vascular leakage, and pharmacologic or genetic blockade of this pathway sensitized mice to LPS-induced lethality. Signaling-selective aPC variants rescued mice with defective PC activation from vascular leakage and lethality. Defects in the aPC pathway were fully compensated by sphingosine 1 phosphate receptor 3 (S1P3) deficiency or by selective agonists of the S1P receptor 1 (S1P1), indicating that PAR1 signaling contributes to setting the tone for the vascular S1P1/S1P3 balance. Thus, the activating proteases and selectivity in coupling to S1P receptor subtypes determine vascular PAR1 signaling specificity in systemic inflammatory response syndromes in vivo. PMID:19141861

  17. Get Screened

    MedlinePLUS

    ... Also, many screening tests are covered under the Affordable Care Act , the health care reform law passed in 2010. Depending on ... Find out which services are covered by the Affordable Care Act . Find out which ... health center near you to learn more . Pages: 1 ...

  18. Screen Animals

    E-print Network

    McMahon, Laura

    2014-01-01

    Screen animals – Introduction Laura McMahon From the protocinematic sequencing of Eadweard Muybridge’s horse and Etienne-Jules Marey’s cat to the proliferation of animal images on video-sharing platforms such as YouTube, the ontologies...

  19. Back to the future: revisiting HIV-1 lethal mutagenesis

    PubMed Central

    Dapp, Michael J.; Patterson, Steven E.; Mansky, Louis M.

    2012-01-01

    The concept of eliminating HIV-1 infectivity by elevating the viral mutation rate was first proposed over a decade ago, even though the general concept had been conceived earlier for RNA viruses. Lethal mutagenesis was originally viewed as a novel chemotherapeutic approach for treating HIV-1 infection in which use of a viral mutagen would over multiple rounds of replication lead to the lethal accumulation of mutations, rendering the virus population non infectious – known as the slow mutation accumulation model. There have been limitations in obtaining good efficacy data with drug leads, leaving some doubt into clinical translation. More recent studies of the APOBEC3 proteins as well as new progress in the use of nucleoside analogs for inducing lethal mutagenesis have helped to refocus attention on rapid induction of HIV-1 lethal mutagenesis in a single or limited number of replication cycles leading to a rapid mutation accumulation model. PMID:23195922

  20. Exploiting synthetic lethal interactions for targeted cancer therapy

    E-print Network

    Jiang, Hai

    Emerging data suggests that synthetic lethal interactions between mutated oncogenes/tumor suppressor genes and molecules involved in DNA damage signaling and repair can be therapeutically exploited to preferentially kill ...

  1. Mechanism by Which Caffeine Potentiates Lethality of Nitrogen Mustard

    Microsoft Academic Search

    Ching C. Lau; Arthur B. Pardee

    1982-01-01

    Caffeine is synergistic with many DNA-damaging agents in increasing lethality to mammalian cells. The mechanism is not well understood. Our results show that caffeine potentiates the lethality of the nitrogen mustard 2-chloro-N-(2-chloroethyl)-N-methylethanamine (HN2) by inducing damaged cells to undergo mitosis before properly repairing lesions in their DNA. Treatment with low doses of HN2 (0.5 mu M for 1 hr) caused

  2. Late-acting dominant lethal genetic systems and mosquito control

    PubMed Central

    Phuc, Hoang Kim; Andreasen, Morten H; Burton, Rosemary S; Vass, Céline; Epton, Matthew J; Pape, Gavin; Fu, Guoliang; Condon, Kirsty C; Scaife, Sarah; Donnelly, Christl A; Coleman, Paul G; White-Cooper, Helen; Alphey, Luke

    2007-01-01

    Background Reduction or elimination of vector populations will tend to reduce or eliminate transmission of vector-borne diseases. One potential method for environmentally-friendly, species-specific population control is the Sterile Insect Technique (SIT). SIT has not been widely used against insect disease vectors such as mosquitoes, in part because of various practical difficulties in rearing, sterilization and distribution. Additionally, vector populations with strong density-dependent effects will tend to be resistant to SIT-based control as the population-reducing effect of induced sterility will tend to be offset by reduced density-dependent mortality. Results We investigated by mathematical modeling the effect of manipulating the stage of development at which death occurs (lethal phase) in an SIT program against a density-dependence-limited insect population. We found late-acting lethality to be considerably more effective than early-acting lethality. No such strains of a vector insect have been described, so as a proof-of-principle we constructed a strain of the principal vector of the dengue and yellow fever viruses, Aedes (Stegomyia) aegypti, with the necessary properties of dominant, repressible, highly penetrant, late-acting lethality. Conclusion Conventional SIT induces early-acting (embryonic) lethality, but genetic methods potentially allow the lethal phase to be tailored to the program. For insects with strong density-dependence, we show that lethality after the density-dependent phase would be a considerable improvement over conventional methods. For density-dependent parameters estimated from field data for Aedes aegypti, the critical release ratio for population elimination is modeled to be 27% to 540% greater for early-acting rather than late-acting lethality. Our success in developing a mosquito strain with the key features that the modeling indicated were desirable demonstrates the feasibility of this approach for improved SIT for disease control. PMID:17374148

  3. Lethal and sublethal effects of cypermethrin to Hypsiboas pulchellus tadpoles

    Microsoft Academic Search

    M. Gabriela Agostini; Guillermo S. Natale; Alicia E. Ronco

    2010-01-01

    The study of the effects of the insecticide cypermethrin (CY) technical grade and its Sherpa® commercial formulation on Hypsiboas pulchellus tadpoles assessing lethality, behavior, growth, and abnormalities under standardized laboratory conditions is reported. Observed\\u000a behaviors were identified and categorized by means of a ranking system according to the loss of mobility. Results of acute\\u000a lethal effects indicate higher potency for

  4. Exploiting synthetic lethal interactions for targeted cancer therapy

    PubMed Central

    Reinhardt, H. Christian; Jiang, Hai; Hemann, Michael T.; Yaffe, Michael B.

    2011-01-01

    Emerging data suggests that synthetic lethal interactions between mutated oncogenes/tumor suppressor genes and molecules involved in DNA damage signaling and repair can be therapeutically exploited to preferentially kill tumor cells. in this review, we discuss the concept of synthetic lethality, and describe several recent examples in which this concept was successfully implemented to target tumor cells in culture, in mouse models, and in human cancer patients. PMID:19755856

  5. Internal Lethal Concentrations of Halobenzenes with Fish ( Gambusia affinis)

    Microsoft Academic Search

    Yupadee Chaisuksant; Qiming Yu; Des Connell

    1997-01-01

    The internal lethal concentration is a potential measure of toxicity which could be usefully applied in environmental toxicology and risk assessment. Using halobenzenes, which are common environmental contaminants, and represented test compounds, experiments were conducted in aquaria with the mosquitofish (Gambusia affinis). The average internal lethal concentration (ILC50) for four representative halohydrocarbons, 1,4-diBB, 1,2,3-triCB, 1,2,4-triBB, and pentaCB, were consistent with

  6. Protection against Anthrax Lethal Toxin Challenge by Genetic Immunization with a Plasmid Encoding the Lethal Factor Protein

    Microsoft Academic Search

    BRIAN M. PRICE; ADRIANE L. LINER; STEPHEN H. LEPPLA; ALFRED MATECZUN; DARRELL R. GALLOWAY

    2001-01-01

    The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB\\/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids (aa) 10 to 254) of Bacillus anthracis

  7. Newborn Screening

    NSDL National Science Digital Library

    American Association for the Advancement of Science (; )

    2005-03-07

    There's been a lot of talk recently about national testing standards for students. But now, some doctors are calling for national testing standards for newborns. In some states, every newborn baby gets a blood test that can detect more than thirty serious diseases. But in other states, it's a very different story. According to geneticist Piero Rinaldo of the Mayo Clinic, just having a state senator with an affected child could make the difference. Now, he and dozens of other experts have developed uniform standards for newborn screening. And they're calling on the federal government to adopt them. This Science update details many of the obstacles by which individual states and the U.S. government face when challenged with newborn screening practices. Additional links for further inquiry are provided.

  8. 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.

  9. Interference from ordinarily used solvents in the outcomes of Artemia salina lethality test.

    PubMed

    Geethaa, Sahgal; Thavamany, Priscilla Jayanthi; Chiew, Siah Poh; Thong, Ong Ming

    2013-10-01

    Methanol, ethanol, Tween 20 and dimethyl sulfoxide (DMSO) are widely used as dissolving agents in Artemia salina lethality test (aka brine shrimp lethality test [BSLT]) to screen the pharmaceutical properties of natural products. Nevertheless, there is lack of toxicity level of these solvents against brine shrimp. High concentration of these organic solvent might be toxic for this zoology invertebrate and interfere in the experimental outcomes. To avoid this, permissible concentration of the solvents used in BSLT was identified. BSLT was performed to evaluate the toxicity effect of Tween 20, methanol, ethanol and DMSO at 24 h post-treatment time point against A. salina. The suggested maximum working concentration (v/v) for DMSO, methanol, ethanol was found to be 1.25% and that for Tween 20 was 0.16%. LC50 for the solvents were 8.5% (DMSO), 6.4% (methanol), 3.4% (ethanol) and 2.5% (Tween 20). The findings have shown a toxicity level among the solvents in descending order as Tween 20 > ethanol > methanol > DMSO. DMSO is a safer solvent to be used in BSLT compared with other tested solvents, whereas Tween 20 has been shown to be the most stringent solvent among the tested solvents. The findings are resourcefully useful to avoid interference of solvents in the assessment of natural products using BSLT. PMID:24350047

  10. Lethal and sub-lethal effects of faecal deltamethrin residues on dung-feeding insects.

    PubMed

    Mann, C M; Barnes, S; Offer, B; Wall, R

    2015-06-01

    Endectocides administered to livestock to facilitate pest and parasite control may be excreted in the faeces at concentrations that are toxic to coprophagous insects, including species of ecological importance. Although much research has focused on the effects of macrocyclic lactones, relatively less attention has been given to any similar impacts of the widely used pyrethroid insecticides. Here, the effects of faecal residues of the pyrethroid deltamethrin after application to Holstein-Friesian cattle in a proprietary pour-on formulation are examined. Freshly dropped dung was collected 1, 3, 5 and 7?days after treatment and from an untreated control group. In laboratory bioasssays, female Lucilia sericata (Diptera: Calliphoridae) blow flies matured significantly smaller egg batches and had a lower percentage of eggs hatch after feeding on dung collected for up to 5?days after treatment, compared with flies feeding on dung from untreated cattle. In the field, artificial dung pats were constructed from the collected dung and left on pastureland for 7?days before being retrieved and searched for insects. Significantly more adult Diptera emerged from the faeces of untreated cattle than from the dung of treated cattle collected on days 1 and 3 after treatment. Adult Coleoptera were found in lower numbers in the dung of treated animals compared with control dung, suggesting a repellent effect. The results indicate that deltamethrin residues in cattle faeces have a range of lethal and sub-lethal effects on dung-feeding insects for up to a week after treatment, but that the precise duration and nature of toxicity varies depending on the sensitivity of the insect in question. PMID:25594879

  11. Quadruple screen test

    MedlinePLUS

    ... screen; Multiple marker screening; AFP plus; Triple screen test; AFP maternal; MSAFP; 4-marker screen ... This test is most often done between the 15th and 22nd weeks of the pregnancy. It is most accurate ...

  12. Are High-Lethality Suicide Attempters With Bipolar Disorder a Distinct Phenotype?

    Microsoft Academic Search

    Maria A. Oquendo; Juan Jose Carballo; Namita Rajouria; Dianne Currier; Adrienne Tin; Jessica Merville; Hanga C. Galfalvy; Leo Sher; Michael F. Grunebaum; Ainsley K. Burke; J. John Mann

    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

  13. [Performance indicators in screening programmes].

    PubMed

    Májek, O; Ngo, O; Daneš, J; Zavoral, M; Dvo?ák, V; Klimeš, D; Dušek, L

    2014-01-01

    Breast, colorectal and cervical cancer screening programmes make it possible to decrease the population mortality rates of these diseases. How-ever, complex standards of the quality of care must be introduced and followed in order to maintain a favourable ratio between the benefits and risks arising from population-wide screening programmes. Such programmes should be organized and population-based, ensuring that quality control is performed at all levels. This review introduces the system of quality control in the Czech cancer screening programmes, and provides specific examples of performance indicators that are usable and/ or being used in these programmes. Cancer screening programmes in the Czech Republic are equipped with a comprehensive information background which involves monitoring of the cancer burden in the population, monitoring of the screening process based on clinical data, and monitoring of the screening process based on administrative data. In particular, the specific performance indicators describe the success rate of take?up of the target population, ability of the screening test to reveal (sensitivity) or to exclude (specificity) the screened condition, correct employment of subsequent diagnostic methods or treatment of detected cancers or precancerous lesions where applicable. In the Czech breast cancer screening programme, these indicators are routinely used in order to monitor the individual centres; in both colorectal and cervical cancer screening programmes, these indicators are used to monitor the entire programme, whereas the system of quality control for individual centres is under continuous development. A project of personalized invitations was launched in 2014, and its results are regularly evaluated in cooperation with the Czech National Reference Centre and the Ministry of Health of the Czech Republic. PMID:25494895

  14. A screen for modifiers of RacGAP(84C) gain-of-function in the Drosophila eye revealed the LIM kinase Cdi\\/TESK1 as a downstream effector of Rac1 during spermatogenesis

    Microsoft Academic Search

    Karine Raymond; Evelyne Bergeret; Amélie Avet-Rochex; Ruth Griffin-Shea; Marie-Odile Fauvarque

    2004-01-01

    In Drosophila, RotundRacGAP\\/RacGAP(84C) is critical to retinal organisation and spermatogenesis. We show that eye-directed expression of RacGAP(84C) or its GTPase activating protein (GAP) domain induces a dominant rough eye phenotype which we used as a starting point in a gain-of-function screen to identify new partners of RacGAP(84C). Proteins known to function in Ras, Rho and Rac signalling were identified confirming

  15. Bacterial community composition of three candidate insect vectors of palm phytoplasma (Texas Phoenix Palm Decline and Lethal Yellowing).

    PubMed

    Powell, Christopher M; Hail, Daymon; Potocnjak, Julia; Hanson, J Delton; Halbert, Susan H; Bextine, Blake R

    2015-02-01

    Texas Phoenix Palm Decline (TPPD) and Lethal Yellowing are two phytoplasma-linked diseases in palms. The phytoplasma causing TPPD is thought to be transmitted by three putative planthopper vectors, Ormenaria rufifascia, Omolicna joi, and Haplaxius crudus. These insects have been morphologically and molecularly described, and have screened positive for Candidatus Phytoplasma palmae. Individuals from each species were subjected to 16S bacterial community sequencing using the Roche 454 platform, providing new information regarding the previously unexplored bacterial communities present in putative vectors. PMID:25298076

  16. THE RELATIVE INFLUENCE OF SEX OF PROGENY ON THE LETHAL EXPRESSION OF THE SONLESS GENE IN DROSOPHILA MELANOGASTER

    Microsoft Academic Search

    JAMES J. COLAIANNE; A. E. BELL

    The sex-linked maternally influenced lethal gene, sonless (snl), in D. melanoggnster was shown to affect the survival of daughters as well as sons of snl\\/snl mothers but to a lesser degree. Interaction studies of sonless with the sex altering mutants transformer (tra) and doublesex (dsz) revealed that any alteration toward increased masculinity of progeny from snl\\/snl females re- duced their

  17. Lethal effects of short-wavelength visible light on insects

    PubMed Central

    Hori, Masatoshi; Shibuya, Kazuki; Sato, Mitsunari; Saito, Yoshino

    2014-01-01

    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. PMID:25488603

  18. 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.

  19. Identification of Novel Host-Targeted Compounds That Protect From Anthrax Lethal Toxin-Induced Cell Death

    PubMed Central

    Slater, Louise H.; Hett, Erik C.; Mark, Kevin; Chumbler, Nicole M.; Patel, Deepa; Lacy, D. Borden; Collier, R. John; Hung, Deborah T.

    2013-01-01

    Studying how pathogens subvert the host to cause disease has contributed to the understanding of fundamental cell biology. Bacillus anthracis, the causative agent of anthrax, produces the virulence factor lethal toxin to disarm host immunity and cause pathology. We conducted a phenotypic small molecule screen to identify inhibitors of lethal toxin-induced macrophage cell death and used an ordered series of secondary assays to characterize the hits and determine their effects on cellular function. We identified a structurally diverse set of small molecules that act at various points along the lethal toxin pathway, including inhibitors of endocytosis; natural product inhibitors of organelle acidification (e.g. the botulinum neurotoxin inhibitor, toosendanin); and a novel proteasome inhibitor, 4MNB (4-methoxy-2-[2-(5-methoxy-2-nitrosophenyl)ethyl]-1-nitrosobenzene). Many of the compounds, including three drugs approved for use in humans, also protected against the related Clostridium difficile toxin TcdB, further demonstrating their value as novel tools for perturbation and study of toxin biology and host cellular processes, and highlighting potential new strategies for intervening on toxin-mediated diseases. PMID:23343607

  20. Lethal Waterhouse-Friderichsen syndrome in posttraumatic asplenia.

    PubMed

    Locker, G J; Wagner, A; Peter, A; Staudinger, T; Marosi, C; Rintelen, C; Knapp, S; Malzer, R; Weiss, K; Metnitz, P

    1995-10-01

    We herein report a case of fulminant lethal Waterhouse-Friderichsen syndrome in an elderly female patient seven years after posttraumatic splenectomy. In contrast to various reports, this patient had not been vaccinated against Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenzae, respectively, although infections with these microorganisms are known to cause the main lethal diseases in asplenic patients. Again, we recommend obligatory vaccinations against the mentioned bacteria for it is known that this decreases the risk of fatal septic events in these patients. To optimize prevention, it is imperative to vaccinate patients undergoing splenectomy before discharge from hospital. PMID:7473978

  1. 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.

  2. Possible involvement of genes on the Q chromosome of Nicotiana tabacum in expression of hybrid lethality and programmed cell death during interspecific hybridization to Nicotiana debneyi

    Microsoft Academic Search

    Takahiro Tezuka; Tsutomu Kuboyama; Toshiaki Matsuda; Wataru Marubashi

    2007-01-01

    Hybrid seedlings from the cross between Nicotiana tabacum, an allotetraploid composed of S and T subgenomes, and N. debneyi die at the cotyledonary stage. This lethality involves programmed cell death (PCD). We carried out reciprocal crosses between\\u000a the two progenitors of N. tabacum, N. sylvestris and N. tomentosiformis, and N. debneyi to reveal whether only the S subgenome in N.

  3. Newborn Screening

    PubMed Central

    Pitt, James J

    2010-01-01

    Early detection of many disorders, mainly inherited, is feasible with population-wide analysis of newborn dried blood spot samples. Phenylketonuria was the prototype disorder for newborn screening (NBS) and early dietary treatment has resulted in vastly improved outcomes for this disorder. Testing for primary hypothyroidism and cystic fibrosis (CF) was later added to NBS programs following the development of robust immunoassays and molecular testing. Current CF testing usually relies on a combined immunoreactive trypsin/mutation detection strategy. Multiplex testing for approximately 25 inborn errors of metabolism using tandem mass spectrometry is a relatively recent addition to NBS. The simultaneous introduction of many disorders has caused some re-evaluation of the traditional guidelines for NBS, because very rare disorders or disorders without good treatments can be included with minimal effort. NBS tests for many other disorders have been developed, but these are less uniformly applied or are currently considered developmental. This review focuses on Australasian NBS practices. PMID:20498829

  4. Odontoid metastasis: a potential lethal complication.

    PubMed

    Rayan, Faizal; Mukundan, Cibu; Shukla, D D; Barrington, R L

    2009-12-01

    Nearly one third of cervical spine metastasis has a primary breast malignancy. Patients with cervical metastasis have higher mortality due to advanced stage of the malignancy. Treatment is palliative to relieve pain, prevent pathological fracture, improve mobility and function, and prolong survival. We describe a 40-year-old woman with a history of breast cancer who presented with neck and shoulder pain of 1 week duration with no neurological deficit. Following clinical examination, radiographs taken of the cervical spine was normal. Radiographs repeated 3 weeks later revealed a large lytic lesion of the odontoid occupying 70-80% of the peg. Further investigation including magnetic resonance imaging and bone scan showed no further spinal lesions. She underwent cyclical radiotherapy with complete resolution of the odontoid peg lesion and clinically was asymptomatic at 2 years. Metastatic lesions of the odontoid are atypical, and this case reinforces the necessity of early detection to evade disastrous consequences. PMID:19921483

  5. 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.

  6. Revealed Preference

    Microsoft Academic Search

    Hal R. Varian

    This is a survey of revealed preference analysis focusing on the period since Samuelson's seminal development of the topic with emphasis on empirical applications. It was prepared for Samuelsonian Economics and the 21st Century, edited by Michael Szenberg.

