Genetic screens to identify new Notch pathway mutants in Drosophila.

Science.gov (United States)

Giagtzoglou, Nikolaos

2014-01-01

Notch signaling controls a wide range of developmental processes, including proliferation, apoptosis, and cell fate specification during both development and adult tissue homeostasis. The functional versatility of the Notch signaling pathway is tightly linked with the complexity of its regulation in different cellular contexts. To unravel the complexity of Notch signaling, it is important to identify the different components of the Notch signaling pathway. A powerful strategy to accomplish this task is based on genetic screens. Given that the developmental context of signaling is important, these screens should be customized to specific cell populations or tissues. Here, I describe how to perform F1 clonal forward genetic screens in Drosophila to identify novel components of the Notch signaling pathway. These screens combine a classical EMS (ethyl methanesulfonate) chemical mutagenesis protocol along with clonal analysis via FRT-mediated mitotic recombination. These F1 clonal screens allow rapid phenotypic screening within clones of mutant cells induced at specific developmental stages and in tissues of interest, bypassing the pleiotropic effects of isolated mutations. More importantly, since EMS mutations have been notoriously difficult to map to specific genes in the past, I briefly discuss mapping methods that allow rapid identification of the causative mutations.

Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

Science.gov (United States)

Pyne, C; Bognar, A L

1992-03-01

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.

High-throughput screening to identify inhibitors of lysine demethylases.

Science.gov (United States)

Gale, Molly; Yan, Qin

2015-01-01

Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several High-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the High-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors.

Screening of chemical compound libraries identified new anti-Toxoplasma gondii agents.

Science.gov (United States)

Adeyemi, Oluyomi Stephen; Sugi, Tatsuki; Han, Yongmei; Kato, Kentaro

2018-02-01

Toxoplasma gondii is the etiological agent of toxoplasmosis, a common parasitic disease that affects nearly one-third of the human population. The primary infection can be asymptomatic in healthy individuals but may prove fatal in immunocompromised individuals. Available treatment options for toxoplasmosis patients are limited, underscoring the urgent need to identify and develop new therapies. Non-biased screening of libraries of chemical compounds including the repurposing of well-characterized compounds is emerging as viable approach to achieving this goal. In the present investigation, we screened libraries of natural product and FDA-approved compounds to identify those that inhibited T. gondii growth. We identified 32 new compounds that potently inhibit T. gondii growth. Our findings are new and promising, and further strengthen the prospects of drug repurposing as well as the screening of a wide range of chemical compounds as a viable source of alternative anti-parasitic therapeutic agents.

Structural analysis of dihydrofolate reductases enables rationalization of antifolate binding affinities and suggests repurposing possibilities.

Science.gov (United States)

Bhosle, Amrisha; Chandra, Nagasuma

2016-03-01

Antifolates are competitive inhibitors of dihydrofolate reductase (DHFR), a conserved enzyme that is central to metabolism and widely targeted in pathogenic diseases, cancer and autoimmune disorders. Although most clinically used antifolates are known to be target specific, some display a fair degree of cross-reactivity with DHFRs from other species. A method that enables identification of determinants of affinity and specificity in target DHFRs from different species and provides guidelines for the design of antifolates is currently lacking. To address this, we first captured the potential druggable space of a DHFR in a substructure called the 'supersite' and classified supersites of DHFRs from 56 species into 16 'site-types' based on pairwise structural similarity. Analysis of supersites across these site-types revealed that DHFRs exhibit varying extents of dissimilarity at structurally equivalent positions in and around the binding site. We were able to explain the pattern of affinities towards chemically diverse antifolates exhibited by DHFRs of different site-types based on these structural differences. We then generated an antifolate-DHFR network by mapping known high-affinity antifolates to their respective supersites and used this to identify antifolates that can be repurposed based on similarity between supersites or antifolates. Thus, we identified 177 human-specific and 458 pathogen-specific antifolates, a large number of which are supported by available experimental data. Thus, in the light of the clinical importance of DHFR, we present a novel approach to identifying differences in the druggable space of DHFRs that can be utilized for rational design of antifolates. © 2016 Federation of European Biochemical Societies.

Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase

Directory of Open Access Journals (Sweden)

O'Neil Michael T

2008-04-01

Full Text Available Abstract Background In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions. Methods The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network. Results Synonymous and non-synonymous single nucleotide polymorphisms (SNPs within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel. SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu. Conclusion It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts

Identifying Relationships between High-Risk Sexual Behaviors and Screening Positive for Chlamydia and Gonorrhea in School-Wide Screening Events

Science.gov (United States)

Salerno, Jennifer; Darling-Fisher, Cindy; Hawkins, Nicole M.; Fraker, Elizabeth

2013-01-01

Background: This article describes a school-wide sexually transmitted infection (STI) screening to identify adolescent high-risk sexual behaviors, STI history/incidence, and presence of chlamydia and gonorrhea, and examines relationships between high-risk behaviors and screening positive for chlamydia and gonorrhea in an alternative high school…

Small molecule screening identifies targetable zebrafish pigmentation pathways

DEFF Research Database (Denmark)

Colanesi, Sarah; Taylor, Kerrie L; Temperley, Nicholas D

2012-01-01

Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish and investig......Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish...... and investigate the effects of a few of these compounds in further detail. We identified and confirmed 57 compounds that altered pigment cell patterning, number, survival, or differentiation. Additional tissue targets and toxicity of small molecules are also discussed. Given that the majority of cell types...

City and County Solar PV Training Program, Module 2: Screening and Identifying PV Projects

Energy Technology Data Exchange (ETDEWEB)

Elgqvist, Emma M [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

2018-04-09

When screening and identifying PV projects, cities and counties should understand the different factors that impact the technical and economic potential of a PV project, the steps of the PV screening process, and how to use REopt Lite to screen a site for PV and storage project potential.

High-throughput matrix screening identifies synergistic and antagonistic antimalarial drug combinations

Science.gov (United States)

Mott, Bryan T.; Eastman, Richard T.; Guha, Rajarshi; Sherlach, Katy S.; Siriwardana, Amila; Shinn, Paul; McKnight, Crystal; Michael, Sam; Lacerda-Queiroz, Norinne; Patel, Paresma R.; Khine, Pwint; Sun, Hongmao; Kasbekar, Monica; Aghdam, Nima; Fontaine, Shaun D.; Liu, Dongbo; Mierzwa, Tim; Mathews-Griner, Lesley A.; Ferrer, Marc; Renslo, Adam R.; Inglese, James; Yuan, Jing; Roepe, Paul D.; Su, Xin-zhuan; Thomas, Craig J.

2015-01-01

Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy. PMID:26403635

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

International Nuclear Information System (INIS)

Price, E.M.; Smith, P.L.; Klein, T.E.; Freisheim, J.H.

1987-01-01

N/sup α/-(4-Amino-4-deoxy-10-methylpteroyl)-N/sup epsilon/-(4-azido-5-[ 125 I]iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the 125 I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme

A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

International Nuclear Information System (INIS)

Yashiroda, Yoko; Okamoto, Reika; Hatsugai, Kaori; Takemoto, Yasushi; Goshima, Naoki; Saito, Tamio; Hamamoto, Makiko; Sugimoto, Yoshikazu; Osada, Hiroyuki; Seimiya, Hiroyuki; Yoshida, Minoru

2010-01-01

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

Rigorous Screening Technology for Identifying Suitable CO2 Storage Sites II

Energy Technology Data Exchange (ETDEWEB)

George J. Koperna Jr.; Vello A. Kuuskraa; David E. Riestenberg; Aiysha Sultana; Tyler Van Leeuwen

2009-06-01

This report serves as the final technical report and users manual for the 'Rigorous Screening Technology for Identifying Suitable CO2 Storage Sites II SBIR project. Advanced Resources International has developed a screening tool by which users can technically screen, assess the storage capacity and quantify the costs of CO2 storage in four types of CO2 storage reservoirs. These include CO2-enhanced oil recovery reservoirs, depleted oil and gas fields (non-enhanced oil recovery candidates), deep coal seems that are amenable to CO2-enhanced methane recovery, and saline reservoirs. The screening function assessed whether the reservoir could likely serve as a safe, long-term CO2 storage reservoir. The storage capacity assessment uses rigorous reservoir simulation models to determine the timing, ultimate storage capacity, and potential for enhanced hydrocarbon recovery. Finally, the economic assessment function determines both the field-level and pipeline (transportation) costs for CO2 sequestration in a given reservoir. The screening tool has been peer reviewed at an Electrical Power Research Institute (EPRI) technical meeting in March 2009. A number of useful observations and recommendations emerged from the Workshop on the costs of CO2 transport and storage that could be readily incorporated into a commercial version of the Screening Tool in a Phase III SBIR.