  7. Rational screening strategies.

    PubMed

    Huntington, Mark K; Thanel, Fredric H

    2010-11-01

    Screening for disease can significantly improve both individual and public health. Many different screening interventions have been proposed for a wide range of conditions. This article reviews the elements that make up an effective clinical screening test and discusses what it means to effectively detect and treat a condition, risks and benefits of screening and the measurement of outcomes from screening. PMID:21117518

  8. Regulation of apoptosis by lethal cytokines in human mesothelial cells

    Microsoft Academic Search

    Marina Penélope Catalan; Dolores Subirá; Ana Reyero; Rafael Selgas; Arturo Ortiz-Gonzalez; Jesús Egido; Alberto Ortiz

    2003-01-01

    Regulation of apoptosis by lethal cytokines in human mesothelial cells.BackgroundDysregulation of peritoneal cell death may contribute to the complications of peritoneal dialysis (PD). Chronic peritoneal dialysis and acute peritonitis are both associated with loss of mesothelial cells. In addition, acute peritonitis is characterized by sudden changes in the number of peritoneal leukocytes. However, the factors regulating peritoneal cell survival are

  9. ACUTE LETHALITY OF COPPER, CADMIUM, AND ZINC TO NORTHERN SQUAWFISH

    EPA Science Inventory

    Flow-through acute toxicity tests on juvenile northern squawfish (Ptychocheilus oregonensis) were conducted with copper, cadmium, and zinc. The 96-hour median lethal concentrations were 18 micrograms/liter for copper, 1,104 micrograms/liter for cadmium, and 3,693 micrograms/liter...

  10. 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…

  11. Anthrax Toxin Receptor 2Dependent Lethal Toxin Killing In Vivo

    E-print Network

    Sejnowski, Terrence J.

    Anthrax Toxin Receptor 2­Dependent Lethal Toxin Killing In Vivo Heather M. Scobie1,2 , Darran J Jolla, California, United States of America Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA

  12. Hyperinsulinaemia: A prospective risk factor for lethal clinical prostate cancer

    Microsoft Academic Search

    Jan Hammarsten; Benkt Högstedt

    2005-01-01

    Previous studies have suggested that hyperinsulinaemia and other components of metabolic syndrome are risk factors for clinical prostate cancer. This prospective study tested the hypothesis that hyperinsulinaemia and other components of metabolic syndrome are risk factors for lethal clinical prostate cancer. The clinical, haemodynamic, anthropometric, metabolic and insulin profile at baseline in men who had died from clinical prostate cancer

  13. Effect of sublethal and lethal temperature on plant cells.

    PubMed

    Daniell, J W; Chappell, W E; Couch, H B

    1969-12-01

    Soybean, Glycine max L., and elodea, Elodea canadensis Michx, leaves were exposed to sublethal and lethal temperatures and examined by light microscopy. Loss of chlorophyll and swollen chloroplasts were observed in cells of elodea leaves exposed to sublethal temperatures. At the thermal death point of leaf cells of elodea and soybean, there was a disorganization of the tonoplast membrane, plasmalemma, and chloroplast membranes. Approximately 40% of the cells in elodea and 50% of the cells in soybean leaves exhibited oriteria of cell death when exposed to a temperature which induced necrotic leaf tissue. Plasmolysis of leaf cells of elodea and soybean occurred at lethal temperatures, but did not appear to be the primary cause of cellular death. The primary effect of lethal temperatures on the leaf cells used in these experiments is disintegration of the cellular membranes.Following exposure of attached elodea leaves to lethal temperatures, changes in leaf cells were periodically observed with a light microscope. In low temperature treatments, (43 through 52 degrees ), the percentages of cells exhibiting criteria of death 12 days after treatment did not change from the percentages determined immediately after treatment. All treatments above 52 degrees resulted in 40% or more of the cells exhibiting criteria of cell death immediately after treatment. In these treatments, this resulted in all cells exhibiting criteria of death on the fourth day after treatment. PMID:16657257

  14. 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...

  15. 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

  16. Cardiotoxic and Lethal Effects of Listeria monocytogenes Hemolysin

    PubMed Central

    Kingdon, G. Charles; Sword, C. P.

    1970-01-01

    Cardiotoxic and lethal effects of Listeria monocytogenes hemolysin were studied in CD-1 mice injected with varying doses of hemolysin. Intravenous injection of 100 complete hemolytic units (CHU) caused 100% lethality within 4 to 5 min. Doses ranging from 40 to 50 CHU caused death of approximately 50% of the animals. Adrenergic blocking agents and antihistamine failed to protect mice against lethality and thereby suggested that death was not due to release of vasoactive agents by hemolysin. Plasma levels of creatine phosphokinase increased after intravenous administration of hemolysin and suggested myopathy, possibly of the myocardium. Electrocardiograms from hemolysin-treated mice indicated serious alterations in heart rate and rhythm, suggesting damage to contractile and pacemaker cardiac tissue. In addition, there were indications of increased potassium levels influencing the heart. Presumably, death was due to functional damage to heart muscle and electrical arrest. The cardiotoxic and lethal effects could be prevented by prior incubation of hemolysin with cholesterol, heating, or failure to reactivate the preparation with cysteine. PMID:16557744

  17. Analysis of Ebola Glycoprotein Sequences from Strains of Varying Lethality

    E-print Network

    Analysis of Ebola Glycoprotein Sequences from Strains of Varying Lethality Biochem 218 Spring 2002 Tammy Doukas tdoukas@stanford.edu I. Background and Significance Ebola hemorrhagic fever is a disease in humans, chimpanzees, and monkeys, caused by infection with Ebola virus, and associated with high

  18. Physicians' Attitudes About Involvement in Lethal Injection for Capital Punishment

    Microsoft Academic Search

    Neil Farber; Elizabeth B. Davis; Joan Weiner; Janine Jordan; E. Gil Boyer; Peter A. Ubel

    2000-01-01

    Background: Physicians could play various roles in car- rying out capital punishment via lethal injection. Medi- cal societies like the American Medical Association (AMA) and American College of Physicians have established which roles are acceptable and which are disallowed. No one has explored physicians' attitudes toward their po- tential roles in this process. Methods: We surveyed physicians about how accept-

  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. Podophyllum hexandrum-Mediated Survival Protection and Restoration of Other Cellular Injuries in Lethally Irradiated Mice

    PubMed Central

    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 LD100, 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. PMID:19553386

  1. Exosomes from Plasmodium yoelii-Infected Reticulocytes Protect Mice from Lethal Infections

    PubMed Central

    Martin-Jaular, Lorena; Nakayasu, Ernesto S.; Ferrer, Mireia; Almeida, Igor C.; del Portillo, Hernando A.

    2011-01-01

    Exosomes are 30–100-nm membrane vesicles of endocytic origin that are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. While initial studies suggested that the role of exosomes was limited to the removal of proteins during the maturation of reticulocytes to erythrocytes, recent studies indicate that they are produced by different types of cells and are involved in promoting inter-cellular communication and antigen presentation. Here, we describe the isolation and characterization of exosomes from peripheral blood of BALB/c mice infected with the reticulocyte-prone non-lethal Plasmodium yoelii 17X strain. Importantly, proteomic analysis revealed the presence of parasite proteins in these vesicles. Moreover, immunization of mice with purified exosomes elicited IgG antibodies capable of recognizing P. yoelii-infected red blood cells. Furthermore, lethal challenge of immunized mice with the normocyte-prone lethal P. yoelii 17XL strain caused a significant attenuation in the course of parasitaemia, increased survival time, and altered the cell tropism to reticulocytes. These results were obtained also when the exosomes were isolated from a P. yoelii-infected reticulocyte culture indicating that reticulocyte-derived exosomes carry antigens and are involved in immune modulation. Moreover, inclusion of CpG ODN 1826 in exosome immunizations elicited IgG2a and IgG2b antibodies and promoted survival, clearance of parasites and subsequent sterile protection of 83% of the animals challenged with P. yoelli 17XL. To our knowledge, this is the first report of immune responses elicited by exosomes derived from reticulocytes opening new avenues for the modulation of anti-malaria responses. PMID:22046311

  2. 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

  3. TOPS: a versatile software tool for statistical analysis and visualization of combinatorial gene-gene and gene-drug interaction screens

    PubMed Central

    2014-01-01

    Background Measuring the impact of combinations of genetic or chemical perturbations on cellular fitness, sometimes referred to as synthetic lethal screening, is a powerful method for obtaining novel insights into gene function and drug action. Especially when performed at large scales, gene-gene or gene-drug interaction screens can reveal complex genetic interactions or drug mechanism of action or even identify novel therapeutics for the treatment of diseases. The result of such large-scale screen results can be represented as a matrix with a numeric score indicating the cellular fitness (e.g. viability or doubling time) for each double perturbation. In a typical screen, the majority of combinations do not impact the cellular fitness. Thus, it is critical to first discern true "hits" from noise. Subsequent data exploration and visualization methods can assist to extract meaningful biological information from the data. However, despite the increasing interest in combination perturbation screens, no user friendly open-source program exists that combines statistical analysis, data exploration tools and visualization. Results We developed TOPS (Tool for Combination Perturbation Screen Analysis), a Java and R-based software tool with a simple graphical user interface that allows the user to import, analyze, filter and plot data from double perturbation screens as well as other compatible data. TOPS was designed in a modular fashion to allow the user to add alternative importers for data formats or custom analysis scripts not covered by the original release. We demonstrate the utility of TOPS on two datasets derived from functional genetic screens using different methods. Dataset 1 is a gene-drug interaction screen and is based on Luminex xMAP technology. Dataset 2 is a gene-gene short hairpin (sh)RNAi screen exploring the interactions between deubiquitinating enzymes and a number of prominent oncogenes using massive parallel sequencing (MPS). Conclusions TOPS provides the benchtop scientist with a free toolset to analyze, filter and visualize data from functional genomic gene-gene and gene-drug interaction screens with a flexible interface to accommodate different technologies and analysis algorithms in addition to those already provided here. TOPS is freely available for academic and non-academic users and is released as open source. PMID:24712852

  4. 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. PMID:24736744

  5. 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

  6. Disruption of the rice plastid ribosomal protein s20 leads to chloroplast developmental defects and seedling lethality.

    PubMed

    Gong, Xiaodi; Jiang, Quan; Xu, Jianlong; Zhang, Jianhui; Teng, Sheng; Lin, Dongzhi; Dong, Yanjun

    2013-10-01

    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

  7. Skin Cancer Screening

    MedlinePLUS

    ... Dictionary Search for Clinical Trials NCI Publications Español Skin Cancer Screening (PDQ®) Skin Cancer Screening Key Points for This Section Tests ... trials is available from the NCI Web site . Skin exams are used to screen for skin cancer. ...

  8. Mental Health Screening Center

    MedlinePLUS

    Mental Health Screening Center These online screening tools are not a substitute for consultation with a health professional. ... you have any concerns, see your doctor or mental health professional. Depression This screening form was developed from ...

  9. Health Screenings and Immunizations

    MedlinePLUS

    ... your primary doctor. Blood Tests – A Common Screening Method (Source: National Heart, Lung, and Blood Institute) Click ... tests, see What Are Blood Tests? Other Screening Methods Doctors can't screen for all diseases and ...

  10. Lung Cancer Screening

    MedlinePLUS

    Lung Cancer Screening Key Points for This Section Tests are used to screen for different types of cancer. ... if they decrease the risk of dying from lung cancer. The following screening tests have been studied to ...

  11. Colorectal Cancer Screening

    MedlinePLUS

    ... colonoscopy DNA stool test Tests are used to screen for different types of cancer. Some screening tests ... from the disease. Four tests are used to screen for colorectal cancer: Fecal occult blood test A ...

  12. Toxicological screening

    PubMed Central

    Parasuraman, S.

    2011-01-01

    Toxicity testing of new compounds is essential for drug development process. The preclinical toxicity testing on various biological systems reveals the species-, organ- and dose- specific toxic effects of an investigational product. The toxicity of substances can be observed by (a) studying the accidental exposures to a substance (b) in vitro studies using cells/ cell lines (c) in vivo exposure on experimental animals. This review mainly focuses on the various experimental animal models and methods used for toxicity testing of substances. The pre-clinical toxicity testing helps to calculate “No Observed Adverse Effect Level” which is needed to initiate the clinical evaluation of investigational products. PMID:21772764

  13. Structure and other chemical characterizations of gila toxin, a lethal toxin from lizard venom.

    PubMed

    Datta, G; Tu, A T

    1997-12-01

    The complete primary structure of a lethal toxin, horridum toxin, from the venom of the lizard, Heloderma horridum horridum, was determined by Edman degradation. The amino acid sequence was deduced by overlapping peptide fragments generated by chemical and enzymatic digestions. Horridum toxin causes hemorrhage in internal organs and particularly in the eye, leading to exophthalmia, an effect that has not been observed for other toxins. It is a glycoprotein with a total of 210 residues. Examination of the primary sequence revealed that horridum toxin has considerable homology to tissue-type kallikrein and trypsin. Furthermore, synthetic substrates for trypsin, such as tosyl-L-arginine methyl ester, benzoyl-L-arginine ethyl ester and other p-nitroanilide substrates, were hydrolyzed. The toxin released bradykinin upon hydrolysis of kininogen. This enzymatic behavior is similar to that of plasma kallikrein: however, the presence of a characteristic "kallikrein-like" loop at 91-100 (GTIYNCNYVN) in the primary structure and other features similar to tissue kallikrein suggest that horridum toxin is more like tissue kallikrein. This toxin degraded all three chains of fibrinogen but did not form a clot, which suggests that it is different from thrombin. Moreover, it differs from another lethal factor from H. horridum horridum, gila toxin, which has 245 amino acid residues and does not cause exophthalmia. PMID:9440045

  14. Malignant gangliocytic paraganglioma of the duodenum with distant metastases and a lethal course

    PubMed Central

    Li, Bin; Li, Yang; Tian, Xiao-Ying; Luo, Bo-Ning; Li, Zhi

    2014-01-01

    Gangliocytic paraganglioma (GP) is rare and has been regarded as benign in general with a good prognosis. We present a patient with duodenal GP showing a malignant and lethal clinical course. A 47-year-old male patient was found to have a duodenal tumor and enlarged regional lymph nodes. The patient initially underwent a pancreaticoduodenectomy to resect the tumor and involved lymph nodes completely. Histological and immunohistochemical analyses showed findings typical of GP. However, the distant metastatic lesions in the liver and pelvic cavity were rapidly observed after surgery. The patient underwent chemotherapy and radiotherapy, as well as a second surgery to partly remove the metastatic mass in the pelvic cavity. The histological examination revealed no significant difference in histological features between the primary duodenal tumor and the metastatic pelvic mass. However, the patient finally died of the tumor due to the recurrence of the residual pelvic lesion and increased liver mass. To our knowledge, this is the first report of lethal GP with multifocal metastases. Our case confirms that GP should be regarded as a malignant potential tumor with behavior code of “1”, rather than a benign tumor of “0”. PMID:25386095

  15. Lethal and sublethal effects of imidacloprid, after chronic exposure, on the insect model Drosophila melanogaster.

    PubMed

    Charpentier, Gaël; Louat, Fanny; Bonmatin, Jean-Marc; Marchand, Patrice A; Vanier, Fanny; Locker, Daniel; Decoville, Martine

    2014-04-01

    Neonicotinoids are subjected to vigilance because of environmental contaminations and deleterious effects on bees. Imidacloprid (IMI) is one of the most representative insecticides of this family. At chronic exposure, concentration-effect relationships are non linear. An insect model should allow a better description of this toxicity. We compared the lethal concentration 50% (LC50) of IMI for a Drosophila-field strain, after acute and chronic exposure. Relative to the acute LC50, the chronic LC50 was lowered by a factor of 29 for males (1.3 mM/45 ?M), 52 for larvae (157 ?M/3 ?M) and more than 172 for females (>3.1 mM/18 ?M). Chronic exposure also revealed significant lethal and sublethal effects, at concentrations 3-5 orders of magnitude lower than the chronic LC50. Mean mortalities reached 28% (at 3.91 nM) and 27% (at 39.1 nM) for females and males, respectively. Fecundity decreased of 16% at 1.96 nM. Mating increased of 30% at 0.391 nM. The LOEC (lowest observed effect concentration: 0.391 nM) was 46?000 times lower than the chronic LC50 for males; it was 115?000 times lower than the chronic LC50 for females. This study illuminates effects that neonicotinoids can induce at very low concentrations. This is of particular interest for nontarget insects and for insect dependent species. PMID:24588730

  16. Erythropoiesis Suppression Is Associated with Anthrax Lethal Toxin-Mediated Pathogenic Progression

    PubMed Central

    Chang, Hsin-Hou; Wang, Tsung-Pao; Chen, Po-Kong; Lin, Yo-Yin; Liao, Chih-Hsien; Lin, Ting-Kai; Chiang, Ya-Wen; Lin, Wen-Bin; Chiang, Chih-Yu; Kau, Jyh-Hwa; Huang, Hsin-Hsien; Hsu, Hui-Ling; Liao, Chi-Yuan; Sun, Der-Shan

    2013-01-01

    Anthrax is a disease caused by the bacterium Bacillus anthracis, which results in high mortality in animals and humans. Although some of the mechanisms are already known such as asphyxia, extensive knowledge of molecular pathogenesis of this disease is deficient and remains to be further investigated. Lethal toxin (LT) is a major virulence factor of B. anthracis and a specific inhibitor/protease of mitogen-activated protein kinase kinases (MAPKKs). Anthrax LT causes lethality and induces certain anthrax-like symptoms, such as anemia and hypoxia, in experimental mice. Mitogen-activated protein kinases (MAPKs) are the downstream pathways of MAPKKs, and are important for erythropoiesis. This prompted us to hypothesize that anemia and hypoxia may in part be exacerbated by erythropoietic dysfunction. As revealed by colony-forming cell assays in this study, LT challenges significantly reduced mouse erythroid progenitor cells. In addition, in a proteolytic activity-dependent manner, LT suppressed cell survival and differentiation of cord blood CD34+-derived erythroblasts in vitro. Suppression of cell numbers and the percentage of erythroblasts in the bone marrow were detected in LT-challenged C57BL/6J mice. In contrast, erythropoiesis was provoked through treatments of erythropoietin, significantly ameliorating the anemia and reducing the mortality of LT-treated mice. These data suggested that suppressed erythropoiesis is part of the pathophysiology of LT-mediated intoxication. Because specific treatments to overcome LT-mediated pathogenesis are still lacking, these efforts may help the development of effective treatments against anthrax. PMID:23977125

  17. Antibody-mediated protection in mice with lethal intracerebral Cryptococcus neoformans infection.

    PubMed Central

    Mukherjee, J; Pirofski, L A; Scharff, M D; Casadevall, A

    1993-01-01

    The fungus Cryptococcus neoformans is an important opportunistic pathogen for patients with AIDS. C. neoformans infections frequently involve the brain and are often fatal. In the setting of AIDS C. neoformans infections are incurable and new treatment strategies are urgently needed. Passive administration of antibody is a potential therapeutic option for the prevention and treatment of C. neoformans. The IgG1 murine monoclonal antibody 2H1 to the capsular polysaccharide of C. neoformans was studied for its ability to modify the course of lethal intracerebral cryptococcal infection in mice. Intraperitoneal administration of antibody 2H1 resulted in small, yet significant, prolongations in the average survival of mice given intracerebral infection and reduced the number of C. neoformans colonies in brain tissue. Histopathological examination of brain tissues revealed a diffuse cryptococcal meningitis with fewer organisms in the brains of mice that received antibody 2H1 than in the control group. Thus, systemic administration of a monoclonal antibody can modify the course of lethal intracerebral C. neoformans infection in mice by prolonging survival and decreasing fungal burden in brain tissues. PMID:8475112

  18. Purification and biophysical characterization of the core protease domain of anthrax lethal factor

    SciTech Connect

    Gkazonis, Petros V.; Dalkas, Georgios A.; Chasapis, Christos T. [Department of Pharmacy, University of Patras, GR-26504 Patras (Greece)] [Department of Pharmacy, University of Patras, GR-26504 Patras (Greece); Vlamis-Gardikas, Alexios [Department of Biochemistry, Foundation for Biomedical Research (BRFAA), Academy of Athens, GR-11527 Athens (Greece)] [Department of Biochemistry, Foundation for Biomedical Research (BRFAA), Academy of Athens, GR-11527 Athens (Greece); Bentrop, Detlef [Institute of Physiology II, University of Freiburg, D-79108 Freiburg (Germany)] [Institute of Physiology II, University of Freiburg, D-79108 Freiburg (Germany); Spyroulias, Georgios A., E-mail: G.A.Spyroulias@upatras.gr [Department of Pharmacy, University of Patras, GR-26504 Patras (Greece)

    2010-06-04

    Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90 kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn{sup 2+} binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site are essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF{sub 672-776}) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ({sup 1}H, {sup 13}C, {sup 15}N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn{sup 2+}, suitable for high resolution structural analysis by NMR.

  19. Using photopigment biomarkers to quantify sub-lethal effects of petroleum pollution on natural phytoplankton assemblages

    SciTech Connect

    Swistak, J.; Pinckney, J.; Piehler, M.; Paerl, H. [Univ. of North Carolina, Morehead City, NC (United States). Inst. of Marine Sciences

    1995-12-31

    Although much work has been undertaken to determine the toxicity of petroleum pollutants to phytoplankton, most studies have used pure cultures to monitor growth of selected phytoplankton species. Fewer have considered the net effect on entire microalgal communities. Using high performance liquid chromatography (HPLC) to characterize diagnostic microalgal pigments, the authors were able to simultaneously assess sub-lethal pollutant effects on entire communities as well as on individual phytoplankton functional groups. Incubations of natural water samples with diesel fuel, an important contributor to coastal petroleum pollution, revealed significant changes in photopigments and relative abundance of taxonomic groups at sub-lethal concentrations. Differential rates of change of indicator pigment concentrations suggest a range of sensitivity among phytoplankton groups. In preliminary experiments, cyanobacteria exhibited the greatest overall tolerance to the diesel fuel concentrations tested, while cryptomonads displayed the most sensitivity. The authors are currently evaluating the responses of seasonal phytoplankton populations from 3 sites exposed to varied levels of petroleum pollution. HPLC will be used to characterize phytoplankton populations and to determine if the most abundant groups are also the most tolerant of diesel fuel. Preliminary experiments indicate that diesel fuel pollution may modify the structure and function of phytoplankton communities and subsequently alter the trophodynamics of impacted systems.

  20. HTST Milk Processing: Evaluating the Thermal Lethality inside Plate Heat Exchangers

    Microsoft Academic Search

    2004-01-01

    In the design of HTST processes, the lethality inside the plate heat exchanger and the heat loss at the holding tube are usually neglected. Thus, the thermal processing becomes more intensive than required and nutrient and sensorial losses may occur. In this work, the overall thermal lethality in milk pasteurization is evaluated by taking into account the lethality throughout the

  1. Toxicological screening in trauma

    PubMed Central

    Carrigan, T; Field, H; Illingworth, R; Gaffney, P; Hamer, D

    2000-01-01

    Objectives—To determine the prevalence and patterns of alcohol and drug use in patients with major trauma. Methods—Consecutive trauma patient enrolment, 24 hours a day, was envisaged with anonymised patient data on gender, age band, and mechanism of injury collected. The study group had surplus plasma quantitatively analysed for ethanol concentration, and urine samples were initially screened, via immunoassay, for opiates, cannabinoids, amphetamines, benzodiazepines, cocaine, and methadone. Confirmation and specification of individual positive results was then performed using thin layer or gas-liquid chromatography. Drugs of treatment given in the resuscitation room, if subsequently detected in the urine samples, were excluded from the final results. Results—There were 116 eligible trauma patients assessed and treated in the resuscitation room over a six month period, of which 93 (80%) were enrolled. Altogether 27% of this trauma population had plasma ethanol concentrations greater than 80 mg/dl. There was a significantly higher prevalence of alcohol intoxication in the group not involved in a road traffic accident (RTA) compared with the group who were involved in a RTA. Initial screening of urine for drugs revealed a prevalence of 51%. After 12 exclusions due to iatrogenic administration of opiates, the final confirmed prevalence was 35% in this trauma population. The individual drug prevalence was 13% for cannabinoids, 11% for codeine, 8% for morphine, 6% for amphetamine, 6% for benzodiazepines, 3% for cocaine, 1% for dihydrocodeine, and 1% for methadone. Conclusions—There is a notable prevalence of drug and alcohol use in this British accident and emergency trauma population. A significantly higher prevalence for alcohol intoxication was found in the non-RTA group compared with the RTA group. The patterns of drug usage detected reflect local influences and less cocaine use is seen compared with American studies. The association between alcohol, drugs, and trauma, together with ethically acceptable methods of screening, are discussed. PMID:10658989

  2. The lethal and sub-lethal consequences of entomopathogenic nematode infestation and exposure for adult pine weevils, Hylobius abietis (Coleoptera: Curculionidae)

    Microsoft Academic Search

    R. D. Girling; D. Ennis; A. B. Dillon; C. T. Griffin

    2010-01-01

    Entomopathogenic nematodes (EPN) frequently kill their host within 1–2days, and interest in EPN focuses mainly on their lethality. However, insects may take longer to die, or may fail to die despite being infected, but little is known about the effects of EPN infection on insects, other than death. Here we investigate both lethal and sub-lethal effects of infection by two

  3. Georgia Revealed

    NSDL National Science Digital Library

    OneWorld Journeys.com and Washingtonpost.com present Georgia Revealed: Searching for the Soul of the Caucasus. The site showcases a Georgia expedition that occurred April 16-29, the first of three explorations OneWorldJourneys.com have planned this year. Wilderness and nature photographers, journalists, and technicians collaborate here to bring users on their journey through the Caucasus Mountains Region of the Country of Georgia. Georgia Revealed not only features daily journal entries (text, streaming video and audio, and photographs) of the expedition, but also has sections providing background on history, travel, culture, and more. Altogether, this is a very well organized, educational site. We look forward to the next expedition to the Sonoran Desert.

  4. America Revealed

    NSDL National Science Digital Library

    2013-04-22

    The tagline on the America Revealed website says it all: "America Revealed explores the hidden patterns and rhythms that make America work." A remarkable series from PBS, the show talks about everything from how fresh seafood is sourced to how farmers combat crop pests. The Stories section includes a collage of images that, when scrolled over, provide accounts from a variety of people and industries. First-time visitors might want to watch the "Introduction to Manufacturing" series, which explores items that are made in the United States. Visitors can also use the Map section to look for stories of note from around the country, from Long Island to Southern California. The Teachers area includes ten lesson plans and links to additional resources. Finally, visitors can click on the Episodes area to watch complete episodes of the program.

  5. Recovery of plants from “Near-Lethal” stress

    Microsoft Academic Search

    A. M. Shirazi; L. H. Fuchigami

    1993-01-01

    This study reports on the dieback and recovery of red-osier dogwood, Cornus sericea L. plants from “near-lethal” (NL, sublethal) stress after varying lengths of post-stress environment (PSE). Intact dormant stems were subjected to 47° C for one hour during either October, November or December, and then placed into either constant 0° C or 23° C (dark condition) or kept under

  6. Acute Lethality of Copper, Cadmium, and Zinc to Northern Squawfish

    Microsoft Academic Search

    James D. Andros; Ronald R. Garton

    1980-01-01

    Flow-through acute toxicity tests on juvenile northern squawfish (Ptychocheilus oregonensis) were conducted with copper, cadmium, and zinc. The 96-hour median lethal concentrations were 18 ?g\\/liter for copper, 1,104 ?g\\/liter for cadmium, and 3,693 ?g\\/liter for zinc in 12 C water. These values, when compared to those for chinook salmon and steelhead parr tested under similar conditions, show that the northern

  7. Acute lethality of copper, cadmium, and zinc to northern squawfish

    Microsoft Academic Search

    JAMES D. ANDROS; RONALD R. GARTON

    1980-01-01

    Flow-through acute toxicity tests on juvenile northern squawfish (Ptychocheilus oregonensis) were conducted with copper, cadmium, and zinc. The 96-hour median lethal concentrations were 18 ..mu..g\\/liter for copper, 1104 ..mu..g\\/liter for cadmium, and 3693 ..mu..g\\/liter for zinc in 12°C water. These values, when compared to those for chinook salmon and steelhead parr tested under similar conditions, show that the northern squawfish

  8. Aortocolic fistula, a lethal cause of lower gastrointestinal bleeding

    Microsoft Academic Search

    Samuel E. Wilson; Milton L. Owens

    1976-01-01

    Summary  Aortocolic fistula occurs with spontaneous upture of aortic and iliac aneurysms into the sigmoid colon, or due to involvement\\u000a of the aneurysmal wall by acute diverticulitis. In the eight cases reviewed, this complication proved uniformly lethal, although\\u000a sufficient clinical findings were present for diagnosis, and adequate time was available for a planned therapeutic approach.\\u000a Lower gastrointestinal bleeding in the patient

  9. Lethal seizures predicted after aminophylline therapy in cocaine abusers

    Microsoft Academic Search

    Maciej Gasior; Jesse T Ungard; Jeffrey M Witkin

    2000-01-01

    Mice with a history of chronic (10 days), but not acute, treatment with a non-convulsant dose of cocaine showed increased sensitivity (P<0.001) to the toxic effects of aminophylline (seizures, lethality) relative to controls even days after the cessation of cocaine treatment. The present finding suggests that individuals with a history of cocaine use may be at increased risk for convulsive

  10. [Screening for diabetic retinopathy

    Microsoft Academic Search

    W. W. Hartstra; F. Holleman; J. B. Hoekstra; R. O. Schlingemann

    2007-01-01

    Concurrent with the increasing incidence of diabetes mellitus, the incidence of diabetic retinopathy is also rising. Timely recognition with the aid of screening, followed by laser therapy, can prevent the greater part of the resulting visual impairment and blindness. However, many patients with diabetes are not screened or not screened adequately. The necessary screening frequency is annually or biannually, depending

  11. Intact alternation performance in high lethality suicide attempters.

    PubMed

    Keilp, John G; Wyatt, Gwinne; Gorlyn, Marianne; Oquendo, Maria A; Burke, Ainsley K; John Mann, J

    2014-09-30

    Suicide attempters often perform poorly on tasks linked to ventral prefrontal cortical (VPFC) function. Object Alternation (OA) - a VPFC probe - has not been used in these studies. In this study, currently depressed medication-free past suicide attempters whose most severe attempt was of high (n=31) vs. low (n=64) lethality, 114 medication-free depressed non-attempters, and 86 non-patients completed a computerized OA task. Participants also completed comparison tasks assessing the discriminant validity of OA (Wisconsin Card Sort), its concurrent validity relative to tasks associated with past attempt status (computerized Stroop task, Buschke Selective Reminding Test), and its construct validity as a VPFC measure (Go-No Go and Iowa Gambling Task). Against expectations, high lethality suicide attempters - the majority of whom used non-violent methods in their attempts with some planning - outperformed other depressed groups on OA, with no group differences observed on Wisconsin Card Sort. Despite intact performance on OA, past attempters exhibited deficits on the Stroop and Buschke. OA performance was associated with performance on Go-No Go and Iowa Gambling, confirming that OA measures a similar construct. VPFC dysfunction may not be a characteristic of all suicide attempters, especially those who make more carefully planned, non-violent - though potentially lethal - attempts. PMID:24878299

  12. Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery.

    PubMed

    Cho, Hongbaek; Uehara, Tsuyoshi; Bernhardt, Thomas G

    2014-12-01

    Penicillin and related beta-lactams comprise one of our oldest and most widely used antibiotic therapies. These drugs have long been known to target enzymes called penicillin-binding proteins (PBPs) that build the bacterial cell wall. Investigating the downstream consequences of target inhibition and how they contribute to the lethal action of these important drugs, we demonstrate that beta-lactams do more than just inhibit the PBPs as is commonly believed. Rather, they induce a toxic malfunctioning of their target biosynthetic machinery involving a futile cycle of cell wall synthesis and degradation, thereby depleting cellular resources and bolstering their killing activity. Characterization of this mode of action additionally revealed a quality control function for enzymes that cleave bonds in the cell wall matrix. The results thus provide insight into the mechanism of cell wall assembly and suggest how best to interfere with the process for future antibiotic development. PMID:25480295

  13. 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 ?g/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 ?g/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 consequences in the entire ecosystem. The biomarker activities measured in daphnids and fish seemed to be useful tools in the assessment of trichlorfon effects, especially ChE activity which was the most sensitive biomarker tested for all organisms. Trichlorfon was teratogenic for zebrafish embryos leading to anomalies in the absorption of the yolk sac, spine bending and pericardial oedemas. The present research suggests that further work is urgently needed in order to monitor environmental concentrations of trichlorfon and to test the long term effects of environmentally realistic concentrations of this compound. PMID:21473847

  14. Risks of Cervical Cancer Screening

    MedlinePLUS

    ... Trials NCI Publications Español Cervical Cancer Screening (PDQ®) Risks of Cervical Cancer Screening Key Points for This ... your need for screening tests. Screening tests have risks. Decisions about screening tests can be difficult. Not ...

  15. Germline mutations in RYR1 are associated with foetal akinesia deformation sequence/lethal multiple pterygium syndrome.

    PubMed

    McKie, Arthur B; Al-Saedi, Atif; Vogt, Julie; Stuurman, Kyra E; Weiss, Marjan M; Shakeel, Hassan; Tee, Louise; Morgan, Neil V; Nikkels, Peter G J; van Haaften, Gijs; Park, Soo-Mi; van der Smagt, Jasper J; Bugiani, Marianna; Maher, Eamonn R

    2014-12-01

    IntroductionFoetal akinesia deformation sequence syndrome (FADS) is a genetically heterogeneous disorder characterised by the combination of foetal akinesia and developmental defects which may include pterygia (joint webbing). Traditionally multiple pterygium syndrome (MPS) has been divided into two forms: prenatally lethal (LMPS) and non-lethal Escobar type (EVMPS) types. Interestingly, FADS, LMPS and EVMPS may be allelic e.g. each of these phenotypes may result from mutations in the foetal acetylcholine receptor gamma subunit gene (CHRNG). Many cases of FADS and MPS do not have a mutation in a known FADS/MPS gene and we undertook molecular genetic studies to identify novel causes of these phenotypes.ResultsAfter mapping a novel locus for FADS/LMPS to chromosome 19, we identified a homozygous null mutation in the RYR1 gene in a consanguineous kindred with recurrent LMPS pregnancies. Resequencing of RYR1 in a cohort of 66 unrelated probands with FADS/LMPS/EVMPS (36 with FADS/LMPS and 30 with EVMPS) revealed two additional homozygous mutations (in frame deletions). The overall frequency of RYR1 mutations in probands with FADS/LMPS was 8.3%.ConclusionsOur findings report, for the first time, a homozygous RYR1 null mutation and expand the range of RYR1-related phenotypes to include early lethal FADS/LMPS. We suggest that RYR1 mutation analysis should be performed in cases of severe FADS/LMPS even in the absence of specific histopathological indicators of RYR1-related disease. PMID:25476234

  16. Effect of peripheral lymphoid cells on the incidence of lethal graft versus host disease following allogeneic mouse bone marrow transplantation

    SciTech Connect

    Almaraz, R.; Ballinger, W.; Sachs, D.H.; Rosenberg, S.A.

    1983-02-01

    Experiments were performed to study the role of circulating lymphoid cells in the incidence of lethal graft versus host disease (GVHD) in radiation-induced fully allogeneic mouse chimeras. The incidence of GVHD was reduced significantly in BALB/c leads to C57BL/6 radiation chimeras if bone marrow donors were exsanguinated immediately prior to marrow harvest. Chimeras resulting from the injection of bone marrow from bled donors exhibited only donor cells in spleen, bone marrow and peripheral blood and normal levels of Thy 1+ and Ia+ cells were found in each of these lymphoid compartments. The addition of as few as 3 X 10(4) peripheral mononuclear cells to the marrow from exsanguinated donors uniformly led to lethal GVHD. /sup 51/Cr-labeled cell traffic studies revealed that prior exsanguination of marrow donors led to about a 70% reduction in the number of circulating mononuclear cells contaminating the bone marrow at the time of marrow harvest. This decrease in contaminating peripheral cells was calculated to be in the appropriate range to account for the decreased GVHD seen when marrow from exsanguinated donors was used. It thus appears that peripheral cells contaminating marrow can be an important factor in causing lethal GVHD in allogeneic radiation chimeras.

  17. Plasma Screen Floating Mount

    SciTech Connect

    Eakle, Robert F. (New Ellenton, SC); Pak, Donald J. (Martine, GA)

    2004-10-26

    A mounting system for a flat display screen, particularly a plasma display screen, suspends the screen separately in each of the x-, y- and z-directions. A series of frames located by linear bearings and isolated by springs and dampers allows separate controlled movement in each axis. The system enables the use of relatively larger display screens in vehicles in which plasma screen are subject to damage from vibration.

  18. Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases

    PubMed Central

    Timms, Richard T.; Duncan, Lidia M.; Tchasovnikarova, Iva A.; Antrobus, Robin; Smith, Duncan L.; Dougan, Gordon; Weekes, Michael P.; Lehner, Paul J.

    2013-01-01

    The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system. PMID:24278019

  19. Blockade of liver macrophages by gadolinium chloride reduces lethality in endotoxemic rats-analysis of mechanisms of lethality in endotoxemia

    Microsoft Academic Search

    Masayuki Yamamoto; Hiroshi Kohno; Jun Itakura; Hideki Fujii; Yoshiro Matsumoto

    1994-01-01

    We investigated the effects of gadolinium chlo- ride (GdCl3 ? 6H20), which blocks phagocytosis by liver macrophages, on the mortality, blood tumor necrosis fac- tor (TNF) levels, and hepatotoxicity in a lethal endotoxic shock rat model system (iO mg\\/kg body weight lipopoly- saccharide (LPS) intravenously). With administration of GdC13, twice at 0.5 or 5 mg\\/kg, the survival rate 24 h

  20. Investigation of Phenolic Profiles, Cytotoxic Potential and Phytochemical Screening of Different Extracts of Drynaria quercifolia J. Smith (Leaves)

    PubMed Central

    Runa, Jannatul Ferdous; Hossain, Marjan; Hasanuzzaman, Md.; Ali, Md. Ramjan

    2013-01-01

    Purpose: The present study is aimed to evaluate phenolic profiles, cytotoxic activity and phytochemical screening of different extracts of Drynaria quercifolia leaves. Methods: The dried and powder leaves were extracted with methanol at room temperature and the concentrated methanolic extract was fractionated by the modified Kupchan partitioning method to provide pet-ether, carbon tetrachloride, chloroform and aqueous soluble fractions. Phenolic profiles were determined by using Folin-Ciocalteau reagent, which results were expressed in gallic acid equivalent (mg of GAE/g of sample). Phytochemical properties of different extractives of plant materials were tested by the method of Trease and Evans. Brine shrimp lethality bioassay was used to investigate the cytotoxic potential of D. quercifolia. Results: The phytochemical screening revealed the potent source of different phytochemical constituents on different extractives including alkaloid, glycosides, tannin, saponins, proteins and amino acids, flavonoids, triterpenes, phenols, phytosterols and carbohydrate. In the determination of phenolic profiles, different extractives showed a significant content of phenolic compounds ranging from 103.43 -132.23 mg of GAE/g of extractive. Compared to vincristine sulfate different extractives of plant materials demonstrated moderate cytotoxic potential (having LC50 of 12.45 ?g/ml, 13.02 ?g/ml 15.83 ?g/ml, 14.95 ?g/ml and 7.612 ?g/ml, respectively). Conclusion: It is concluded from this study that D. quercifolia is an excellent source of phenolic content and phytoconstitutes as well as possesses moderate cytotoxic activity. PMID:24312880

  1. Species Origin of Genomic Factors in Nicotiana nudicaulis Watson Controlling Hybrid Lethality in Interspecific Hybrids between N. nudicaulis Watson and N. tabacum L

    PubMed Central

    Liu, Hongshuo; Marubashi, Wataru

    2014-01-01

    Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis×N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris×N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris×N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla×N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla×N. tabacum and N. sylvestris×N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis. PMID:24806486

  2. Digital Measures screen changes 1 Digital Measures screen changes 2

    E-print Network

    Barrash, Warren

    Digital Measures screen changes 1 #12; Digital Measures screen changes 2 #12; Digital Measures screen changes 3 #12; Digital Measures screen changes 4 #12; Digital Measures screen changes 5 #12; Digital Measures screen changes 6 #12; Digital Measures screen changes 7 #12

  3. Georgians Revealed

    NSDL National Science Digital Library

    What was life like during the Georgian era in Britain? During the period between 1714 and 1830, cities and towns were transformed, conspicuous consumption became the pastime of the emerging middle classes, and gardening and shopping for leisure became commonplace. This digital companion to the British Library's "Georgians Revealed" exhibit brings together some of the key books and newspapers from the period, along with details about guided tours through the physical exhibitions, a Georgian London walking tour, and more. For those unable to view the exhibit in person, this companion site provides brief but detailed narratives on interesting facets of the exhibit, including dancing with the Georgians and celebrity culture. The site is rounded out by an excellent timeline of key events from the time of George I (1714-1727) to George IV (1820-1830) accompanied by vivid illustrations and portraiture.

  4. Two Cases of Lethal Complications Following Ultrasound-Guided Percutaneous Fine-Needle Biopsy of the Liver

    SciTech Connect

    Drinkovic, Ivan; Brkljacic, Boris [Department of Radiology, Ultrasonic Center, Medical School of the University of Zagreb, University Hospital 'Merkur', Zajceva 19, 10000 Zagreb (Croatia)

    1996-09-15

    Two cases with lethal complications are reported among 1750 ultrasound (US)-guided percutaneous fine-needle liver biopsies performed in our department. The first patient had angiosarcoma of the liver which was not suspected after computed tomography (CT) and US studies had been performed. The other patient had hepatocellular carcinoma in advanced hepatic cirrhosis. Death was due to bleeding in both cases. Pre-procedure laboratory tests did not reveal the existence of major bleeding disorders in either case. Normal liver tissue was interposed in the needle track between the liver capsule and the lesions which were targeted.

  5. Border Screening for SARS

    PubMed Central

    King, Arlene; de Jong, Dick; Bodie-Collins, Margaret; Squires, Susan G.; Tam, Theresa WS

    2005-01-01

    With the rapid international spread of severe acute respiratory syndrome (SARS) from March through May 2003, Canada introduced various measures to screen airplane passengers at selected airports for symptoms and signs of SARS. The World Health Organization requested that all affected areas screen departing passengers for SARS symptoms. In spite of intensive screening, no SARS cases were detected. SARS has an extremely low prevalence, and the positive predictive value of screening is essentially zero. Canadian screening results raise questions about the effectiveness of available screening measures for SARS at international borders. PMID:15705315

  6. Lethal and sublethal effects of marine sediment extracts on fish cells and chromosomes

    NASA Astrophysics Data System (ADS)

    Landolt, Marsha L.; Kocan, Richard M.