High-throughput screen of drug repurposing library identifies inhibitors of Sarcocystis neurona growth

Directory of Open Access Journals (Sweden)

Gregory D. Bowden

2018-04-01

Full Text Available The apicomplexan parasite Sarcocystis neurona is the primary etiologic agent of equine protozoal myeloencephalitis (EPM, a serious neurologic disease of horses. Many horses in the U.S. are at risk of developing EPM; approximately 50% of all horses in the U.S. have been exposed to S. neurona and treatments for EPM are 60–70% effective. Advancement of treatment requires new technology to identify new drugs for EPM. To address this critical need, we developed, validated, and implemented a high-throughput screen to test 725 FDA-approved compounds from the NIH clinical collections library for anti-S. neurona activity. Our screen identified 18 compounds with confirmed inhibitory activity against S. neurona growth, including compounds active in the nM concentration range. Many identified inhibitory compounds have well-defined mechanisms of action, making them useful tools to study parasite biology in addition to being potential therapeutic agents. In comparing the activity of inhibitory compounds identified by our screen to that of other screens against other apicomplexan parasites, we found that most compounds (15/18; 83% have activity against one or more related apicomplexans. Interestingly, nearly half (44%; 8/18 of the inhibitory compounds have reported activity against dopamine receptors. We also found that dantrolene, a compound already formulated for horses with a peak plasma concentration of 37.8 ± 12.8 ng/ml after 500 mg dose, inhibits S. neurona parasites at low concentrations (0.065 μM [0.036–0.12; 95% CI] or 21.9 ng/ml [12.1–40.3; 95% CI]. These studies demonstrate the use of a new tool for discovering new chemotherapeutic agents for EPM and potentially providing new reagents to elucidate biologic pathways required for successful S. neurona infection. Keywords: Drug repurposing, High-throughput screen, Sarcocystis neurona, Equine protozoal myeloencephalitis

Screening and syndromic approaches to identify gonorrhea and chlamydial infection among women.

Science.gov (United States)

Sloan, N L; Winikoff, B; Haberland, N; Coggins, C; Elias, C

2000-03-01

The standard diagnostic tools to identify sexually transmitted infections are often expensive and have laboratory and infrastructure requirements that make them unavailable to family planning and primary health-care clinics in developing countries. Therefore, inexpensive, accessible tools that rely on symptoms, signs, and/or risk factors have been developed to identify and treat reproductive tract infections without the need for laboratory diagnostics. Studies were reviewed that used standard diagnostic tests to identify gonorrhea and cervical chlamydial infection among women and that provided adequate information about the usefulness of the tools for screening. Aggregation of the studies' results suggest that risk factors, algorithms, and risk scoring for syndromic management are poor indicators of gonorrhea and chlamydial infection in samples of both low and high prevalence and, consequently, are not effective mechanisms with which to identify or manage these conditions. The development and evaluation of other approaches to identify gonorrhea and chlamydial infections, including inexpensive and simple laboratory screening tools, periodic universal treatment, and other alternatives must be given priority.

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library.

Science.gov (United States)

Macdonald, Spencer S; Patel, Ankoor; Larmour, Veronica L C; Morgan-Lang, Connor; Hallam, Steven J; Mark, Brian L; Withers, Stephen G

2018-03-02

Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to N -acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Viral load in children with congenital cytomegalovirus infection identified on newborn hearing screening.

Science.gov (United States)

Kawada, Jun-ichi; Torii, Yuka; Kawano, Yoshihiko; Suzuki, Michio; Kamiya, Yasuko; Kotani, Tomomi; Kikkawa, Fumitaka; Kimura, Hiroshi; Ito, Yoshinori