    1984-03-01

    The cost of conducting conventional chronic bioassays with every potentially toxic compound found in marine ecosystems is prohibitive; therefore short-term toxicity tests which can be used for rapid screening were developed. The tests employ cultured fish cells to measure lethal, sublethal or genotoxic effects of pure compounds and complex mixtures. The sensitivity of these tests has been proven under laboratory conditions; the following study used two of these tests, the anaphase aberration test and a cytotoxicity assay, under field conditions. Sediment was collected from 97 stations within Puget Sound, Washington. Serial washings of the sediment in methanol and dichloromethane yielded an organic extract which was dried, dissolved in DMSO and incubated as a series of dilutions with rainbow trout gonad (RTG-2) cells. The toxic effects of the extract were measured by examining the rate of cell proliferation and the percentage of damaged anaphase figures. Anaphase figures were considered to be abnormal if they exhibited non-disjunctions, chromosome fragments, or chromosome bridges. A second cell line (bluegill fry, BF-2) was also tested for cell proliferation and was included because, unlike the RTG-2 cell line, it contains little or no mixed function oxygenase activity. Of 97 stations tested, 35 showed no genotoxic activity, 42 showed high genotoxic activity (P?.01) and the remainder were intermediate. Among the toxic sites were several deep water stations adjacent to municipal sewage outfalls and four urban waterways contaminated by industrial and municipal effluents. Extracts from areas that showed genotoxic effects also inhibited cell proliferation and were cytotoxic to RTG-2 cells. Few effects were noted in the MFO deficient BF-2 cells. Short term in vitro tests provide aquatic toxicologists with a versatile and cost effective tool for screening complex environments. Through these tests one can identify compounds or geographic regions that exhibit high cytotoxic or genotoxic potential.

  7. EVALUATING THE PREDICTIVE VALIDITY OF SUICIDAL INTENT AND MEDICAL LETHALITY IN YOUTH

    PubMed Central

    Sapyta, Jeffrey; Goldston, David B.; Erkanli, Alaattin; Daniel, Stephanie S.; Heilbron, Nicole; Mayfield, Andrew; Treadway, S. Lyn

    2012-01-01

    Objectives To examine whether suicidal intent and medical lethality of past suicide attempts are predictive of future attempts, the association between intent and lethality, and the consistency of these characteristics across repeated attempts among youth. Method Suicide attempts in a 15-year prospective study of 180 formerly psychiatrically hospitalized adolescents (Mage at hospitalization = 14.83; 51% female; 80% Caucasian) were characterized using the Subjective Intent Rating Scale and Lethality of Attempt Rating Scale. Anderson-Gill recurrent events survival models and generalized estimating equations were used to assess predictive validity. Generalized linear models were used to examine stability of characteristics across attempts. Results Neither intent nor lethality from the most recent attempt predicted future attempts. The highest level of intent and most severe lethality of attempts during the follow-up predicted subsequent attempts, but the degree to which highest intent and most severe lethality contributed to prediction after considering methods of suicide attempts, past number of attempts, or psychiatric diagnoses was mixed. Across successive attempts, there was little consistency in reported characteristics. Intent and lethality were related to each other only for attempts occurring in early adulthood. Conclusions Highest intent and lethality were better predictors of future attempts than intent and lethality of the most recent attempt. However, these characteristics should only be considered as predictors within the context of other factors. For youth, clinicians should not infer true intent from the lethality of attempts, nor assume that characteristics of future suicide attempts will be similar to previous attempts. PMID:22250854

  8. Rifaximin diminishes neutropenia following potentially lethal whole-body radiation.

    PubMed

    Jahraus, Christopher D; Schemera, Bettina; Rynders, Patricia; Ramos, Melissa; Powell, Charles; Faircloth, John; Brawner, William R

    2010-07-01

    Terrorist attacks involving radiological or nuclear weapons are a substantial geopolitical concern, given that large populations could be exposed to potentially lethal doses of radiation. Because of this, evaluating potential countermeasures against radiation-induced mortality is critical. Gut microflora are the most common source of systemic infection following exposure to lethal doses of whole-body radiation, suggesting that prophylactic antibiotic therapy may reduce mortality after radiation exposure. The chemical stability, easy administration and favorable tolerability profile of the non-systemic antibiotic, rifaximin, make it an ideal potential candidate for use as a countermeasure. This study evaluated the use of rifaximin as a countermeasure against low-to-intermediate-dose whole-body radiation in rodents. Female Wistar rats (8 weeks old) were irradiated with 550 cGy to the whole body and were evaluated for 30 d. Animals received methylcellulose, neomycin (179 mg/kg/d) or variably dosed rifaximin (150-2000 mg/kg/d) one hour after irradiation and daily throughout the study period. Clinical assessments (e.g. body weight) were made daily. On postirradiation day 30, blood samples were collected and a complete blood cell count was performed. Animals receiving high doses of rifaximin (i.e. 1000 or 2000 mg/kg/d) had a greater increase in weight from the day of irradiation to postirradiation day 30 compared with animals that received placebo or neomycin. For animals with an increase in average body weight from irradiation day within 80-110% of the group average, methylcellulose rendered an absolute neutrophil count (ANC) of 211, neomycin rendered an ANC of 334, rifaximin 300 mg/kg/d rendered an ANC of 582 and rifaximin 1000 mg/kg/d rendered an ANC of 854 (P = 0.05 for group comparison). Exposure to rifaximin after near-lethal whole-body radiation resulted in diminished levels of neutropenia. PMID:20558844

  9. A Limited Number of Caenorhabditis Elegans Genes Are Readily Mutable to Dominant, Temperature-Sensitive Maternal-Effect Embryonic Lethality

    PubMed Central

    Mitenko, N. L.; Eisner, J. R.; Swiston, J. R.; Mains, P. E.

    1997-01-01

    Dominant gain-of-function mutations can give unique insights into the study of gene function. In addition, gain-of-function mutations, unlike loss-of-function alleles, are not biased against the identification of genetically redundant loci. To identify novel genetic functions active during Caenorhabditis elegans embryogenesis, we have collected a set of dominant temperature-sensitive maternal-effect embryonic lethal mutations. In a previous screen, we isolated eight such mutations, distributed among six genes. In the present study, we describe eight new dominant mutations that identify only three additional genes, yielding a total of 16 dominant mutations found in nine genes. Therefore, it appears that a limited number of C. elegans genes mutate to this phenotype at appreciable frequencies. Five of the genes that we identified by dominant mutations have loss-of-function alleles. Two of these genes may lack loss-of-function phenotypes, indicating that they are nonessential and so may represent redundant loci. Loss-of-function mutations of three other genes are associated with recessive lethality, indicating nonredundancy. PMID:9409829

  10. Anti-bacterial activity and brine shrimp lethality bioassay of methanolic extracts of fourteen different edible vegetables from Bangladesh

    PubMed Central

    Ullah, M. Obayed; Haque, Mahmuda; Urmi, Kaniz Fatima; Zulfiker, Abu Hasanat Md.; Anita, Elichea Synthi; Begum, Momtaj; Hamid, Kaiser

    2013-01-01

    Objective To investigate the antibacterial and cytotoxic activity of fourteen different edible vegetables methanolic extract from Bangladesh. Methods The antibacterial activity was evaluated using disc diffusion assay method against 12 bacteria (both gram positive and gram negative). The plant extracts were also screened for cytotoxic activity using the brine shrimp lethality bioassay method and the lethal concentrations (LC50) were determined at 95% confidence intervals by analyzing the data on a computer loaded with “Finney Programme”. Results All the vegetable extracts showed low to elevated levels of antibacterial activity against most of the tested strains (zone of inhibition=5-28 mm). The most active extract against all bacterial strains was from Xanthium indicum which showed remarkable antibacterial activity having the diameter of growth inhibition zone ranging from 12 to 28 mm followed by Alternanthera sessilis (zone of inhibition=6-21 mm). All extracts exhibited considerable general toxicity towards brine shrimps. The LC50 value of the tested extracts was within the range of 8.447 to 60.323 µg/mL with respect to the positive control (vincristine sulphate) which was 0.91 µg/mL. Among all studied extracts, Xanthium indicum displayed the highest cytotoxic effect with LC50 value of 8.447 µg/mL. Conclusions The results of the present investigation suggest that most of the studied plants are potentially good source of antibacterial and anticancer agents. PMID:23570009

  11. Intimate partner homicide: new insights for understanding lethality and risks.

    PubMed

    Sheehan, Brynn E; Murphy, Sharon B; Moynihan, Mary M; Dudley-Fennessey, Erin; Stapleton, Jane G

    2015-02-01

    Research on covictims, family members, and close friends who have lost loved ones to intimate partner homicide (IPH) is a neglected area of study. We conducted phenomenological interviews with covictims to gain insights into risk and lethality, examined affidavits from criminal case files, and reviewed news releases. The data uncovered acute risk factors prior to the homicide, identified changes in the perpetrators' behavior and the perpetrators' perceived loss of control over the victim, and described barriers that victims faced when attempting to gain safety. Findings suggest that recognizing acute risk factors is an important area for future IPH research. PMID:25540257

  12. Acute lethality of copper, cadmium, and zinc to northern squawfish

    SciTech Connect

    Andros, J.D.; Garton, R.R.

    1980-03-01

    Flow-through acute toxicity tests on juvenile northern squawfish (Ptychocheilus oregonensis) were conducted with copper, cadmium, and zinc. The 96-hour median lethal concentrations were 18 ..mu..g/liter for copper, 1104 ..mu..g/liter for cadmium, and 3693 ..mu..g/liter for zinc in 12/sup 0/C water. These values, when compared to those for chinook salmon and steelhead parr tested under similar conditions, show that the northern squawfish are more tolerant than the two salmonids to zinc and cadmium but equally sensitive to copper.

  13. Palliative care of the infant with lethal anomalies.

    PubMed

    Leuthner, Steven R

    2004-06-01

    Although many families of infants with prenatally diagnosed lethal anomalies may receive counseling by clinicians with perinatal, neonatal, or genetic expertise and deliver their babies in a tertiary care center, pediatricians may be called on to support and care for these infants. Their involvement may begin prenatally, at the time of delivery, in the newborn nursery, or upon discharge home from a NICU. The goal of this article is to help the general pediatrician gain some comfort in knowing which cases might be considered and provide some tools and ideas for counseling a family from any point of involvement. PMID:15157596

  14. Revealing Rembrandt

    PubMed Central

    Parker, Andrew J.

    2014-01-01

    The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI). Our results emphasized the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt's portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings. PMID:24795552

  15. The Revealer

    NSDL National Science Digital Library

    2005-01-01

    For those interested in insightful and critical analysis of issues regarding religion and its portrayal in the media, it can be difficult to sift and winnow through the myriad of material offered on the web. Jointly sponsored by the New York University Department of Journalism and New York Universityâ??s Center for Religion and Media, The Revealer is a well-thought out review of just such matters, and one that will be of great interest to persons with a penchant for the subject. The review is divided into three playful headings: Today, Timely, and Timeless. As might be expected, the Today section culls media coverage from that particular day. The Timely section offers links to media coverage of particularly germane issues and events, while the Timeless area offers some exclusive commentaries on photography and the occult and the relationship between science and religion. Finally, visitors can zero in on the religion of their choice by looking through the material as organized by faith, such as Hinduism, Paganism, and Christianity.

  16. Glutamate receptor pathology is present in the hippocampus following repeated sub-lethal soman exposure in the absence of spatial memory deficits.

    PubMed

    Johnson, Erik A; Daugherty, Kelly S; Gallagher, Sarah J; Moran, Anita V; DeFord, S Michelle

    2008-01-01

    Much is still unknown about the long-term effects of repeated, sub-lethal exposure to organophosphorus (OP) nerve agents, such as soman (GD), on learning and memory tasks and related protein expression in the hippocampus. In the present study, guinea pigs were exposed to sub-lethal doses of GD for 10 days and cognitive performance assessed using the Morris water maze (MWM) up to 88 days post-exposure to investigate spatial learning. Additionally, hippocampal lysates were probed for cytoskeletal, synaptic and glutamate receptor proteins using Western blot analyses. No significant difference in MWM performance was observed between repeated sub-lethal GD exposed and saline control groups. However, Western blot analyses revealed significant changes in glutamate receptor protein immunoreactivity for subunits GluR2, NMDAR1, NMDAR2a and NMDAR2b in the hippocampi of GD-exposed guinea pigs. Levels of GluR2, NMDAR2a and NMDAR2b increased by 3 months post-initial exposure and returned to control levels by 6 months while NMDAR1 decreased by 6 months. No significant differences in neurofilament medium (NFM), neurofilament light (NFL) or synaptophysin densitometry were detected and alpha-II-spectrin proteolytic breakdown was also absent. These results reveal that repeated, sub-lethal exposure to GD affects glutamate receptor subunit expression but does not affect cytoskeletal protein immunoreactivity or the proteolytic state in the hippocampus. Though these changes do not affect spatial memory, they may contribute to other cognitive deficits previously observed following sub-lethal OP exposure. PMID:17942156

  17. National Lung Screening Trial

    Cancer.gov

    Information about the National Lung Screening Trial (NLST), a research study sponsored by the National Cancer Institute that used low-dose helical CT scans or chest X-ray to screen men and women at risk for lung cancer.

  18. Stretching Screens and Imaginations.

    ERIC Educational Resources Information Center

    Douthwaite, Shelaugh

    1983-01-01

    Secondary students utilize a simplified technique to make silk screen prints, which can be printed onto T-shirts. The only materials needed from art suppliers are a few squeegees and a few yards of polyester screen mesh. (RM)

  19. Periodic Screening Evaluation

    Cancer.gov

    Statistical Software Periodic Screening Evaluation (Written by Stuart G. Baker) New Approach (Simplified Approximation): See Baker SG. Evaluating periodic cancer screening without a randomized control group: a simplified design and analysis. In: Duffy

  20. Lung Cancer Screening

    PubMed Central

    Gutierrez, Antonio; Suh, Robert; Abtin, Fereidoun; Genshaft, Scott; Brown, Kathleen

    2013-01-01

    Lung cancer is the leading cause of cancer death. Although smoking prevention and cessation programs have decreased lung cancer mortality, there remains a large at-risk population. Dismal long-term survival rates persist despite improvements in diagnosis, staging, and treatment. Early efforts to identify an effective screening test have been unsuccessful. Recent advances in multidetector computed tomography have allowed screening studies using low-dose computed tomography (LDCT) to be performed. This set the stage for the National Lung Screening Trial that found that annual LDCT screening benefits individuals at high risk for lung cancer. An understanding of the harmful effects of lung cancer screening is required to help maximize the benefits and decrease the risks of a lung cancer screening program. Although many questions remain regarding LDCT screening, a comprehensive lung cancer screening program of high-risk individuals will increase detection of preclinical and potentially curable disease, creating a new model of lung cancer surveillance and management. PMID:24436526

  1. International Cancer Screening Network

    Cancer.gov

    Skip to Main Content Search International Cancer Screening Network Sponsored by the National Cancer Institute Working Together to Evaluate Cancer Screening and Improve Outcomes Internationally About the ICSN Overview Participating Countries Contact

  2. Screening for Prostate Cancer

    MedlinePLUS

    Screening for Prostate Cancer: A Guidance Statement From the Clinical Guidelines Committee of the American College of Physicians The full report is titled “Screening for Prostate Cancer: A Guidance Statement From the Clinical Guidelines ...

  3. Prostate Cancer Screening

    MedlinePLUS

    The prostate is the gland below a man's bladder that produces fluid for semen. Cancer screening is looking for ... treat. There is no standard screening test for prostate cancer. Researchers are studying different tests to find ...

  4. Video Screen Capture Basics

    ERIC Educational Resources Information Center

    Dunbar, Laura

    2014-01-01

    This article is an introduction to video screen capture. Basic information of two software programs, QuickTime for Mac and BlueBerry Flashback Express for PC, are also discussed. Practical applications for video screen capture are given.

  5. Newborn screening tests

    MedlinePLUS

    ... March of Dimes and the American College of Medical Genetics suggest more than two dozen additional tests. The ... Saunders Elsevier; 2011:chap 707. American College of Medical Genetics Newborn Screening Expert Group. Newborn screening: toward a ...

  6. Lung Cancer Screening with Low Dose CT

    PubMed Central

    Caroline, Chiles

    2014-01-01

    SUMMARY The announcement of the results of the NLST, showing a 20% reduction in lung-cancer specific mortality with LDCT screening in a high risk population, marked a turning point in lung cancer screening. This was the first time that a randomized controlled trial had shown a mortality reduction with an imaging modality aimed at early detection of lung cancer. Current guidelines endorse LDCT screening for smokers and former smokers ages 55 to 74, with at least a 30 pack year smoking history. Adherence to published algorithms for nodule follow-up is strongly encouraged. Future directions for screening research include risk stratification for selection of the screening population, and improvements in the diagnostic follow-up for indeterminate pulmonary nodules. As with screening for other malignancies, screening for lung cancer with LDCT has revealed that there are indolent lung cancers which may not be fatal. More research is necessary if we are to maximize the risk-benefit ratio in lung cancer screening. PMID:24267709

  7. Revealing Mercury

    NASA Astrophysics Data System (ADS)

    Prockter, L. M.; Solomon, S. C.; Head, J. W.; Watters, T. R.; Murchie, S. L.; Robinson, M. S.; Chapman, C. R.; McNutt, R. L.

    2009-04-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft, developed under NASA's Discovery Program, launched in August 2004. En route to insertion into orbit about Mercury in 2011, MESSENGER flies by Mercury three times. The first and second of these encounters were accomplished in January and October of 2008. These flybys viewed portions of Mercury's surface that were not observed by Mariner 10 during its reconnaissance of somewhat less than half of the planet in 1974-1975. All MESSENGER instruments operated during each flyby and returned a wealth of new data. Many of the new observations were focused on the planet's geology, including monochrome imaging at resolutions as high as 100 m/pixel, multispectral imaging in 11 filters at resolutions as high as 500 m/pixel, laser altimetry tracks extending over several thousands of kilometers, and high-resolution spectral measurements of several types of terrain. Here we present an overview of the first inferences on the global geology of Mercury from the MESSENGER observations. Whereas evidence for volcanism was equivocal from Mariner 10 data, the new MESSENGER images and altimetry provide compelling evidence that volcanism was widespread and protracted on Mercury. Color imaging reveals three common spectral units on the surface: a higher-reflectance, relatively red material occurring as a distinct class of smooth plains, typically with distinct embayment relationships interpreted to indicate volcanic emplacement; a lower-reflectance, relatively blue material typically excavated by impact craters and therefore inferred to be more common at depth; and a spectrally intermediate terrain that constitutes much of the uppermost crust. Three more minor spectral units are also seen: fresh crater ejecta, reddish material associated with rimless depressions interpreted to be volcanic centers, and high-reflectance deposits seen in some crater floors. Preliminary measurements of crater size-frequency distribution suggest that smooth plains on Mercury's surface range in age from the end of the period of heavy impact bombardment to as young as perhaps 1 billion years; these ongoing measurements are helping to elucidate the volcanic history of the planet. Mercury's global tectonic history is also revealed by the MESSENGER image and laser altimeter data. Significant evidence for global contraction was seen in Mariner 10 images in the form of widespread lobate scarps. The MESSENGER images show that contractional features are the dominant tectonic landform globally, and the inferred average contractional strain is at least one third greater than previously inferred from Mariner 10 observations. Only three exceptions to the dominance of contractional deformation have been found to date: extensional troughs that include prominent basin-radial systems documented in two basins, the Pantheon Fossae within the 1500-km-diameter Caloris basin and a similar set of features within a newly-imaged 700-km-diameter basin, and a circumferential trough system within the smaller, younger Raditladi basin. That these extensional tectonic features are rare on Mercury, and that they are not seen within basins elsewhere in the Solar System, pose important constraints on the thermal and mechanical evolution of Mercury's interior.

  8. Revealing Things

    NSDL National Science Digital Library

    Revealing Things is the Smithsonian Institution's first specifically web based exhibit; both the content and design of the site are fascinating. This work in progress is a prototype of a future, more fully-developed exhibit. It concentrates on "common, everyday objects to tell stories about people, their cultures, and the meanings they associate with their possessions." Items discussed include a 1937 chemistry set, a Vietnam memorial offering, a duckpin bowling ball, an early TV, and a celery vase, among many others. Organized according to theme, era, and object, the exhibit is presented in a new pop-up browser window. Within that window, navigation takes place via "maplets," a connected series of moving colored labels representing the three ways that the exhibit is organized. Users can move slider bars to effect the placement of the labels, and search on terms to create their own thematic or object-based exhibit. When the cursor is placed over an object label, scrolling text introduces it. Alternatively, the site can be navigated via a series of icons that run down the middle of the exhibition page. When an icon is clicked, the series of icons may rearrange. Each exhibit contains a photo of the object, along with written commentary on it. In addition, sound is sometimes available to play period music, or render out loud the exhibition text. The most fully-developed object at this time is "Patched Bellbottoms." Users are advised to read the help files on both the main page and the exhibit page for navigation tips. The exhibit is a fascinating precursor of what could be a new way to interactively view museum exhibits, allowing the user to cast off the restraints of a linear orientation. Note that the exhibit is extremely browser and bandwidth intensive.

  9. Bacillus anthracis’ lethal toxin induces broad transcriptional responses in human peripheral monocytes

    PubMed Central

    2012-01-01

    Background Anthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system. Results Western Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500?ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT. Conclusion We conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte’s normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway. PMID:22747600

  10. Host Responses to Sepsis Vary in Different Low-Lethality Murine Models

    PubMed Central

    Gentile, Lori F.; Nacionales, Dina C.; Lopez, M. Cecilia; Vanzant, Erin; Cuenca, Angela; Szpila, Benjamin E.; Cuenca, Alex G.; Joseph, Anna; Moore, Frederick A.; Leeuwenburgh, Christiaan; Baker, Henry V.; Moldawer, Lyle L.; Efron, Philip A.