2015-04-01

Congenital cytomegalovirus (CMV) infection is the most common non-genetic cause of sensorineural hearing loss (SNHL) in children. However, congenital SNHL without other clinical abnormalities is rarely diagnosed as CMV-related in early infancy. The aim of this study was to identify and treat patients with congenital CMV-related SNHL or CMV-related clinical abnormalities other than SNHL. The association between CMV load and SNHL was also evaluated. Newborns who had abnormal hearing screening results or other clinical abnormalities were screened for congenital CMV infection by PCR of saliva or urine specimens, and identified infected patients were treated with valganciclovir (VGCV) for 6 weeks. The CMV load of patients with or without SNHL was compared at regular intervals during as well as after VGCV treatment. Of 127 infants with abnormal hearing screening results, and 31 infants with other clinical abnormalities, CMV infection was identified in 6 and 3 infants, respectively. After VGCV treatment, 1 case had improved hearing but the other 5 SNHL cases had little or no improvement. Among these 9 patients with or without SNHL at 1 year of age, there was no significant difference in CMV blood or urine load at diagnosis, but both were significantly higher in patients with SNHL during VGCV treatment. Selective CMV screening of newborns having an abnormal hearing screening result would be a reasonable strategy for identification of symptomatic congenital CMV infection. Prolonged detection of CMV in blood could be a risk factor for SNHL. Copyright © 2015 Elsevier B.V. All rights reserved.

Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities

DEFF Research Database (Denmark)

Radic-Sarikas, Branka; Tsafou, Kalliopi P; Emdal, Kristina B.

2017-01-01

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any...... associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents...... including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong...

Brief screening questionnaires to identify problem drinking during pregnancy: a systematic review.

Science.gov (United States)

Burns, Ethel; Gray, Ron; Smith, Lesley A

2010-04-01

Although prenatal screening for problem drinking during pregnancy has been recommended, guidance on screening instruments is lacking. We investigated the sensitivity, specificity and predictive value of brief alcohol screening questionnaires to identify problem drinking in pregnant women. Electronic databases from their inception to June 2008 were searched, as well as reference lists of eligible papers and related review papers. We sought cohort or cross-sectional studies that compared one or more brief alcohol screening questionnaire(s) with reference criteria obtained using structured interviews to detect 'at-risk' drinking, alcohol abuse or dependency in pregnant women receiving prenatal care. Five studies (6724 participants) were included. In total, seven instruments were evaluated: TWEAK (Tolerance, Worried, Eye-opener, Amnesia, Kut down), T-ACE [Take (number of drinks), Annoyed, Cut down, Eye-opener], CAGE (Cut down, Annoyed, Guilt, Eye-opener], NET (Normal drinker, Eye-opener, Tolerance), AUDIT (Alcohol Use Disorder Identification Test), AUDIT-C (AUDIT-consumption) and SMAST (Short Michigan Alcohol Screening Test). Study quality was generally good, but lack of blinding was a common weakness. For risk drinking sensitivity was highest for T-ACE (69-88%), TWEAK (71-91%) and AUDIT-C (95%), with high specificity (71-89%, 73-83% and 85%, respectively). CAGE and SMAST performed poorly. Sensitivity of AUDIT-C at score >or=3 was high for past year alcohol dependence (100%) or alcohol use disorder (96%) with moderate specificity (71% each). For life-time alcohol dependency the AUDIT at score >or=8 performed poorly. T-ACE, TWEAK and AUDIT-C show promise for screening for risk drinking, and AUDIT-C may also be useful for identifying alcohol dependency or abuse. However, their performance as stand-alone tools is uncertain, and further evaluation of questionnaires for prenatal alcohol use is warranted.

A Drosophila Genome-Wide Screen Identifies Regulators of Steroid Hormone Production and Developmental Timing

DEFF Research Database (Denmark)

Thomas Danielsen, E.; E. Møller, Morten; Yamanaka, Naoki

2016-01-01

Steroid hormones control important developmental processes and are linked to many diseases. To systematically identify genes and pathways required for steroid production, we performed a Drosophila genome-wide in vivo RNAi screen and identified 1,906 genes with potential roles in steroidogenesis...... and developmental timing. Here, we use our screen as a resource to identify mechanisms regulating intracellular levels of cholesterol, a substrate for steroidogenesis. We identify a conserved fatty acid elongase that underlies a mechanism that adjusts cholesterol trafficking and steroidogenesis with nutrition...... and developmental programs. In addition, we demonstrate the existence of an autophagosomal cholesterol mobilization mechanism and show that activation of this system rescues Niemann-Pick type C1 deficiency that causes a disorder characterized by cholesterol accumulation. These cholesterol-trafficking mechanisms...

Fragment-based design of symmetrical bis-benzimidazoles as selective inhibitors of the trimethoprim-resistant, type II R67 dihydrofolate reductase.