    2014-01-01

    Introduction Animal models for the study of sepsis are being increasingly scrutinized, despite their essential role for early translational research. In particular, recent studies have suggested that at the level of the leukocyte transcriptome, murine models of burns, trauma and endotoxemia markedly differ from their human equivalents, and are only weakly similar amongst themselves. We compared the plasma cytokine and leukocyte transcriptome responses between two different low-lethality murine models of polymicrobial intra-abdominal sepsis. Methods Six to ten week male C57BL/6j mice underwent either the ‘gold standard’ cecal ligation and puncture (CLP) model of intra-abdominal sepsis or administration of a cecal slurry (CS), where cecal contents are injected intraperitoneally. Surviving mice were euthanized at two hours, one or three days after sepsis. Results The murine leukocyte transcriptomic response to the CLP and CS models of sepsis was surprisingly dissimilar at two hours, one, and three days after sepsis. The Pearson correlation coefficient for the maximum change in expression for the entire leukocyte transcriptome that changed significantly over time (n?=?19,071) was R?=?0.54 (R2?=?0.297). The CS model resulted in greater magnitude of early inflammatory gene expression changes in response to sepsis with associated increased production of inflammatory chemokines and cytokines. Two hours after sepsis, CLP had more significant expression of genes associated with IL-10 signaling pathways, whereas CS had greater expression of genes related to CD28, apoptosis, IL-1 and T-cell receptor signaling. By three days, the changes in gene expression in both sepsis models were returning to baseline in surviving animals. Conclusion These analyses reveal that the murine blood leukocyte response to sepsis is highly dependent on which model of intra-abdominal sepsis is employed, despite their similar lethality. It may be difficult to extrapolate findings from one murine model to another, let alone to human sepsis. PMID:24788351

  11. Antibiotics induce redox-related physiological alterations as part of their lethality

    PubMed Central

    Dwyer, Daniel J.; Belenky, Peter A.; Yang, Jason H.; MacDonald, I. Cody; Martell, Jeffrey D.; Takahashi, Noriko; Chan, Clement T. Y.; Lobritz, Michael A.; Braff, Dana; Schwarz, Eric G.; Ye, Jonathan D.; Pati, Mekhala; Vercruysse, Maarten; Ralifo, Paul S.; Allison, Kyle R.; Khalil, Ahmad S.; Ting, Alice Y.; Walker, Graham C.; Collins, James J.

    2014-01-01

    Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. PMID:24803433

  12. Antibiotics induce redox-related physiological alterations as part of their lethality.

    PubMed

    Dwyer, Daniel J; Belenky, Peter A; Yang, Jason H; MacDonald, I Cody; Martell, Jeffrey D; Takahashi, Noriko; Chan, Clement T Y; Lobritz, Michael A; Braff, Dana; Schwarz, Eric G; Ye, Jonathan D; Pati, Mekhala; Vercruysse, Maarten; Ralifo, Paul S; Allison, Kyle R; Khalil, Ahmad S; Ting, Alice Y; Walker, Graham C; Collins, James J

    2014-05-20

    Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. PMID:24803433

  13. Biological Screening of Medicinal Plants Collected from Eastern Ghats of India Using Artemia salina (Brine Shrimp Test)

    Microsoft Academic Search

    Alluri V. Krishnaraju; Tayi V. N. Rao; Dodda Sundararaju; Mulabagal Vanisree; Hsin-Sheng Tsay; Gottumukkala V. Subbaraju

    Medicinal plants constitute important components of flora and are widely distributed in different regions of India. Based on ethnomedical significance, we have collected several me- dicinal plants used in traditional medicine from Eastern Ghats of India and evaluated for their biological activity. In the present study, a method utilizing brine shrimp (Artemia salina Leach) lethality was used to screen medicinal

  14. Improvement in Screening Radiologists' Performance in an Organized Screening Program

    Cancer.gov

    Improvement in Screening Radiologists’ Performance in an Organized Screening Program Nancy A. T. Wadden, MD, FRCPC Gregory Doyle, BSc, MBA Breast Screening Program for Newfoundland and Labrador Canada Background • Breast Screening Program for Newfoundland

  15. How many loci on the X-chromosome of Drosophila melanogaster can mutate to recessive lethals

    SciTech Connect

    Abrahamson, S. (Univ. of Wisconsin, Madison); Wuergler, F.E.; DeJongh, C.; Meyer, H.U.

    1980-01-01

    The sensitivity of the sex-linked recessive lethal test is due to the fact that a very large number of loci are included in the mutation study. From extensive studies on the spontaneous sex-linked recessive lethal frequency and spontaneous specific locus mutation rates, it is possible to derive an estimate of the number of loci included in the recessive lethal test. The average number derived from three estimates on male and female germ cells in 563 loci. A second independent approach derives from published data which analyzed short regions of the genome and the proportion of loci within these regions which mutate to lethality. This analysis suggests that 830 loci are potentially lethal mutables. We describe the reasons for concluding that 600 to 800 loci of the approximately 1000 loci on the X-chromosome are involved in the X-linked recessive lethal test.

  16. Abundant genetic variability in Drosophila simulans for hybrid female lethality in interspecific crosses to Drosophila melanogaster.

    PubMed

    Gérard, Pierre R; Presgraves, Daven C

    2012-02-01

    Intrinsic postzygotic reproductive isolation is thought to result from the substitution of multiple harmless or beneficial genetic differences between species that are incidentally deleterious when combined in species hybrids, causing hybrid sterility or inviability. Genetic variability for hybrid sterility or inviability phenotypes is, however, rarely assessed in natural populations. Here, we assess variation for Drosophila simulans-encoded maternal factor(s) that cause lethality in D. simulans-Drosophila melanogaster F(1) hybrid females. First, we survey genetic variability in the strength of D. simulans-mediated maternal effect hybrid lethality among 37 geographic and laboratory isolates. We find abundant variability in the strength of maternal effect hybrid lethality, ranging from complete lethality to none. Second, we assess maternal effect hybrid lethality for a subset of wild isolates made heterozygous with two so-called hybrid rescue strains. The results suggest that the D. simulans maternal effect hybrid lethality involves a diversity of alleles and/or multiple loci. PMID:22353244

  17. Equation of state and fragmentation issues in computational lethality analysis

    SciTech Connect

    Trucano, T.G.

    1993-07-01

    The purpose of this report is to summarize the status of computational analysis of hypervelocity impact lethality in relatively nontechnical terms from the perspective of the author. It is not intended to be a review of the technical literature on the problems of concern. The discussion is focused by concentrating on two phenomenology areas which are of particular concern in computational impact studies. First, the material`s equation of state, specifically the treatment of expanded states of metals undergoing shock vaporization, is discussed. Second, the process of dynamic fragmentation is addressed. In both cases, the context of the discussion deals with inaccuracies and difficulties associated with numerical hypervelocity impact simulations. Laboratory experimental capabilities in hypervelocity impact for impact velocities greater than 10.0 km/s are becoming increasingly viable. This paper also gives recommendations for experimental thrusts which utilize these capabilities that will help to resolve the uncertainties in the numerical lethality studies that are pointed out in the present report.

  18. A virulent parasite can provide protection against a lethal parasitoid.

    PubMed

    Sternberg, Eleanore D; Lefèvre, Thierry; Rawstern, Amanda H; de Roode, Jacobus C

    2011-03-01

    Hosts often become infected with multiple parasite strains or species. Previous work has shown that the outcome of infections with multiple parasite strains or species often differs significantly from that of single infections, making them a potentially important factor in determining the prevalence and spread of disease. Here we show that infection with a virulent parasite increases host survival during later exposure to a lethal parasitoid. Specifically, when monarch butterfly larvae (Danaus plexippus) are inoculated with the virulent protozoan parasite Ophryocystis elektroscirrha and then attacked by the lethal parasitoid fly Lespesia archippivora, survival is higher than when the larvae are exposed to the parasitoid only. This is potentially a result of the protozoan's requirement for host survival to obtain between-host transmission. Our findings suggest that a virulent parasite can play a protective role for its host and indicate that parasites can act as mutualists depending on the presence of other parasites. We emphasize the importance of considering infection in an ecological context, including the presence of competing parasites. PMID:21145987

  19. Effectiveness of lethal, directed wolf-depredation control in Minnesota

    USGS Publications Warehouse

    Harper, E.K.; Paul, W.J.; Mech, L.D.; Weisberg, S.

    2008-01-01

    Wolf (Canis lupus) depredations on livestock in Minnesota, USA, are an economic problem for many livestock producers, and depredating wolves are lethally controlled. We sought to determine the effectiveness of lethal control through the analysis of data from 923 government-verified wolf depredations from 1979 to 1998. We analyzed the data by 1) assessing the correlations between the number of wolves killed in response to depredations with number of depredations the following year at state and local levels, and 2) the time to the next depredation. No analysis indicated that trapping wolves substantially reduced the following year's depredations at state or local levels. However, more specific analyses indicated that in certain situations, killing wolves was more effective than no action (i.e., not trapping). For example, trapping and killing adult males decreased the re-depredation risk. At sheep farms, killing wolves was generally effective. Attempting to trap, regardless of the results, seemed more effective at reducing depredations than not trapping, suggesting that mere human activity near depredation sites might deter future depredations.

  20. Injury risk assessment of non-lethal projectile head impacts.

    PubMed

    Oukara, Amar; Nsiampa, Nestor; Robbe, Cyril; Papy, Alexandre

    2014-01-01

    Kinetic energy non-lethal projectiles are used to impart sufficient effect onto a person in order to deter uncivil or hazardous behavior with a low probability of permanent injury. Since their first use, real cases indicate that the injuries inflicted by such projectiles may be irreversible and sometimes lead to death, especially for the head impacts. Given the high velocities and the low masses involved in such impacts, the assessment approaches proposed in automotive crash tests and sports may not be appropriate. Therefore, there is a need of a specific approach to assess the lethality of these projectiles. In this framework, some recent research data referred in this article as "force wall approach" suggest the use of three lesional thresholds (unconsciousness, meningeal damages and bone damages) that depend on the intracranial pressure. Three corresponding critical impact forces are determined for a reference projectile. Based on the principle that equal rigid wall maximal impact forces will produce equal damage on the head, these limits can be determined for any other projectile. In order to validate the consistence of this innovative method, it is necessary to compare the results with other existing assessment methods. This paper proposes a comparison between the "force wall approach" and two different head models. The first one is a numerical model (Strasbourg University Finite Element Head Model-SUFEHM) from Strasbourg University; the second one is a mechanical surrogate (Ballistics Load Sensing Headform-BLSH) from Biokinetics. PMID:25400712

  1. Less-lethal munitions as extended-range impact weapons

    NASA Astrophysics Data System (ADS)

    Hubbs, Ken

    1997-01-01

    With the proliferation of 'suicide by cop' incidents, the concept of less lethal (LL) impact munitions has definitely caught on. There is much to be said for sterile laboratory testing and wound ballistic studies, but having 'real world' operational data is invaluable. Two years ago, a data base was set up to collect this information. The data base continues to grow with incidents from a cross the country and others pursued internationally. Indications are that LL munitions deliver a similar amount of force as conventional police impact weapons i.e., police batons, PR-24's, nunchakus, etc. One advantage over conventional impact weapons, is that LL munitions can be used at much greater distances from a suspect or crown of rioters. This gave rise to the term: extended range impact weapons. Having the ability to examine numerous cases in which these LL munitions have been successfully used for the resolution of critical incidents, is beneficial in evaluating the application and defending the usage of these force options. This paper examines 187 less lethal shootings and discusses such things as: the distance the munitions were fired, the types of injuries sustained by the targeted suspect, the body area of impact, what, if any weapons the suspect was armed with, and the type of incident requiring police response.

  2. HLA-A2-Restricted Protection against Lethal Lymphocytic Choriomeningitis? †

    PubMed Central

    Botten, Jason; Whitton, J. Lindsay; Barrowman, Polly; Sidney, John; Whitmire, Jason K.; Alexander, Jeff; Ting, Joey P. C.; Bui, Huynh-Hoa; Sette, Alessandro; Buchmeier, Michael J.

    2007-01-01

    The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8+ T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides—nucleoprotein NP69-77, glycoprotein precursor GPC10-18, GPC447-455, and zinc-binding protein Z49-58—that displayed high-affinity binding (?275 nM) to HLA-A*0201, induced CD8+ T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC447-455), after peptide immunization of HLA-A*0201 mice, induced CD8+ T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV. PMID:17166907

  3. Injury Risk Assessment of Non-Lethal Projectile Head Impacts

    PubMed Central

    Oukara, Amar; Nsiampa, Nestor; Robbe, Cyril; Papy, Alexandre

    2014-01-01

    Kinetic energy non-lethal projectiles are used to impart sufficient effect onto a person in order to deter uncivil or hazardous behavior with a low probability of permanent injury. Since their first use, real cases indicate that the injuries inflicted by such projectiles may be irreversible and sometimes lead to death, especially for the head impacts. Given the high velocities and the low masses involved in such impacts, the assessment approaches proposed in automotive crash tests and sports may not be appropriate. Therefore, there is a need of a specific approach to assess the lethality of these projectiles. In this framework, some recent research data referred in this article as “force wall approach” suggest the use of three lesional thresholds (unconsciousness, meningeal damages and bone damages) that depend on the intracranial pressure. Three corresponding critical impact forces are determined for a reference projectile. Based on the principle that equal rigid wall maximal impact forces will produce equal damage on the head, these limits can be determined for any other projectile. In order to validate the consistence of this innovative method, it is necessary to compare the results with other existing assessment methods. This paper proposes a comparison between the “force wall approach” and two different head models. The first one is a numerical model (Strasbourg University Finite Element Head Model-SUFEHM) from Strasbourg University; the second one is a mechanical surrogate (Ballistics Load Sensing Headform-BLSH) from Biokinetics. PMID:25400712

  4. Amifostine alleviates radiation-induced lethal small bowel damage via promotion of 14-3-3?-mediated nuclear p53 accumulation

    PubMed Central

    Chen, Yu-Min; Chen, Yi-Fan; Wang, Chung-Chi; Lin, I-Hui; Huang, Yu-Jie; Yang, Kuender D.

    2014-01-01

    Amifostine (AM) is a radioprotector that scavenges free radicals and is used in patients undergoing radiotherapy. p53 has long been implicated in cell cycle arrest for cellular repair after radiation exposure. We therefore investigated the protective p53-dependent mechanism of AM on small bowel damage after lethal whole-abdominal irradiation (WAI). AM increased both the survival rate of rats and crypt survival following lethal 18 Gy WAI. The p53 inhibitor PFT-? compromised AM-mediated effects when administered prior to AM administration. AM significantly increased clonogenic survival in IEC-6 cells expressing wild type p53 but not in p53 knockdown cells. AM significantly increased p53 nuclear accumulation and p53 tetramer expression before irradiation through the inhibition of p53 degradation. AM inhibited p53 interactions with MDM2 but enhanced p53 interactions with 14-3-3?. Knockdown of 14-3-3? also compromised the effect of AM on clonogenic survival and p53 nuclear accumulation in IEC-6 cells. For the first time, our data reveal that AM alleviates lethal small bowel damage through the induction of 14-3-3? and subsequent accumulation of p53. Enhancement of the p53/14-3-3? interaction results in p53 tetramerization in the nucleus that rescues lethal small bowel damage. PMID:25230151

  5. The Effect of Non-Lethal Weapons on Police Officer Safety

    Microsoft Academic Search

    Alex Yuskavage

    Between 1990 and 2000, there was an increase in the use of non-lethal weapons and a decline in the number and severity of attacks on police officers. Using longitudinal data on several hundred U.S. police agencies, I investigate the relationship between police officer safety and the adoption of non-lethal weapons. I find that the adoption of non-lethal chemical weapons had

  6. Impulsivity, aggression and brain structure in high and low lethality suicide attempters with borderline personality disorder.

    PubMed

    Soloff, Paul; White, Richard; Diwadkar, Vaibhav A

    2014-06-30

    Impulsivity and aggressiveness are trait dispositions associated with the vulnerability to suicidal behavior across diagnoses. They are associated with structural and functional abnormalities in brain networks involved in regulation of mood, impulse and behavior. They are also core characteristics of borderline personality disorder (BPD), a disorder defined, in part, by recurrent suicidal behavior. We assessed the relationships between personality traits, brain structure and lethality of suicide attempts in 51 BPD attempters using multiple regression analyses on structural MRI data. BPD was diagnosed by the Diagnostic Interview for Borderline Patients-revised, impulsivity by the Barratt Impulsiveness Scale (BIS), aggression by the Brown-Goodwin Lifetime History of Aggression (LHA), and high lethality by a score of 4 or more on the Lethality Rating Scale (LRS). Sixteen High Lethality attempters were compared to 35 Low Lethality attempters, with no significant differences noted in gender, co-morbidity, childhood abuse, BIS or LHA scores. Degree of medical lethality (LRS) was negatively related to gray matter volumes across multiple fronto-temporal-limbic regions. Effects of impulsivity and aggression on gray matter volumes discriminated High from Low Lethality attempters and differed markedly within lethality groups. Lethality of suicide attempts in BPD may be related to the mediation of these personality traits by specific neural networks. PMID:24656768

  7. Risks of Endometrial Cancer Screening

    MedlinePLUS

    ... Trials NCI Publications Español Endometrial Cancer Screening (PDQ®) Risks of Endometrial Cancer Screening Key Points for This ... caused by the test itself. Screening tests have risks. Decisions about screening tests can be difficult. Not ...

  8. Risks of Skin Cancer Screening

    MedlinePLUS

    ... Trials NCI Publications Español Skin Cancer Screening (PDQ®) Risks of Skin Cancer Screening Key Points for This ... A biopsy may cause scarring. Screening tests have risks. Decisions about screening tests can be difficult. Not ...

  9. Risks of Colorectal Cancer Screening

    MedlinePLUS

    ... Trials NCI Publications Español Colorectal Cancer Screening (PDQ®) Risks of Colorectal Cancer Screening Key Points for This ... testing Sigmoidoscopy Colonoscopy Virtual colonoscopy Screening tests have risks. Decisions about screening tests can be difficult. Not ...

  10. DISPLAY SCREEN EQUIPMENT REGULATIONS 1992

    E-print Network

    Screen Equipment can be defined as any conventional cathode ray tube screen and other display screen and possibly mental stress. These conditions are not unique to working with display screen equipment

  11. Risks of Breast Cancer Screening

    MedlinePLUS

    Risks of Breast Cancer Screening Key Points for This Section Screening tests have risks. The risks of breast cancer screening tests include ... risk of dying from cancer . The risks of breast cancer screening tests include the following: Finding breast cancer ...

  12. Screening colonoscopy in Australia.

    PubMed

    Singh, Rajvinder; Mangira, Dileep; Kawano, Hiroshi; Matsuda, Takashia

    2015-04-01

    Colorectal cancer (CRC) is a global epidemic predominantly affecting Western countries. It is the second leading cause of cancer-related deaths in Australia with one in 12 Australians affected by this condition by the age of 85 years. Appropriate preventive measures by screening followed by colonoscopy can detect cancer and precancerous lesions, which are potentially curable. The National Bowel Cancer Screening Program (NBCSP) is a national screening program implemented by the Australian Government aimed at reducing morbidity and mortality from bowel cancer by actively recruiting and screening the target population. The long-term goal of the program is to include the at-risk population (50-74 years of age) in a biennial screening program. Newer technologies could have a potential role in screening programs by enhancing adenoma detection rates. However, until more evidence is available, improving screening uptake and bowel preparation strategies are the prime focus in reducing CRC-related morbidity and mortality. PMID:25523496

  13. Mode of action of Pseudomonas fluorescens strain CL145A, a lethal control agent of dreissenid mussels (Bivalvia: Dreissenidae).

    PubMed

    Molloy, Daniel P; Mayer, Denise A; Giamberini, Laure; Gaylo, Michael J

    2013-05-01

    Pseudomonas fluorescens strain CL145A (Pf-CL145A) has demonstrated promise as an efficacious and selective agent for the control of macrofouling Dreissena spp. mussels. Herein, we report trials to investigate the mode of action of this biocontrol agent against Dreissena polymorpha, the zebra mussel. Exposure to dead Pf-CL145A cells achieved the same temporal pattern and percentage mussel mortality as did live cells, thereby excluding infection as the possible lethal mode of action. Histological analysis revealed pathologies consistent with the cause of death being intoxicating natural products associated with Pf-CL145A cells. Irrespective of whether the mussels were exposed to live or dead Pf-CL145A cells, examination of tissues from histological sections revealed that: (1) at the end of the 24-h treatment period there was massive hemocyte infiltration into the lumina of both the digestive gland and stomach; and (2) mussel deaths occurred following lysis and necrosis of the digestive gland and sloughing of stomach epithelium. These trials provide strong evidence that the lethal mode of action of Pf-CL145A is intoxication. PMID:23313118

  14. Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches*

    PubMed Central

    Zakharova, Maria Yu.; Kuznetsov, Nikita A.; Dubiley, Svetlana A.; Kozyr, Arina V.; Fedorova, Olga S.; Chudakov, Dmitry M.; Knorre, Dmitry G.; Shemyakin, Igor G.; Gabibov, Alexander G.; Kolesnikov, Alexander V.

    2009-01-01

    Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca2+ and Mn2+. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors. PMID:19359249

  15. A homoallelic Gly[sup 317] [yields] Asp mutation in ALPL causes the perinatal (lethal) form of hypophosphatasia in Canadian mennonites

    SciTech Connect

    Greenberg, C.R.; Taylor, C.D.; Haworth, J.C.; Seargeant, L.E.; Phillipps, S.; Triggs-Raine, B.; Chodirker, B.N. (Univ. of Manitoba, Winnepeg (Canada))

    1993-07-01

    The authors have discovered a single homoallelic nucleotide substitution as the putative cause of the perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Previous linkage and haplotype analysis in this population suggested that a single mutational event was responsible for this autosomal recessive form of hypophosphatasia. The mutation is a guanosine-to-adenosine substitution at nucleotide position 1177 in exon 10 of the tissue nonspecific (liver/bone/kidney) alkaline phosphatase gene. This Gly[sup 317] [yields] Asp mutation segregates exclusively with the heterozygote phenotype previously assigned by biochemical testing (maximum combined lod score of 18.24 at [theta] = 0.00). This putative disease-causing mutation has not been described in controls nor in other non-Mennonite probands with both lethal and nonlethal forms of hypophosphatasia studied to date. This Gly[sup 317] [yields] Asp mutation changes a polar glycine to an acidic aspartate at amino acid position 317 within the highly conserved active site region of the 507-amino-acid polypeptide. Carrier screening for this lethal mutation in a high-risk population is now feasible. 15 refs., 2 figs.

  16. Screening for Breast Cancer

    PubMed Central

    Elmore, Joann G.; Armstrong, Katrina; Lehman, Constance D.; Fletcher, Suzanne W.

    2011-01-01

    Context Breast cancer screening in community practices may be different from that in randomized controlled trials. New screening modalities are becoming available. Objectives To review breast cancer screening, especially in the community and to examine evidence about new screening modalities. Data Sources and Study Selection English-language articles of randomized controlled trials assessing effectiveness of breast cancer screening were reviewed, as well as meta-analyses, systematic reviews, studies of breast cancer screening in the community, and guidelines. Also, studies of newer screening modalities were assessed. Data Synthesis All major US medical organizations recommend screening mammography for women aged 40 years and older. Screening mammography reduces breast cancer mortality by about 20% to 35% in women aged 50 to 69 years and slightly less in women aged 40 to 49 years at 14 years of follow-up. Approximately 95% of women with abnormalities on screening mammograms do not have breast cancer with variability based on such factors as age of the woman and assessment category assigned by the radiologist. Studies comparing full-field digital mammography to screen film have not shown statistically significant differences in cancer detection while the impact on recall rates (percentage of screening mammograms considered to have positive results) was unclear. One study suggested that computer-aided detection increases cancer detection rates and recall rates while a second larger study did not find any significant differences. Screening clinical breast examination detects some cancers missed by mammography, but the sensitivity reported in the community is lower (28% to 36%) than in randomized trials (about 54%). Breast self-examination has not been shown to be effective in reducing breast cancer mortality, but it does increase the number of breast biopsies performed because of false-positives. Magnetic resonance imaging and ultrasound are being studied for screening women at high risk for breast cancer but are not recommended for screening the general population. Sensitivity of magnetic resonance imaging in high-risk women has been found to be much higher than that of mammography but specificity is generally lower. Effect of the magnetic resonance imaging on breast cancer mortality is not known. A balanced discussion of possible benefits and harms of screening should be undertaken with each woman. Conclusions In the community, mammography remains the main screening tool while the effectiveness of clinical breast examination and self-examination are less. New screening modalities are unlikely to replace mammography in the near future for screening the general population. PMID:15755947

  17. Defective Kernel Mutants of Maize. I. Genetic and Lethality Studies

    PubMed Central

    Neuffer, M. G.; Sheridan, William F.

    1980-01-01

    A planting of 3,919 M1 kernels from normal ears crossed by EMS-treated pollen produced 3,461 M1 plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive. PMID:17249053

  18. Functional wiring of the yeast kinome revealed by global analysis of genetic network motifs

    PubMed Central

    Sharifpoor, Sara; van Dyk, Dewald; Costanzo, Michael; Baryshnikova, Anastasia; Friesen, Helena; Douglas, Alison C.; Youn, Ji-Young; VanderSluis, Benjamin; Myers, Chad L.; Papp, Balázs; Boone, Charles; Andrews, Brenda J.

    2012-01-01

    A combinatorial genetic perturbation strategy was applied to interrogate the yeast kinome on a genome-wide scale. We assessed the global effects of gene overexpression or gene deletion to map an integrated genetic interaction network of synthetic dosage lethal (SDL) and loss-of-function genetic interactions (GIs) for 92 kinases, producing a meta-network of 8700 GIs enriched for pathways known to be regulated by cognate kinases. Kinases most sensitive to dosage perturbations had constitutive cell cycle or cell polarity functions under standard growth conditions. Condition-specific screens confirmed that the spectrum of kinase dosage interactions can be expanded substantially in activating conditions. An integrated network composed of systematic SDL, negative and positive loss-of-function GIs, and literature-curated kinase–substrate interactions revealed kinase-dependent regulatory motifs predictive of novel gene-specific phenotypes. Our study provides a valuable resource to unravel novel functional relationships and pathways regulated by kinases and outlines a general strategy for deciphering mutant phenotypes from large-scale GI networks. PMID:22282571

  19. The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization? §

    PubMed Central

    Pfaff, Kathleen L.; Straub, Christian T.; Chiang, Ken; Bear, Daniel M.; Zhou, Yi; Zon, Leonard I.

    2007-01-01

    A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly. PMID:17576815

  20. Complex physiological traits as biomarkers of the sub-lethal toxicological effects of pollutant exposure in fishes.

    PubMed

    McKenzie, D J; Garofalo, E; Winter, M J; Ceradini, S; Verweij, F; Day, N; Hayes, R; van der Oost, R; Butler, P J; Chipman, J K; Taylor, E W

    2007-11-29

    Complex physiological traits, such as routine aerobic metabolic rate or exercise performance, are indicators of the functional integrity of fish that can reveal sub-lethal toxicological effects of aquatic pollutants. These traits have proved valuable in laboratory investigations of the sub-lethal effects of heavy metals, ammonia and various xenobiotics. It is not known, however, whether they can also function as biomarkers of the complex potential range of effects upon overall functional integrity caused by exposure to mixtures of chemicals in polluted natural environments. The current study used portable swimming respirometers to compare exercise performance and respiratory metabolism of fish exposed in cages for three weeks to either clean or polluted sites on three urban European river systems: the river Lambro, Milan, Italy; the rivers Blythe, Cole and Tame, Birmingham, UK; and the river Amstel, Amsterdam, The Netherlands. The UK and Italian rivers were variously polluted with high levels of both bioavailable heavy metals and organics, and the Amstel by mixtures of bioavailable organics at high concentrations. In both the UK and Italy, indigenous chub (Leuciscus cephalus) exposed to clean or polluted sites swam equally well in an initial performance test, but the chub from polluted sites could not repeat this performance after a brief recovery interval. These animals were unable to raise the metabolic rate and allocate oxygen towards exercise in the second trial, an effect confirmed in successive campaigns in Italy. Swimming performance was therefore a biomarker indicator of pollutant exposure in chub exposed at these sites. Exposure to polluted sites on the river Amstel did not affect the repeat swimming performance of cultured cloned carp (Cyprinus carpio), indicating either a species-specific tolerance or relative absence of heavy metals. However, measurements of oxygen uptake during swimming revealed increased rates of routine aerobic metabolism in both chub and carp at polluted sites in all of the rivers studied, indicating a sub-lethal metabolic loading effect. Therefore, the physiological traits of exercise performance and metabolic rate have potential as biomarkers of the overall sub-lethal toxic effects of exposure to complex mixtures of pollutants in rivers, and may also provide insight into why fish do not colonize some polluted environments. PMID:17475615

  1. Hydroxychloroquine retinopathy screening

    PubMed Central

    Semmer, Anne E; Lee, Michael S; Harrison, Andrew R; Olsen, Timothy W

    2010-01-01

    Aim To compare current hydroxychloroquine retinopathy screening practices with the published 2002 American Academy of Ophthalmology (AAO) Preferred Practice Patterns (PPP). Methods A multiple-choice survey was distributed to 105 ophthalmologists to assess current screening practices and knowledge of patient risk factors. Results were compared with the PPP guidelines. A cost analysis of the PPP and survey paradigms was conducted. Results Sixty-seven (64%) of 105 surveys were completed. The majority (90%) of physicians screen for hydroxychloroquine retinopathy with either central automated threshold perimetry or Amsler grid as recommended by the PPP. Most survey respondents could not correctly identify the evidence-based risk factors. The majority screen more frequently than recommended: 87% screen high-risk patients and 94% screen low-risk patients more frequently than recommended in the PPP. The increased screening frequency of low-risk patients translates into an excess of $44 million in the first five years of therapy. If all patients were screened using exact PPP paradigm, savings could exceed $150 million every 10 years. Conclusions Ophthalmologists currently screen for hydroxychloroquine retinopathy correctly, however, their lack of familiarity with evidence-based guidelines may result in excessive follow up. Increasing awareness and implementation of the PPP could potentially reduce hydroxychloroquine retinopathy screening costs significantly. PMID:18829634

  2. Automated Groundwater Screening

    SciTech Connect

    Taylor, Glenn A.; Collard, Leonard, B.

    2005-10-31

    The Automated Intruder Analysis has been extended to include an Automated Ground Water Screening option. This option screens 825 radionuclides while rigorously applying the National Council on Radiation Protection (NCRP) methodology. An extension to that methodology is presented to give a more realistic screening factor for those radionuclides which have significant daughters. The extension has the promise of reducing the number of radionuclides which must be tracked by the customer. By combining the Automated Intruder Analysis with the Automated Groundwater Screening a consistent set of assumptions and databases is used. A method is proposed to eliminate trigger values by performing rigorous calculation of the screening factor thereby reducing the number of radionuclides sent to further analysis. Using the same problem definitions as in previous groundwater screenings, the automated groundwater screening found one additional nuclide, Ge-68, which failed the screening. It also found that 18 of the 57 radionuclides contained in NCRP Table 3.1 failed the screening. This report describes the automated groundwater screening computer application.

  3. Dominant lethal test in rats treated with some plant extracts.

    PubMed

    Aritajat, S; Kaweewat, K; Manosroi, J; Manosroi, A

    2000-01-01

    The present study was undertaken to investigate the toxic effect of aqueous extracts of Aegle marmelos (AM), Stevia rebaudiana (SR), Pouteria cambodiana (PC) and Clausena excavata (CE) on rats by dominant lethal test. The data of 8-week treatment suggested that none of the extracts adversely affected male body and testicular weights as well as cauda epididymal sperm counts. No notable changes in sperm morphology and motility were observed. On the other hand, sperm count in the CE group was significantly higher as compared to both control and other treatment groups. There were no abnormal changes in the number of implantation sites, number of viable fetuses and number of dead fetuses in females mated with plant extract-treated males relative to controls. Based on these results, it could be concluded that all the investigated plant extracts have no toxic effect on male rat reproduction and progeny outcome. PMID:11414451

  4. Launcher barrel for the lethality test system railgun

    SciTech Connect

    Sims, J.R.; Christensen, K.E.; Cummings, C.E.; Calkins, N.C.

    1986-01-01

    A reusable plasma armature railgun barrel design is described. This barrel was designed and is being fabricated at the Los Alamos National Laboratory for use in a kinetic energy lethality test system. The performance goals for this barrel are (1) that it be able to withstand the loads generated when accelerating a 30-g projectile to a velocity of 15 km/s without sustaining permanent damage, (2) that it have multiple shot capability, and, (3) that it be capable of being repaired and/or modified. The barrel consists of a multipiece modular core contained in an outer structural shell. The core assembly, composed of rails and insulators, is accessible for repair or replacement. The outer structural shell is designed to allow access to the core and is used to preload the core compressively. The barrel design incorporates various features that will allow the use of stronger and stiffer materials such as structural ceramics and reinforced coppers as they become available.

  5. Non-lethal laser dazzling as a personnel countermeasure

    NASA Astrophysics Data System (ADS)

    Shannon, David C.

    2013-10-01

    Optical distraction is likely one of the original and simpler optical countermeasure concepts with a technology history dating back to the 1800's. The objective is to distract or suppress either equipment or personnel with optical radiation from a safe distance. This paper is intended to review and expand on the concepts presented at the 2012 SPIE Security and Defense meeting; "Non-Lethal Optical Interruption (Dazzling): Technology, Devices, and Scenarios". The information that follows will focus primarily on the technology and techniques associated with the safe laser dazzling of personnel. Key product design guidelines will be highlighted and reviewed. Recent advances in laser technology and their associated impact on hand-held devices will also be discussed. Finally, the author will offer his opinion on the growth rate of military and non-military markets for laser dazzlers.

  6. Batrachochytrium dendrobatidis infection and lethal chytridiomycosis in caecilian amphibians (Gymnophiona).

    PubMed

    Gower, David J; Doherty-Bone, Thomas; Loader, Simon P; Wilkinson, Mark; Kouete, Marcel T; Tapley, Benjamin; Orton, Frances; Daniel, Olivia Z; Wynne, Felicity; Flach, Edmund; Müller, Hendrik; Menegon, Michele; Stephen, Ian; Browne, Robert K; Fisher, Mathew C; Cunningham, Andrew A; Garner, Trenton W J

    2013-06-01

    Batrachochytrium dendrobatidis (Bd) is commonly termed the 'amphibian chytrid fungus' but thus far has been documented to be a pathogen of only batrachian amphibians (anurans and caudatans). It is not proven to infect the limbless, generally poorly known, and mostly soil-dwelling caecilians (Gymnophiona). We conducted the largest qPCR survey of Bd in caecilians to date, for more than 200 field-swabbed specimens from five countries in Africa and South America, representing nearly 20 species, 12 genera, and 8 families. Positive results were recovered for 58 specimens from Tanzania and Cameroon (4 families, 6 genera, 6+ species). Quantities of Bd were not exceptionally high, with genomic equivalent (GE) values of 0.052-17.339. In addition, we report the first evidence of lethal chytridiomycosis in caecilians. Mortality in captive (wild-caught, commercial pet trade) Geotrypetes seraphini was associated with GE scores similar to those we detected for field-swabbed, wild animals. PMID:23677560

  7. Radiation-induced mutagenicity and lethality in Salmonella typhimurium

    SciTech Connect

    Isildar, M.; Bakale, G.

    1983-01-01

    The mutagenic and lethal effects of ionizing radiation on histidine-deficient auxotrophs of Salmonella typhimurium were studied to improve the understanding of radiation damage to DNA. The auxotrophs were divided into two groups - one which is sensitive to base-pair substitutions and another sensitive to frameshifts. These groups were composed of parent-daughter pairs in which the chemical mutagenicity enhancing plasmid, pKM101, is absent in the parent strain and present in the daughter. Co-60 ..gamma..-radiation and 250 kV x-rays were used to irradiate the bacteria. Irradiation of the frameshift - sensitive strains which carry the pKm101 plasmid doubled the absolute number of induced revertants whereas irradiation of the base-pair substitution sensitive strain which also carries the pKm101 plasmid produced nearly no change in the number of induced revertants. A nearly negligible effect on the mutation rate was observed for all parent strains. (ACR)

  8. Aroclor 1254 residues in birds: Lethal levels and loss rates

    USGS Publications Warehouse

    Stickel, W.H.; Stickel, L.F.; Dyrland, R.A.; Hughes, D.L.

    1984-01-01

    Lethal residues of polychlorinated biphenyls (PCBs) were determined experimentally in four species of wild birds (male common grackles (Quiscalus quiscula ), immature female red-winged blackbirds (Agelaius phoeniceus ), adult male brown-headed cowbirds (Molothrus ater ) and immature female starlings (Sturnus vulgaris)) given dietary dosage of 1,500 ppm of Aroclor 1254) until one-half had died, sacrificing the survivors, chemically analyzing the tissues, and comparing results in dead birds and survivors. For all species, residues of 310 ppm or higher in the brain showed increasing likelihood of death from PCB poisoning. Residues in dead birds did not differ among species except for starlings (Sturnus vulgaris ), which averaged slightly lower than the others. However, the species differed in the length of time to 50% mortality and in the levels of PCBs in brains at sacrifice.

  9. DDE in birds: Lethal residues and loss rates

    USGS Publications Warehouse

    Stickel, W.H.; Stickel, L.F.; Dyrland, R.A.; Hughes, D.L.

    1984-01-01

    Lethal brain residues of DDE were determined experimentally in four species of wild birds (male common grackels (Quiscalus quiscula ), immature female red-winged blackbirds (Agelaius phoeniceus ), adult male brown-headed cowbirds (Molathrus ater ), and immature female starlings (Sturnus vulgaris ) given dietary dosage of 1,500 ppm DDE until one-half had died, then sacrificing the survivors, chemically analyzing the tissues, and comparing results in dead birds and survivors. In all species, residues of 300 to 400 ppm of DDE in the brain were considered to show increasing likelihood of death from DDE, confirming results of an earlier study with a single species. Body residues (ppm wet weight) were not diagnostic, overlapping grossly in dead birds and survivors, but averaging higher in survivors.

  10. Pre-Participation Musculoskeletal and Cardiac Screening of Male Athletes in the United Arab Emirates

    PubMed Central

    Alattar, A; Ghani, S; Mahdy, N; Hussain, H; Maffulli, N

    2014-01-01

    This study presents the results of pre-participation musculoskeletal and cardiac screening using the Lausanne recommendations, which include a personal and family history, physical examination and electrocardiography. Cross sectional study using the Lausanne screenings and the European Society of Cardiology (ESC) recommendations carried out at Al-Ahli club in Dubai, United Arab Emirates. 230 male athletes participating in organised sports were included. Exclusion criteria were those under 14 or over 35 years old, females and athletes with established cardiovascular disease. Primary outcome are the results of Lausanne screening with outline of the negative, positive and false positive results and number needed to screen. Secondary outcomes include the results of musculoskeletal and neurological screening. A total of 174 (76%) athletes had a negative screening result. Fifty-four athletes (23%) underwent additional testing. Forty-seven athletes (20.4%) had false positive screening results. Seven athletes (3%) had a positive screening result and four athletes (2%) were restricted from sport. The number of athletes needed to screen to detect one lethal cardiovascular condition was 33 athletes. The Lausanne recommendations are well suited for the United Arab Emirates. The number needed to screen to detect one athlete with serious cardiovascular disease is acceptable at 33. PMID:24809035

  11. An exome sequencing strategy to diagnose lethal autosomal recessive disorders

    PubMed Central

    Ellard, Sian; Kivuva, Emma; Turnpenny, Peter; Stals, Karen; Johnson, Matthew; Xie, Weijia; Caswell, Richard; Lango Allen, Hana

    2015-01-01

    Rare disorders resulting in prenatal or neonatal death are genetically heterogeneous. For some conditions, affected fetuses can be diagnosed by ultrasound scan, but this is not usually possible until mid-gestation. There is often limited fetal DNA available for investigation. We investigated a strategy for diagnosing autosomal recessive lethal disorders in non-consanguineous pedigrees with multiple affected fetuses. Exome sequencing was performed to identify genes where each parent is heterozygous for a rare non-synonymous-coding or splicing variant. Putative pathogenic variants were tested for cosegregation in affected fetuses and unaffected siblings. In eight couples of European ancestry, we found on average 1.75 genes (range 0–4) where both parents were heterozygous for rare potentially deleterious variants. A proof-of-principle study detected heterozygous DYNC2H1 variants in a couple whose five fetuses had short-rib polydactyly. Prospective analysis of two couples with multiple pregnancy terminations for fetal akinesia syndrome was performed and a diagnosis was obtained in both the families. The first couple were each heterozygous for a previously reported GLE1 variant, p.Arg569His or p.Val617Met; both were inherited by their two affected fetuses. The second couple were each heterozygous for a novel RYR1 variant, c.14130-2A>G or p.Ser3074Phe; both were inherited by their three affected fetuses but not by their unaffected child. Biallelic GLE1 and RYR1 disease-causing variants have been described in other cases with fetal akinesia syndrome. We conclude that exome sequencing of parental samples can be an effective tool for diagnosing lethal recessive disorders in outbred couples. This permits early prenatal diagnosis in future pregnancies. PMID:24961629

  12. Antibody-Targeted Lethal Photosensitization of Porphyromonas gingivalis

    PubMed Central

    Bhatti, M.; MacRobert, A.; Henderson, B.; Shepherd, P.; Cridland, J.; Wilson, M.

    2000-01-01

    We have previously demonstrated that Porphyromonas gingivalis is susceptible to killing by toluidine blue O (TBO) when irradiated with light from a helium-neon (HeNe) laser. The aim of this study was to determine whether a TBO-antibody conjugate (Ab-TBO) could be used to specifically target P. gingivalis to lethal photosensitization in the presence of Streptococcus sanguis or human gingival fibroblasts (HGFs). When a mixture of P. gingivalis and S. sanguis was exposed to 4 ?g of TBO/ml and irradiated with HeNe laser light, there were 1.5- and 4.0-log10-unit reductions in the viable counts, respectively. In contrast, when TBO was conjugated with a murine monoclonal antibody against P. gingivalis lipopolysaccharide, the reductions in viable counts of P. gingivalis and S. sanguis amounted to 5.0 and 0.1 log10 units, respectively. Lethal photosensitization of P. gingivalis in the presence of HGFs using unconjugated TBO resulted in a 0.7-log10-unit reduction in P. gingivalis viable counts and a 99% reduction in the incorporation of tritiated thymidine ([3H]Tdr) by the HGFs. In contrast, when the Ab-TBO conjugate was used, there was a 100% reduction in P. gingivalis viable counts but no significant reduction in the incorporation of [3H]Tdr by HGFs. These results demonstrate that specific targeting of P. gingivalis can be achieved using TBO conjugated to a monoclonal antibody raised against a cell surface component of this organism. PMID:10991833

  13. Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue?