Science.gov (United States)

Bastien, Dominic; Ebert, Maximilian C C J C; Forge, Delphine; Toulouse, Jacynthe; Kadnikova, Natalia; Perron, Florent; Mayence, Annie; Huang, Tien L; Vanden Eynde, Jean Jacques; Pelletier, Joelle N

2012-04-12

The continuously increasing use of trimethoprim as a common antibiotic for medical use and for prophylactic application in terrestrial and aquatic animal farming has increased its prevalence in the environment. This has been accompanied by increased drug resistance, generally in the form of alterations in the drug target, dihydrofolate reductase (DHFR). The most highly resistant variants of DHFR are known as type II DHFR, among which R67 DHFR is the most broadly studied variant. We report the first attempt at designing specific inhibitors to this emerging drug target by fragment-based design. The detection of inhibition in R67 DHFR was accompanied by parallel monitoring of the human DHFR, as an assessment of compound selectivity. By those means, small aromatic molecules of 150-250 g/mol (fragments) inhibiting R67 DHFR selectively in the low millimolar range were identified. More complex, symmetrical bis-benzimidazoles and a bis-carboxyphenyl were then assayed as fragment-based leads, which procured selective inhibition of the target in the low micromolar range (K(i) = 2-4 μM). The putative mode of inhibition is discussed according to molecular modeling supported by in vitro tests. © 2012 American Chemical Society

High-throughput screen of drug repurposing library identifies inhibitors of Sarcocystis neurona growth.

Science.gov (United States)

Bowden, Gregory D; Land, Kirkwood M; O'Connor, Roberta M; Fritz, Heather M

2018-04-01

The apicomplexan parasite Sarcocystis neurona is the primary etiologic agent of equine protozoal myeloencephalitis (EPM), a serious neurologic disease of horses. Many horses in the U.S. are at risk of developing EPM; approximately 50% of all horses in the U.S. have been exposed to S. neurona and treatments for EPM are 60-70% effective. Advancement of treatment requires new technology to identify new drugs for EPM. To address this critical need, we developed, validated, and implemented a high-throughput screen to test 725 FDA-approved compounds from the NIH clinical collections library for anti-S. neurona activity. Our screen identified 18 compounds with confirmed inhibitory activity against S. neurona growth, including compounds active in the nM concentration range. Many identified inhibitory compounds have well-defined mechanisms of action, making them useful tools to study parasite biology in addition to being potential therapeutic agents. In comparing the activity of inhibitory compounds identified by our screen to that of other screens against other apicomplexan parasites, we found that most compounds (15/18; 83%) have activity against one or more related apicomplexans. Interestingly, nearly half (44%; 8/18) of the inhibitory compounds have reported activity against dopamine receptors. We also found that dantrolene, a compound already formulated for horses with a peak plasma concentration of 37.8 ± 12.8 ng/ml after 500 mg dose, inhibits S. neurona parasites at low concentrations (0.065 μM [0.036-0.12; 95% CI] or 21.9 ng/ml [12.1-40.3; 95% CI]). These studies demonstrate the use of a new tool for discovering new chemotherapeutic agents for EPM and potentially providing new reagents to elucidate biologic pathways required for successful S. neurona infection. Copyright © 2018. Published by Elsevier Ltd.

Current and emerging screening methods to identify post-head-emergence frost adaptation in wheat and barley.

Science.gov (United States)

Frederiks, T M; Christopher, J T; Harvey, G L; Sutherland, M W; Borrell, A K

2012-09-01

Cereal crops can suffer substantial damage if frosts occur at heading. Identification of post-head-emergence frost (PHEF) resistance in cereals poses a number of unique and difficult challenges. Many decades of research have failed to identify genotypes with PHEF resistance that could offer economically significant benefit to growers. Research and breeding gains have been limited by the available screening systems. Using traditional frost screening systems, genotypes that escape frost injury in trials due to spatial temperature differences and/or small differences in phenology can be misidentified as resistant. We believe that by improving techniques to minimize frost escapes, such 'false-positive' results can be confidently identified and eliminated. Artificial freezing chambers or manipulated natural frost treatments offer many potential advantages but are not yet at the stage where they can be reliably used for frost screening in breeding programmes. Here we describe the development of a novel photoperiod gradient method (PGM) that facilitates screening of genotypes of different phenology under natural field frosts at matched developmental stages. By identifying frost escapes and increasing the efficiency of field screening, the PGM ensures that research effort can be focused on finding genotypes with improved PHEF resistance. To maximize the likelihood of identifying PHEF resistance, we propose that the PGM form part of an integrated strategy to (i) source germplasm;(ii) facilitate high throughput screening; and (iii) permit detailed validation. PGM may also be useful in other studies where either a range of developmental stages and/or synchronized development are desired.