    PubMed Central

    Graci, Jason D.; Harki, Daniel A.; Korneeva, Victoria S.; Edathil, Jocelyn P.; Too, Kathleen; Franco, David; Smidansky, Eric D.; Paul, Aniko V.; Peterson, Blake R.; Brown, Daniel M.; Loakes, David; Cameron, Craig E.

    2007-01-01

    Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses. PMID:17686844

  14. Screening Mobile Devices to Examine Network Health

    Microsoft Academic Search

    Grant A. Jacoby; J. Scot Ransbottom; Thadeus Hickman; Maxwell Potasznik

    2007-01-01

    Screening mobile devices for changes to their local registries is a key vital sign that can provide comprehensive health examinations for the benefit of an enterprise network. Our research reveals that the registry can be modified with very little difficulty to include \\

  15. Turbulent flow through screens

    NASA Technical Reports Server (NTRS)

    Mehta, R. D.

    1984-01-01

    A detailed experimental investigation has been carried out on the effects of different types of screens on turbulent flow, in particular turbulent boundary layers. The effect of a screen on a turbulent boundary layer is to give it a 'new lease of life'. The boundary layer turbulence is reorganized and the thickness reduced, thus making it less susceptible to separation. The aerodynamic properties of plastic screens are found to differ significantly from those of the conventional metal screens, evidently because of differences in the weaving properties. The 'overshoot' in mean velocity profile near the boudnary layer edge is shown to be a result of the effect of screen inclination on pressure drop coefficient. A more accurate formulation for the deflection coefficient of a screen is also proposed.

  16. Current tuberculosis screening practices.

    PubMed Central

    Snider, D E; Anderson, H R; Bentley, S E

    1984-01-01

    Health department officials in all 50 states and 14 major cities responded to a survey questionnaire designed to obtain information about current tuberculosis screening practices. Persons being screened fell into the groups designated as high risk by the American Thoracic Society (ATS) and the Centers for Disease Control (CDC). The methods used for screening were generally those advocated by ATS, CDC, and the Food and Drug Administration (FDA), although chest radiographs continue to be overused. Screening in about one-half of the groups is mandated by law or regulation. There appears to be some confusion about the circumstances in which "two-step" tuberculin testing should be used. Data on the productivity and costs of screening activities were very limited. We encourage those responsible for tuberculosis screening programs to evaluate them, discontinue those which are unproductive, and intensify those which are productive. PMID:6507687

  17. The art and design of genetic screens: zebrafish

    Microsoft Academic Search

    E. Elizabeth Patton; Leonard I. Zon

    2001-01-01

    Inventive genetic screens in zebrafish are revealing new genetic pathways that control vertebrate development, disease and behaviour. By exploiting the versatility of zebrafish, biological processes that had been previously obscured can be visualized and many of the responsible genes can be isolated. Coupled with gene knockdown and overexpression technologies, and small-molecule-induced phenotypes, genetic screens in zebrafish provide a powerful system

  18. Screening for Cancer by Residents in an Internal Medicine Program.

    ERIC Educational Resources Information Center

    Lynch, Garrett R.; Prout, Marianne N.

    1986-01-01

    A study of cancer screening by internal medicine residents in an inner-city clinic revealed that screening was more frequent for male patients, and breast examinations and Pap smears were performed on less than a third of female patients, suggesting a need for more intensive early-detection education of residents. (MSE)

  19. Active versus Passive Screen Time for Young Children

    ERIC Educational Resources Information Center

    Sweetser, Penelope; Johnson, Daniel; Ozdowska, Anne; Wyeth, Peta

    2012-01-01

    In this paper we report some initial findings from our investigations into the Australian Government's Longitudinal Study of Australian Children dataset. It is revealed that the majority of Australian children are exceeding the government's Screen Time recommendations and that most of their screen time is spent as TV viewing, as opposed to video…

  20. Dominant lethal mutations in the plasma membrane H(+)-ATPase gene of Saccharomyces cerevisiae.

    PubMed Central

    Harris, S L; Na, S; Zhu, X; Seto-Young, D; Perlin, D S; Teem, J H; Haber, J E

    1994-01-01

    The plasma membrane H(+)-ATPase of Saccharomyces cerevisiae is an essential protein that is required to establish cellular membrane potential and maintain a normal internal pH. An Asp-378 to Asn substitution at the residue phosphorylated during catalysis is dominant lethal when the pma1-D378N mutation is expressed along with a wild-type plasma membrane H(+)-ATPase (PMA1) gene. Several mutations in the first two putative transmembrane domains are also dominant lethal. However, these dominant lethal mutants often appear to be innocuous, because they are frequently lost by gene conversion to the wild-type sequence during the process of introducing the mutant sequence and subsequently removing the wild-type gene. Loss of the mutation by gene conversion does not occur while introducing recessive lethal mutations. Cells carrying the wild-type PMA1 gene on the chromosome and a dominant lethal mutation under the control of a GAL1 promoter on a centromere-containing plasmid exhibit a galactose-dependent lethality. Indirect immunofluorescence staining using anti-Pma1 antibodies shows that induction of dominant lethal PMA1 mutations leads to the accumulation of a number of intensely staining cytoplasmic structures that are not coincident with the nucleus and its immediately surrounding endoplasmic reticulum. These structures also accumulate the endoplasmic reticulum protein Kar2. Expression of the dominant lethal protein also prevents transport of the wild-type ATPase to the plasma membrane. Images PMID:7937988

  1. Evaluating the Predictive Validity of Suicidal Intent and Medical Lethality in Youth

    ERIC Educational Resources Information Center

    Sapyta, Jeffrey; Goldston, David B.; Erkanli, Alaattin; Daniel, Stephanie S.; Heilbron, Nicole; Mayfield, Andrew; Treadway, S. Lyn

    2012-01-01

    Objectives: To examine whether suicidal intent and medical lethality of past suicide attempts are predictive of future attempts, the association between intent and lethality, and the consistency of these characteristics across repeated attempts among youth. Method: Suicide attempts in a 15-year prospective study of 180 formerly psychiatrically…

  2. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the

    E-print Network

    Lieberman, Judy

    Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens, 2000 (received for review January 24, 2000) Bacillus anthrax lethal toxin can be engineered to deliver compartment of mammalian cells. The engineered anthrax toxin vaccine appears unlikely to induce an antibody

  3. Proteasomes Control Caspase-1 Activation in Anthrax Lethal Toxin-mediated Cell Killing*S

    E-print Network

    Brojatsch, Jürgen

    Proteasomes Control Caspase-1 Activation in Anthrax Lethal Toxin-mediated Cell Killing*S Received York 10461 Activation of caspase-1 through the inflammasome protein Nalp1b controls anthrax lethal. The spore-forming bacterium Bacillus anthracis is the caus- ative agent of anthrax disease (1

  4. Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs

    E-print Network

    Brojatsch, Jürgen

    Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune- evasion

  5. Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin

    Microsoft Academic Search

    Anshu Agrawal; Jai Lingappa; Stephen H. Leppla; Sudhanshu Agrawal; Abdul Jabbar; Conrad Quinn; Bali Pulendran

    2003-01-01

    Anthrax poses a clear and present danger as an agent of biological terrorism. Infection with Bacillus anthracis, the causative agent of anthrax, if untreated can result in rampant bacteraemia, multisystem dysfunction and death. Anthrax lethal toxin (LT) is a critical virulence factor of B. anthracis, which occurs as a complex of protective antigen and lethal factor. Here we demonstrate that

  6. BIOASSAY PROTOCOL FOR LETHAL AND SUBLETHAL EFFECTS OF FUNGAL PATHOGENS ON CHRYSOPERLA CARNEA

    EPA Science Inventory

    This practice describes procedures for evaluating the lethal and sub-lethal effects of exposure to fungal pathogens on larvae and adults of the predatory insect Chrysoperla carnea (Stephens). his practice was developed and tested with the fungal insect pathogen Beauveria bassiana...

  7. Examining the Impact of Psychiatric Diagnosis and Comorbidity on the Medical Lethality of Adolescent "Suicide Attempts"

    ERIC Educational Resources Information Center

    Mc Manama O'Brien, Kimberly H.; Berzin, Stephanie C.

    2012-01-01

    Specific psychiatric diagnoses and comorbidity patterns were examined to determine if they were related to the medical lethality of "suicide attempts" among adolescents presenting to an urban general hospital (N = 375). Bivariate analysis showed that attempters with substance abuse disorders had higher levels of lethality than attempters without…

  8. Improving on Army Field Gauze for Lethal Vascular Injuries: Challenges in Dressing Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accounting for half of all deaths, uncontrolled hemorrhage remains the leading cause of death on the battlefield. Gaining hemostatic control of lethal vascular injuries sustained in combat using topical agents remains a challenge. Recent animal testing using a lethal arterial injury model compared a...

  9. Co-lethality studied as an asset against viral drug escape: the HIV protease case

    PubMed Central

    2010-01-01

    Background Co-lethality, or synthetic lethality is the documented genetic situation where two, separately non-lethal mutations, become lethal when combined in one genome. Each mutation is called a "synthetic lethal" (SL) or a co-lethal. Like invariant positions, SL sets (SL linked couples) are choice targets for drug design against fast-escaping RNA viruses: mutational viral escape by loss of affinity to the drug may induce (synthetic) lethality. Results From an amino acid sequence alignment of the HIV protease, we detected the potential SL couples, potential SL sets, and invariant positions. From the 3D structure of the same protein we focused on the ones that were close to each other and accessible on the protein surface, to possibly bind putative drugs. We aligned 24,155 HIV protease amino acid sequences and identified 290 potential SL couples and 25 invariant positions. After applying the distance and accessibility filter, three candidate drug design targets of respectively 7 (under the flap), 4 (in the cantilever) and 5 (in the fulcrum) amino acid positions were found. Conclusions These three replication-critical targets, located outside of the active site, are key to our anti-escape strategy. Indeed, biological evidence shows that 2/3 of those target positions perform essential biological functions. Their mutational variations to escape antiviral medication could be lethal, thus limiting the apparition of drug-resistant strains. Reviewers This article was reviewed by Arcady Mushegian, Shamil Sunyaev and Claus Wilke. PMID:20565756

  10. The Danger Assessment: Validation of a Lethality Risk Assessment Instrument for Intimate Partner Femicide

    ERIC Educational Resources Information Center

    Campbell, Jacquelyn C.; Webster, Daniel W.; Glass, Nancy

    2009-01-01

    The Danger Assessment (DA) is an instrument designed to assess the likelihood of lethality or near lethality occurring in a case of intimate partner violence. This article describes the development, psychometric validation, and suggestions for use of the DA. An 11-city study of intimate partner femicide used multivariate analysis to test the…

  11. Suicide through stress: A bacterial response to sub-lethal injury in the food environment

    Microsoft Academic Search

    Christine E. R. Dodd; Philip J. Richards; Timothy G. Aldsworth

    2007-01-01

    The response of bacteria to sub-lethal injury is an important aspect of food microbiology as many inimical processes to which bacteria are subjected during processing are non-lethal. For pathogens like Salmonella and Escherichia coli, the difference in injury levels of exponential phase cells compared to their stationary phase counterparts in this regard is well recognised and evident for a variety

  12. Using VAAST to Identify an X-Linked Disorder Resulting in Lethality in Male Infants

    E-print Network

    Yandell, Mark

    ARTICLE Using VAAST to Identify an X-Linked Disorder Resulting in Lethality in Male Infants Due undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed

  13. Composite scintillator screen

    DOEpatents

    Zeman, Herbert D. (1687 Peach St., Memphis, TN 38112)

    1994-01-01

    A scintillator screen for an X-ray system includes a substrate of low-Z material and bodies of a high-Z material embedded within the substrate. By preselecting the size of the bodies embedded within the substrate, the spacial separation of the bodies and the thickness of the screen, the sensitivity of the screen to X-rays within a predetermined energy range can be predicted.

  14. ScreenFlow

    NSDL National Science Digital Library

    2008-01-01

    Capturing images and screen shots can be tricky, and it's nice to hear about new applications that can help out with such tasks. ScreenFlow allows users to create screen recordings and it also includes an array of editing options. Visitors should note that this is a trial version, and that the full-featured version costs $99. This version is compatible with computers running Mac OS X 10.5 Leopard.

  15. Arsenic-induced sub-lethal stress reprograms human bronchial epithelial cells to CD61¯ cancer stem cells

    PubMed Central

    Chang, Qingshan; Chen, Bailing; Thakur, Chitra; Lu, Yongju; Chen, Fei

    2014-01-01

    In the present report, we demonstrate that sub-lethal stress induced by consecutive exposure to 0.25 ?M arsenic (As3+) for six months can trigger reprogramming of the human bronchial epithelial cell (BEAS-2B) to form cancer stem cells (CSCs) without forced introduction of the stemness transcription factors. These CSCs formed from As3+-induced sub-lethal stress featured with an increased expression of the endogenous stemness genes, including Oct4, Sox2, Klf4, Myc, and others that are associated with the pluripotency and self-renewal of the CSCs. Flow cytometry analysis indicated that 90% of the CSC cells are CD61¯, whereas 100% of the parental cells are CD61+. These CD61¯ CSCs are highly tumorigenic and metastatic to the lung in xenotransplantation tests in NOD/SCID Il2r??/? mice. Additional tests also revealed that the CD61¯ CSCs showed a significant decrease in the expression of the genes important for DNA repair and oxidative phosphorylation. To determine the clinical relevance of the above findings, we stratified human lung cancers based on the level of CD61 protein and found that CD61low cancer correlates with poorer survival of the patients. Such a correlation was also observed in human breast cancer and ovarian cancer. Taken together, our findings suggest that in addition to the traditional approaches of enforced introduction of the exogenous stemness circuit transcription factors, sub-lethal stress induced by consecutive low dose As3+ is also able to convert non-stem cells to the CSCs. PMID:24675390

  16. Overdiagnosis in cancer screening.

    PubMed

    Cervera Deval, J; Sentís Crivillé, M; Zulueta, J J

    2014-08-28

    In screening programs, overdiagnosis is defined as the detection of a disease that would have gone undetected without screening when that disease would not have resulted in morbimortality and was treated unnecessarily. Overdiagnosis is a bias inherent in screening and an undesired effect of secondary prevention and improved sensitivity of diagnostic techniques. It is difficult to discriminate a priori between clinically relevant diagnoses and those in which treatment is unnecessary. To minimize the effects of overdiagnosis, screening should be done in patients at risk. PMID:25174786

  17. Searching for Synergies: Matrix Algebraic Approaches for Efficient Pair Screening

    PubMed Central

    Gerlee, Philip; Schmidt, Linnéa; Monsefi, Naser; Kling, Teresia; Jörnsten, Rebecka; Nelander, Sven

    2013-01-01

    Functionally interacting perturbations, such as synergistic drugs pairs or synthetic lethal gene pairs, are of key interest in both pharmacology and functional genomics. However, to find such pairs by traditional screening methods is both time consuming and costly. We present a novel computational-experimental framework for efficient identification of synergistic target pairs, applicable for screening of systems with sizes on the order of current drug, small RNA or SGA (Synthetic Genetic Array) libraries (>1000 targets). This framework exploits the fact that the response of a drug pair in a given system, or a pair of genes' propensity to interact functionally, can be partly predicted by computational means from (i) a small set of experimentally determined target pairs, and (ii) pre-existing data (e.g. gene ontology, PPI) on the similarities between targets. Predictions are obtained by a novel matrix algebraic technique, based on cyclical projections onto convex sets. We demonstrate the efficiency of the proposed method using drug-drug interaction data from seven cancer cell lines and gene-gene interaction data from yeast SGA screens. Our protocol increases the rate of synergism discovery significantly over traditional screening, by up to 7-fold. Our method is easy to implement and could be applied to accelerate pair screening for both animal and microbial systems. PMID:23935877

  18. Combination of hearing screening and genetic screening for deafness-susceptibility genes in newborns

    PubMed Central

    YAO, GEN-DONG; LI, SHOU-XIA; CHEN, DING-LI; FENG, HAI-QIN; ZHAO, SU-BIN; LIU, YONG-JIE; GUO, LI-LI; YANG, ZHI-MING; ZHANG, XIAO-FANG; SUN, CAI-XIA; WANG, ZE-HUI; ZHANG, WEI-YONG

    2014-01-01

    The aim of this study was to determine the clinical significance of the results of screening of newborn hearing and the incidence of deafness-susceptibility genes. One thousand newborn babies in the Handan Center Hospital (Handan, China) underwent screening of hearing and deafness-susceptibility genes. The first screening test was carried out using otoacoustic emissions (OAEs). Babies with hearing loss who failed to pass the initial screening were scheduled for rescreening at 42 days after birth. Cord blood was used for the screening of deafness-susceptibility genes, namely the GJB2, SLC26A4 and mitochondrial 12S rRNA (MTRNR1) genes. Among the 1,000 neonates that underwent the first hearing screening, 25 exhibited left-sided hearing loss, 21 exhibited right-sided hearing loss and 15 cases had binaural hearing loss. After rescreening 42 days later, only one of the initial 61 cases exhibited hearing loss under OAE testing. The neonatal deafness gene tests showed two cases with 1555A>G mutation and two cases with 1494C>T mutation of the MTRNR1 gene. In the SLC26A4 gene screening, four cases exhibited the heterozygous IVS7-2A>G mutation and one case exhibited heterozygous 1226G>A mutation. In the GJB2 gene screening, two cases exhibited the homozygous 427C>T mutation and 10 exhibited the heterozygous 235delC mutation. The genetic screening revealed 21 newborns with mutations in the three deafness-susceptibility genes. The overall carrier rate was 2.1% (21/1,000). The association of hearing and gene screening may be the promising screening strategy for the diagnosis of hearing loss. PMID:24348793

  19. A Nutritional Conditional Lethal Mutant Due to Pyridoxine 5?-Phosphate Oxidase Deficiency in Drosophila melanogaster

    PubMed Central

    Chi, Wanhao; Zhang, Li; Du, Wei; Zhuang, Xiaoxi

    2014-01-01

    The concept of auxotrophic complementation has been proposed as an approach to identify genes in essential metabolic pathways in Drosophila melanogaster. However, it has achieved limited success to date, possibly due to the low probability of finding mutations fit with the chemically defined profile. Instead of using the chemically defined culture media lacking specific nutrients, we used bare minimum culture medium, i.e., 4% sucrose, for adult Drosophila. We identified a nutritional conditional lethal mutant and localized a c.95C > A mutation in the Drosophila pyridoxine 5?-phosphate oxidase gene [dPNPO or sugarlethal (sgll)] using meiotic recombination mapping, deficiency mapping, and whole genome sequencing. PNPO converts dietary vitamin B6 such as pyridoxine to its active form pyridoxal 5?-phosphate (PLP). The missense mutation (sgll95) results in the substitution of alanine to aspartate (p.Ala32Asp). The sgll95 flies survive well on complete medium but all die within 6 d on 4% sucrose only diet, which can be rescued by pyridoxine or PLP supplement, suggesting that the mutation does not cause the complete loss of PNPO activity. The sgll knockdown further confirms its function as the Drosophila PNPO. Because better tools for positional cloning and cheaper whole genome sequencing have made the identification of point mutations much easier than before, alleviating the necessity to pinpoint specific metabolic pathways before gene identification, we propose that nutritional conditional screens based on bare minimum growth media like ours represent promising approaches for discovering important genes and mutations in metabolic pathways, thereby accelerating the establishment of in vivo models that recapitulate human metabolic diseases. PMID:24739647

  20. A nutritional conditional lethal mutant due to pyridoxine 5'-phosphate oxidase deficiency in Drosophila melanogaster.

    PubMed

    Chi, Wanhao; Zhang, Li; Du, Wei; Zhuang, Xiaoxi

    2014-06-01

    The concept of auxotrophic complementation has been proposed as an approach to identify genes in essential metabolic pathways in Drosophila melanogaster. However, it has achieved limited success to date, possibly due to the low probability of finding mutations fit with the chemically defined profile. Instead of using the chemically defined culture media lacking specific nutrients, we used bare minimum culture medium, i.e., 4% sucrose, for adult Drosophila. We identified a nutritional conditional lethal mutant and localized a c.95C > A mutation in the Drosophila pyridoxine 5'-phosphate oxidase gene [dPNPO or sugarlethal (sgll)] using meiotic recombination mapping, deficiency mapping, and whole genome sequencing. PNPO converts dietary vitamin B6 such as pyridoxine to its active form pyridoxal 5'-phosphate (PLP). The missense mutation (sgll(95)) results in the substitution of alanine to aspartate (p.Ala32Asp). The sgll(95) flies survive well on complete medium but all die within 6 d on 4% sucrose only diet, which can be rescued by pyridoxine or PLP supplement, suggesting that the mutation does not cause the complete loss of PNPO activity. The sgll knockdown further confirms its function as the Drosophila PNPO. Because better tools for positional cloning and cheaper whole genome sequencing have made the identification of point mutations much easier than before, alleviating the necessity to pinpoint specific metabolic pathways before gene identification, we propose that nutritional conditional screens based on bare minimum growth media like ours represent promising approaches for discovering important genes and mutations in metabolic pathways, thereby accelerating the establishment of in vivo models that recapitulate human metabolic diseases. PMID:24739647

  1. Lung cancer screening.

    PubMed

    Tanoue, Lynn T; Tanner, Nichole T; Gould, Michael K; Silvestri, Gerard A

    2015-01-01

    The United States Preventive Services Task Force recommends lung cancer screening with low-dose computed tomography (LDCT) in adults of age 55 to 80 years who have a 30 pack-year smoking history and are currently smoking or have quit within the past 15 years. This recommendation is largely based on the findings of the National Lung Screening Trial. Both policy-level and clinical decision-making about LDCT screening must consider the potential benefits of screening (reduced mortality from lung cancer) and possible harms. Effective screening requires an appreciation that screening should be limited to individuals at high risk of death from lung cancer, and that the risk of harm related to false positive findings, overdiagnosis, and unnecessary invasive testing is real. A comprehensive understanding of these aspects of screening will inform appropriate implementation, with the objective that an evidence-based and systematic approach to screening will help to reduce the enormous mortality burden of lung cancer. PMID:25369325

  2. Colorectal Cancer Screening

    Microsoft Academic Search

    Jiann-Hwa Chen; Hans Hsienhong Lin

    2009-01-01

    Colorectal cancer (CRC) was the third-leading cause of cancer death in Taiwan in 2008. The natural history of CRC provides a chance for screening and prevention. Most CRC develops from adenomatous polyps. This progression takes at least 10 years in most people. About 90% of CRC develops after 50 years of age. Screening tests can identify cancers, usually at an

  3. Screening: the legal view.

    PubMed

    Eaden, J; Mayberry, M K; Sherr, A; Mayberry, J F

    2001-05-01

    Screening has become central to the effective prevention of several diseases, but implementation suffers from difficulties with targeting and rates of compliance. Such issues are also complicated by the need to consider legal provisions regarding confidentiality of patients and other human rights issues. Screening has been an inexact science in relation to, e.g., faecal occult blood testing for colorectal cancer, false positive and false negative tests for HIV, and there have been inadequate quality controls in breast cancer screening programmes. The public need to be made aware of what the screening programmes really offer, balanced against the expectations they may have. There needs to be a clearer understanding of the nature of the contractual and other legal rights of patients/consumers as against providers. A positive screening test may carry adverse consequences as well as benefits. It could alert an insurance company to a risk and lead to additional weighting or even outright rejection for life insurance policies. Job prospects may also be affected for employees. The method of informing patients in relation to screening and screening failure has already been considered by the courts. Realistic information about both screening and treatment efficiency needs to be offered to patients so that they can have a real understanding of what can and cannot be achieved by current science. The development of understanding of the human genome makes the need for clearer legislation in this are more urgent. PMID:11429719

  4. Cancer screening decisions.

    PubMed

    McCaul, K D; Tulloch, H E

    1999-01-01

    This review focuses on why people decide to obtain or to avoid screening for cancer. We discuss three topics: (a) physician prompts that may elicit compliant screening behavior, (b) the independent and joint effects of risk perceptions and worry, and (c) the costs and benefits of getting screened. Overall, the data suggest that each of these factors will influence screening. So, for example, people are more likely to seek screening if a physician recommends the behavior, if they feel personally vulnerable and worry a little about cancer, if insurance covers the screening, and if they believe that the test is an effective early detection procedure. Future research needs include studies comparing theories, longitudinal rather than cross-sectional studies, and true experiments. We also need to know more about why physicians are such powerful change agents and the trade-offs of increasing personal risk versus exacerbating worry. Practical recommendations for promoting cancer screening include encouraging physician interventions, explaining risk, and lowering the costs while emphasizing the benefits of screening. PMID:10854458

  5. Touch screen have become

    E-print Network

    Wobbrock, Jacob O.

    Typing { l ABSTRACT Touch screen have become screens pales examine typing physical keybo o inform future Author Keywo Touch-typing, m ACM Classific H.5.2. [Inform nterfaces--inp General Terms Human factors, NTRODUCTIO Touch surfaces he form of m ablets and in Microsoft Surfa ablets and tabl finger text inpu hese

  6. Screen time and children

    MedlinePLUS

    ... screen, such as watching TV, working on a computer, or playing video games. Screen time is sedentary activity, meaning you are ... child eat while watching TV or using the computer. Do not leave the ... as family board games, puzzles, or going for a walk. Keep a ...

  7. Film Screening and Conversation

    E-print Network

    Mathis, Wayne N.

    Film Screening and Conversation 2011 6-9pm Smithsonian Asian Paci c American Program Rasmuson Director John Sayles Film Run Time: 124 minutes Closest Metro: L'Enfant Plaza Related Traveling Exhibition, the Smithsonian Asian Pacific American Program presents a screening of the film Amigo and a conversation

  8. Deletion of MCL-1 causes lethal cardiac failure and mitochondrial dysfunction

    PubMed Central

    Wang, Xi; Bathina, Madhavi; Lynch, John; Koss, Brian; Calabrese, Christopher; Frase, Sharon; Schuetz, John D.; Rehg, Jerold E.; Opferman, Joseph T.

    2013-01-01

    MCL-1 is an essential BCL-2 family member that promotes the survival of multiple cellular lineages, but its role in cardiac muscle has remained unclear. Here, we report that cardiac-specific ablation of Mcl-1 results in a rapidly fatal, dilated cardiomyopathy manifested by a loss of cardiac contractility, abnormal mitochondria ultrastructure, and defective mitochondrial respiration. Strikingly, genetic ablation of both proapoptotic effectors (Bax and Bak) could largely rescue the lethality and impaired cardiac function induced by Mcl-1 deletion. However, while the overt consequences of Mcl-1 loss were obviated by combining with the loss of Bax and Bak, mitochondria from the Mcl-1-, Bax-, and Bak-deficient hearts still revealed mitochondrial ultrastructural abnormalities and displayed deficient mitochondrial respiration. Together, these data indicate that merely blocking cell death is insufficient to completely overcome the need for MCL-1 function in cardiomyocytes and suggest that in cardiac muscle, MCL-1 also facilitates normal mitochondrial function. These findings are important, as specific MCL-1-inhibiting therapeutics are being proposed to treat cancer cells and may result in unexpected cardiac toxicity. PMID:23788622

  9. Bacillus anthracis lethal toxin induces TNF-?–independent hypoxia-mediated toxicity in mice

    PubMed Central

    Moayeri, Mahtab; Haines, Diana; Young, Howard A.; Leppla, Stephen H.

    2003-01-01

    Bacillus anthracis lethal toxin (LT) is the major virulence factor of anthrax and reproduces most of the laboratory manifestations of the disease in animals. We studied LT toxicity in BALB/cJ and C57BL/6J mice. BALB/cJ mice became terminally ill earlier and with higher frequency than C57BL/6J mice. Timed histopathological analysis identified bone marrow, spleen, and liver as major affected organs in both mouse strains. LT induced extensive hypoxia. Crisis was due to extensive liver necrosis accompanied by pleural edema. There was no evidence of disseminated intravascular coagulation or renal dysfunction. Instead, analyses revealed hepatic dysfunction, hypoalbuminemia, and vascular/oxygenation insufficiency. Of 50 cytokines analyzed, BALB/cJ mice showed rapid but transitory increases in specific factors including KC, MCP-1/JE, IL-6, MIP-2, G-CSF, GM-CSF, eotaxin, FasL, and IL-1?. No changes in TNF-? occurred. The C57BL/6J mice did not mount a similar cytokine response. These factors were not induced in vitro by LT treatment of toxin-sensitive macrophages. The evidence presented shows that LT kills mice through a TNF-?–independent, FasL-independent, noninflammatory mechanism that involves hypoxic tissue injury but does not require macrophage sensitivity to toxin. PMID:12952916

  10. New strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice.

    PubMed Central

    Hierholzer, J C; Broderson, J R; Murphy, F A

    1979-01-01

    A new strain of mouse hepatitis virus (MHV) was isolated from pooled gut suspensions from an epizootic of lethal enteritis in newborn mice. Negative-contrast electron microscopy showed an abundance of coronavirus particles in the intestinal contents and intestinal epithelium of moribund mice. We found no other virus in the epizootic. Dams seroconverted to MHV polyvalent antigen and to the agent isolated, but did not develop antibodies to other known mouse pathogens. Virus propagated in NCTC-1469 tissue culture produced enteric disease in suckling mice but not fatal diarrhea; the dams of these mice also developed antibodies to MHV and to the isolates. By complement fixation, single radial hemolysis, and quantal neutralization tests, we found the isolates antigenically most closely related to MHV-S, unilaterally related to MHV-JHM, and more distantly related to MHV-1, MHV-3, MHV-A59, and human coronavirus OC-43. We also studied cross-reactions among the murine and human coronaviruses in detail. Tissues of infected newborn mice were examined by light microscopy, thin-section electron microscopy, and frozen-section indirect immunofluorescence, revealing that viral antigen, virus particles, and pathological changes were limited to the intestinal tract. We have designated our isolates as MHV-S/CDC. Images PMID:222687

  11. Renal Mucormycosis: A Rare and Potentially Lethal Complication of Kidney Transplantation

    PubMed Central

    He, Chun; Cronin, David C.

    2013-01-01

    Renal mucormycosis is a rare and potentially lethal complication of kidney transplantation. We describe two cases of renal mucormycosis following deceased donor kidney transplantation. This is the second report of renal mucormycosis following kidney transplantation in the United States, and the first case of renal mucormycosis infection presumed to be of recipient origin. Case A had an early presentation of mucormycosis isolated to the kidney allograft. He had an unexpected rise in serum creatinine and leukocytosis necessitating allograft biopsy which showed mucormycosis. He underwent transplant nephrectomy on posttransplant day 11, was treated with amphotericin B, and discharged home on posttransplant day 22. Case B had a late presentation of renal mucormycosis, preceded by a cutaneous manifestation. One year after kidney transplantation he had a nonhealing knee ulcer which on biopsy showed cutaneous mucormycosis. Treatment included aggressive debridement and amphotericin B. Allograft biopsy showed mucormycosis, necessitating transplant nephrectomy. He was discharged to a rehabilitation facility and died from noninfectious causes. Review of the published literature of renal mucormycosis cases following kidney transplantation reveals a mortality rate of more than 50%. The key to successful outcome is early recognition, prompt institution of surgical debridement of all infected tissue, and appropriate antifungal therapy. PMID:24251062

  12. Embryonic viability, lipase deficiency, hypertriglyceridemia and neonatal lethality in a novel LMF1-deficient mouse model

    PubMed Central

    2014-01-01

    Background Lipase Maturation Factor 1 (LMF1) is an ER-chaperone involved in the post-translational maturation and catalytic activation of vascular lipases including lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). Mutations in LMF1 are associated with lipase deficiency and severe hypertriglyceridemia indicating the critical role of LMF1 in plasma lipid homeostasis. The currently available mouse model of LMF1 deficiency is based on a naturally occurring truncating mutation, combined lipase deficiency (cld), which may represent a hypomorphic allele. Thus, development of LMF1-null mice is needed to explore the phenotypic consequences of complete LMF1 deficiency. Findings In situ hybridization and qPCR analysis in the normal mouse embryo revealed ubiquitous and high-level LMF1 expression. To investigate if LMF1 was required for embryonic viability, a novel mouse model based on a null-allele of LMF1 was generated and characterized. LMF1-/- progeny were born at Mendelian ratios and exhibited combined lipase deficiency, hypertriglyceridemia and neonatal lethality. Conclusion Our results raise the possibility of a previously unrecognized role for LMF1 in embryonic development, but indicate that LMF1 is dispensable for the viability of mouse embryo. The novel mouse model developed in this study will be useful to investigate the full phenotypic spectrum of LMF1 deficiency. PMID:25302068

  13. A LigA Three-Domain Region Protects Hamsters from Lethal Infection by Leptospira interrogans

    PubMed Central

    Coutinho, Mariana L.; Choy, Henry A.; Kelley, Melissa M.; Matsunaga, James; Babbitt, Jane T.; Lewis, Michael S.; Aleixo, Jose Antonio G.; Haake, David A.

    2011-01-01

    The leptospiral LigA protein consists of 13 bacterial immunoglobulin-like (Big) domains and is the only purified recombinant subunit vaccine that has been demonstrated to protect against lethal challenge by a clinical isolate of Leptospira interrogans in the hamster model of leptospirosis. We determined the minimum number and location of LigA domains required for immunoprotection. Immunization with domains 11 and 12 was found to be required but insufficient for protection. Inclusion of a third domain, either 10 or 13, was required for 100% survival after intraperitoneal challenge with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. As in previous studies, survivors had renal colonization; here, we quantitated the leptospiral burden by qPCR to be 1.2×103 to 8×105 copies of leptospiral DNA per microgram of kidney DNA. Although renal histopathology in survivors revealed tubulointerstitial changes indicating an inflammatory response to the infection, blood chemistry analysis indicated that renal function was normal. These studies define the Big domains of LigA that account for its vaccine efficacy and highlight the need for additional strategies to achieve sterilizing immunity to protect the mammalian host from leptospiral infection and its consequences. PMID:22180800

  14. Declines in the Lethality of Suicide Attempts Explain the Decline in Suicide Deaths in Australia

    PubMed Central

    Spittal, Matthew J.; Pirkis, Jane; Miller, Matthew; Studdert, David M.

    2012-01-01

    Background To investigate the epidemiology of a steep decrease in the incidence of suicide deaths in Australia. Methods National data on suicide deaths and deliberate self-harm for the period 1994–2007 were obtained from the Australian Institute of Health and Welfare. We calculated attempt and death rates for five major methods and the lethality of these methods. Negative binomial regression was used to estimate the size and significance of method-specific time-trends in attempts and lethality. Results Hanging, motor vehicle exhaust and firearms were the most lethal methods, and together accounted for 72% of all deaths. The lethality of motor vehicle exhaust attempts decreased sharply (RR?=?0.94 per year, 95% CI 0.93–0.95) while the motor vehicle exhaust attempt rate changed little; this combination of motor vehicle exhaust trends explained nearly half of the overall decline in suicide deaths. Hanging lethality also decreased sharply (RR?=?0.96 per year, 95% CI 0.956–0.965) but large increases in hanging attempts negated the effect on death rates. Firearm lethality changed little while attempts decreased. Conclusion Declines in the lethality of suicide attempts–especially attempts by motor vehicle exhaust and hanging–explain the remarkable decline in deaths by suicide in Australia since 1997. PMID:22957084

  15. Hemolysis-induced lethality involves inflammasome activation by heme

    PubMed Central

    Dutra, Fabianno F.; Alves, Letícia S.; Rodrigues, Danielle; Fernandez, Patricia L.; de Oliveira, Rosane B.; Golenbock, Douglas T.; Zamboni, Dario S.; Bozza, Marcelo T.

    2014-01-01

    The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1? dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K+ efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release. PMID:25225402

  16. Interferon alfacon-1 protects hamsters from lethal pichinde virus infection.

    PubMed

    Gowen, Brian B; Barnard, Dale L; Smee, Donald F; Wong, Min-Hui; Pace, Anne M; Jung, Kie-Hoon; Winslow, Scott G; Bailey, Kevin W; Blatt, Lawrence M; Sidwell, Robert W

    2005-06-01

    Hemorrhagic fever of arenaviral origin is a frequently fatal infectious disease of considerable priority to the biodefense mission. Historically, the treatment of arenaviral infections with alpha interferons has not yielded favorable results. Here we present evidence that interferon alfacon-1, a nonnaturally occurring bioengineered alpha interferon approved for the treatment of chronic hepatitis C, is active against Pichinde and Tacaribe arenaviruses in cell culture. In the hamster model of Pichinde virus (PCV) infection, interferon alfacon-1 treatment significantly protected animals from death, prolonged the survival of those that eventually died, reduced virus titers, and limited liver damage characteristic of PCV-induced disease. Moreover, interferon alfacon-1 also demonstrated therapeutic activity, to a lesser degree, when the initiation of treatment was delayed up to 2 days post-virus challenge. Despite the observed advantages of interferon alfacon-1 therapy, efforts to stimulate the immune system with the known interferon inducer poly(I:C12U) (Ampligen) offered only limited protection against lethal PCV challenge. Taken together, these data suggest that the increased potency of the bio-optimized interferon alfacon-1 molecule may be critical to the observed antiviral effects. These data are the first report demonstrating efficacious treatment of acute arenaviral disease with alpha interferon therapy, and further study is warranted. PMID:15917537

  17. Rescue of Progeria in Trichothiodystrophy by Homozygous Lethal Xpd Alleles

    PubMed Central

    Andressoo, Jaan-Olle; Jans, Judith; de Wit, Jan; Coin, Frederic; Hoogstraten, Deborah; van de Ven, Marieke; Toussaint, Wendy; Huijmans, Jan; Thio, H. Bing; van Leeuwen, Wibeke J; de Boer, Jan; Egly, Jean-Marc; Hoeijmakers, Jan H. J; van der Horst, Gijsbertus T. J; Mitchell, James R

    2006-01-01

    Although compound heterozygosity, or the presence of two different mutant alleles of the same gene, is common in human recessive disease, its potential to impact disease outcome has not been well documented. This is most likely because of the inherent difficulty in distinguishing specific biallelic effects from differences in environment or genetic background. We addressed the potential of different recessive alleles to contribute to the enigmatic pleiotropy associated with XPD recessive disorders in compound heterozygous mouse models. Alterations in this essential helicase, with functions in both DNA repair and basal transcription, result in diverse pathologies ranging from elevated UV sensitivity and cancer predisposition to accelerated segmental progeria. We report a variety of biallelic effects on organismal phenotype attributable to combinations of recessive Xpd alleles, including the following: (i) the ability of homozygous lethal Xpd alleles to ameliorate a variety of disease symptoms when their essential basal transcription function is supplied by a different disease-causing allele, (ii) differential developmental and tissue-specific functions of distinct Xpd allele products, and (iii) interallelic complementation, a phenomenon rarely reported at clinically relevant loci in mammals. Our data suggest a re-evaluation of the contribution of “null” alleles to XPD disorders and highlight the potential of combinations of recessive alleles to affect both normal and pathological phenotypic plasticity in mammals. PMID:17020410

  18. Immunohistochemical expression of BRCA1 and lethal prostate cancer.

    PubMed

    Fiorentino, Michelangelo; Judson, Gregory; Penney, Kathryn; Flavin, Richard; Stark, Jennifer; Fiore, Christopher; Fall, Katja; Martin, Neil; Ma, Jing; Sinnott, Jennifer; Giovannucci, Edward; Stampfer, Meir; Sesso, Howard D; Kantoff, Philip W; Finn, Stephen; Loda, Massimo; Mucci, Lorelei

    2010-04-15

    BRCA1 functions as a tumor suppressor; recent work suggests that BRCA1 may also induce cell cycle arrest to allow for DNA repair. We hypothesized that BRCA1 expression in prostate tumor tissue may be associated with prostate cancer progression through regulation of the cell cycle. We used immunohistochemistry to evaluate BRCA1 protein expression in archival tumor samples from 393 prostate cancer cases in the Physicians' Health Study. The men were followed prospectively from diagnosis to development of metastases and mortality. Fifteen percent of tumors stained positive for BRCA1. BRCA1-positive tumors had substantially increased tumor proliferation index compared with negative tumors (47.0 Ki67-positive nuclei versus 10.3, P = 0.0016) and were more likely to develop lethal cancer compared with BRCA1-negative tumors (hazard ratio, 4.6; 95% confidence interval, 2.4-8.7). These findings strengthen the hypothesis that BRCA1 plays a role in cell cycle control and show that BRCA1 is a marker of clinical prostate cancer prognosis. Cancer Res; 70(8); 3136-9. (c)2010 AACR. PMID:20388772

  19. Lethal Mutagenesis of HIV with Mutagenic Nucleoside Analogs

    NASA Astrophysics Data System (ADS)

    Loeb, Lawrence A.; Essigmann, John M.; Kazazi, Farhad; Zhang, Jue; Rose, Karl D.; Mullins, James I.

    1999-02-01

    The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA\\cdot DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G -> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

  20. Sticky foam as a less-than-lethal technology

    NASA Astrophysics Data System (ADS)

    Scott, Steven H.

    1997-01-01

    Sandia National Laboratories (SNL) in 1994 completed a project funded by the National Institute of Justice (NIJ) to determine the applicability of sticky foam for correctional applications. Sticky foam is an extremely tacky, tenacious material used to block, entangle, and impair individuals. The NIJ project developed a gun capable of firing multiple shots of sticky foam, tested the gun and sticky foam effectiveness on SNL volunteers acting out prison and law enforcement scenarios, and had the gun and sticky foam evaluated by correctional representatives. Based on the NIJ project work, SNL supported the Marine Corps Mission, Operation United Shield, with sticky foam guns and supporting equipment to assist in the withdrawal of UN Peacekeepers from Somalia. Prior to the loan of the waste disposal, use limitations, use protocol and precautions, emergency facial clean-up, skin clean-up, gun filling, targeting and firing, and gun cleaning. The Marine Corps successfully used the sticky foam guns as part of that operation. This paper describes these recent developments of sticky foam for non-lethal uses and some of the lessons learned from scenario and application testing.