An AICD-based functional screen to identify APP metabolism regulators

Directory of Open Access Journals (Sweden)

Lee Jeremy C

2007-08-01

Full Text Available Abstract Background A central event in Alzheimer's disease (AD is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP, to generate the β-amyloid (Aβ peptide and the APP intracellular domain (AICD. Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered. Results To validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis. Conclusion Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.

Novel high-throughput screening system for identifying STAT3-SH2 antagonists

International Nuclear Information System (INIS)

Uehara, Yutaka; Mochizuki, Masato; Matsuno, Kenji; Haino, Takeharu; Asai, Akira

2009-01-01

Constitutive activation of the oncogenic transcription factor STAT3 frequently occurs in various human malignancies. STAT3 activation involves dimerization via intermolecular pTyr-SH2 interaction. Thus, antagonizing this interaction is a feasible approach to inhibit STAT3 activation for cancer therapy. In order to identify selective STAT3 inhibitors, we developed a biochemical HTS system based on AlphaScreen technology, which measures the abilities of test compounds to antagonize pTyr-SH2 interactions. We screened our chemical libraries using this system and identified 5,15-diphenylporphyrin (5,15-DPP) as a selective STAT3-SH2 antagonist. Selective inhibition of STAT3 nuclear translocation and DNA biding activity was observed in cells treated with 5,15-DPP. IL-6-dependent dimerization of STAT3, c-myc promoter binding and c-myc protein expression were all suppressed by 5,15-DPP, whereas no decrement in either expression or phosphorylation level of STAT3 was observed. Thus, the HTS assay system represented herein may be useful for identifying novel STAT3-SH2 antagonists.

Biosynthetic incorporation of telluromethionine into dihydrofolate reductase and crystallographic analysis of the distribution of tellurium atoms in the protein molecule

Energy Technology Data Exchange (ETDEWEB)

Kunkle, M.G.; Lewinski, K.; Boles, J.O.; Dunlap, R.B.; Odom, J.D.; Lebioda, L. [Univ. of South Carolina, Columbia, SC (United States)

1994-12-01

Recent successes in crystallographic studies of proteins with methionine (Met) residues replaced with SeMet, pioneered by Hendrickson and coworkers, inspired us to replace Met with TeMet in Escherichia coli dihydrofolate reductase (DHFR). E. coli DHFR, which catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, consists of 159 residues, 5 of which are Met. TeMet was incorporated into DHFR using the Met auxotroph, E. coli DL41, carrying the expression vector pWT8 with an IPTG inducible promoter and ampicillin resistance gene. The enzyme was purified by successive chromatography on Q-Sepharose and PHenyl Sepharose resins, yielding milligram quantities of homogeneous enzyme with a specific activity of 40 units/mg. TeMet DHFR exhibits kinetic properties similar to those of wt DHFR. Amino acid analysis indicated 3 authentic Met residues in TeMet DHFR, whereas atomic absorption spectroscopy detected 2 Te per protein molecule. Amino acid sequence analysis results suggested that only authentic Met was present in the first three Met positions (1,16,and 20). Crystals of Te-DHFR were grown in the presence of methotrexate from PEG 4000 and were isomorphous with wt-DHFR crystals grown from ethanol. Difference Fourier maps and restrained least-squares refinement show very little, if any, Te in the first three Met positions: Met{sup 1}, Met{sup 16}, and Met{sup 20}, whereas the occupancy of Te in positions 42 and 92 is 0.64. Apparently, the process of folding, subsequent purification, and crystallization select DHFR molecules with Te in Met{sup 42} and Met{sup 92}. Replacing Met with TeMet provides an internal probe that should facilitate structural and mechanistic studies of proteins.

High-Throughput Screening to Identify Regulators of Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

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Kassir, Yona

2017-01-01

Meiosis and gamete formation are processes that are essential for sexual reproduction in all eukaryotic organisms. Multiple intracellular and extracellular signals feed into pathways that converge on transcription factors that induce the expression of meiosis-specific genes. Once triggered the meiosis-specific gene expression program proceeds in a cascade that drives progress through the events of meiosis and gamete formation. Meiosis-specific gene expression is tightly controlled by a balance of positive and negative regulatory factors that respond to a plethora of signaling pathways. The budding yeast Saccharomyces cerevisiae has proven to be an outstanding model for the dissection of gametogenesis owing to the sophisticated genetic manipulations that can be performed with the cells. It is possible to use a variety selection and screening methods to identify genes and their functions. High-throughput screening technology has been developed to allow an array of all viable yeast gene deletion mutants to be screened for phenotypes and for regulators of gene expression. This chapter describes a protocol that has been used to screen a library of homozygous diploid yeast deletion strains to identify regulators of the meiosis-specific IME1 gene.

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs.

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Karlas, Alexander; Berre, Stefano; Couderc, Thérèse; Varjak, Margus; Braun, Peter; Meyer, Michael; Gangneux, Nicolas; Karo-Astover, Liis; Weege, Friderike; Raftery, Martin; Schönrich, Günther; Klemm, Uwe; Wurzlbauer, Anne; Bracher, Franz; Merits, Andres; Meyer, Thomas F; Lecuit, Marc

2016-05-12

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents.

Accuracy of Brief Screening Tools for Identifying Postpartum Depression Among Adolescent Mothers

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Venkatesh, Kartik K.; Zlotnick, Caron; Triche, Elizabeth W.; Ware, Crystal

2014-01-01

OBJECTIVE: To evaluate the accuracy of the Edinburgh Postnatal Depression Scale (EPDS) and 3 subscales for identifying postpartum depression among primiparous adolescent mothers. METHODS: Mothers enrolled in a randomized controlled trial to prevent postpartum depression completed a psychiatric diagnostic interview and the 10-item EPDS at 6 weeks, 3 months, and 6 months postpartum. Three subscales of the EPDS were assessed as brief screening tools: 3-item anxiety subscale (EPDS-3), 7-item depressive symptoms subscale (EPDS-7), and 2-item subscale (EPDS-2) that resemble the Patient Health Questionnaire-2. Receiver operating characteristic curves and the areas under the curves for each tool were compared to assess accuracy. The sensitivities and specificities of each screening tool were calculated in comparison with diagnostic criteria for a major depressive disorder. Repeated-measures longitudinal analytical techniques were used. RESULTS: A total of 106 women contributed 289 postpartum visits; 18% of the women met criteria for incident postpartum depression by psychiatric diagnostic interview. When used as continuous measures, the full EPDS, EPDS-7, and EPDS-2 performed equally well (area under the curve >0.9). Optimal cutoff scores for a positive depression screen for the EPDS and EPDS-7 were lower (≥9 and ≥7, respectively) than currently recommended cutoff scores (≥10). At optimal cutoff scores, the EPDS and EPDS-7 both had sensitivities of 90% and specificities of >85%. CONCLUSIONS: The EPDS, EPDS-7, and EPDS-2 are highly accurate at identifying postpartum depression among adolescent mothers. In primary care pediatric settings, the EPDS and its shorter subscales have potential for use as effective depression screening tools. PMID:24344102

Testing the woman abuse screening tool to identify intimate partner violence in Indonesia.

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Iskandar, Livia; Braun, Kathryn L; Katz, Alan R

2015-04-01

Intimate Partner Violence (IPV) is a global public health problem. IPV prevalence in Indonesia has been estimated to be less than 1%, based on reported cases. It is likely that IPV prevalence is underreported in Indonesia, as it is in many other countries. Screening for IPV has been found to increase IPV identification, but no screening tools are in use in Indonesia. The aim of this study was to test the translated Woman Abuse Screening Tool (WAST) for detecting IPV in Indonesia. The WAST was tested against a diagnostic interview by a trained psychologist on 240 women attending two Primary Health Centers in Jakarta. IPV prevalence and the reliability, sensitivity, and specificity of the WAST were estimated. Prevalence of IPV by diagnostic interview was 36.3%, much higher than published estimates. The most common forms of IPV identified were psychological (85%) and physical abuse (24%). Internal reliability of the WAST was high (α = .801). A WAST score of 13 (out of 24) is the recommended cutoff for identifying IPV, but only 17% of the Indonesian sample scored 13 or higher. Test sensitivity of the WAST with a cutoff score of 13 was only 41.9%, with a specificity of 96.8%. With a cutoff score of 10, the sensitivity improved to 84.9%, while the specificity decreased to 61.0%. Use of the WAST with a cutoff score of 10 provides good sensitivity and reasonable specificity and would provide a much-needed screening tool for use in Indonesia. Although a lower cutoff would yield a greater proportion of false positives, most of the true cases would be identified, increasing the possibility that women experiencing abuse would receive needed assistance. © The Author(s) 2014.

The comparison of the performance of two screening strategies identifying newly-diagnosed HIV during pregnancy.

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Boer, Kees; Smit, Colette; van der Flier, Michiel; de Wolf, Frank

2011-10-01

In the Netherlands, a non-selective opt-out instead of a selective opt-in antenatal HIV screening strategy was implemented in 2004. In case of infection, screening was followed by prevention of mother-to-child-transmission (PMTCT). We compared the performance of the two strategies in terms of detection of new cases of HIV and vertical transmission. HIV-infected pregnant women were identified retrospectively from the Dutch HIV cohort ATHENA January 2000 to January 2008. Apart from demographic, virological and immunological data, the date of HIV infection in relation to the index pregnancy was established. Separately, all infants diagnosed with HIV born following implementation of the screening program were identified by a questionnaire via the paediatric HIV centres. 162/481 (33.7%) HIV-positive pregnant women were diagnosed with HIV before 2004 and 172/214 (80.3%) after January 2004. Multivariate analysis showed an 8-fold (95% confidence interval 5.47-11.87) increase in the odds of HIV detection during pregnancy after the national introduction of the opt-out strategy. Still, three children born during a 5-year period after July 2004 were infected due to de novo infection in pregnancy. Implementation of a nation-wide screening strategy based upon non-selective opt-out screening followed by effective PMTCT appeared to detect more HIV-infected women for the first time in pregnancy and to reduce vertical transmission of HIV substantially. Nonetheless, still few children are infected because of maternal infection after the first trimester. We propose the introduction of partner screening on HIV as part of the antenatal screening strategy.

Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

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Yuthavong Yongyuth

2011-05-01

Full Text Available Abstract Background The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development. Methods A system to identify mutations in Pfdhfr gene that confer antifolate drug resistance using an animal Plasmodium parasite model was developed. By using error-prone PCR and Plasmodium transfection technologies, libraries of Pfdhfr mutant were generated and then episomally transfected to Plasmodium berghei parasites, from which pyrimethamine-resistant PfDHFR mutants were selected. Results The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic P. berghei, in which endogenous Pbdhfr allele was replaced with the mutant PfdhfrS108N, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine. Conclusion This study demonstrated the power of the transgenic P. berghei system to predict drug-resistant Pfdhfr mutations in an in vivo parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

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Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

2018-04-03

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.

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Anton Simeonov

2008-01-01

Full Text Available Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR and peroxiredoxin (Prx and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 microL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC(50s ranging from micromolar to the assay response limit ( approximately 25 nM. This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump

High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

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Jessica K Leet

Full Text Available Using transgenic zebrafish (fli1:egfp that stably express enhanced green fluorescent protein (eGFP within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1 screening chemicals for cardiovascular toxicity and (2 prioritizing chemicals for future hypothesis

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

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Manguso, Robert T; Pope, Hans W; Zimmer, Margaret D; Brown, Flavian D; Yates, Kathleen B; Miller, Brian C; Collins, Natalie B; Bi, Kevin; LaFleur, Martin W; Juneja, Vikram R; Weiss, Sarah A; Lo, Jennifer; Fisher, David E; Miao, Diana; Van Allen, Eliezer; Root, David E; Sharpe, Arlene H; Doench, John G; Haining, W Nicholas

2017-07-27

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Screening of pharmacologically active small molecule compounds identifies antifungal agents against Candida biofilms

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Takao eWatamoto

2015-12-01

Full Text Available Candida species have emerged as important and common opportunistic human pathogens, particularly in immunocompromised individuals. The current antifungal therapies either have toxic side effects or are insufficiently effect. The aim of this study is develop new small-molecule antifungal compounds by library screening methods using C. albicans, and to evaluate their antifungal effects on Candida biofilms and cytotoxic effects on human cells. Wild-type C. albicans strain SC5314 was used in library screening. To identify antifungal compounds, we screened a small-molecule library of 1,280 pharmacologically active compounds (LOPAC1280TM using an antifungal susceptibility test (AST. To investigate the antifungal effects of the hit compounds, ASTs were conducted using Candida strains in various growth modes, including biofilms. We tested the cytotoxicity of the hit compounds using human gingival fibroblast (hGF cells to evaluate their clinical safety. Only 35 compounds were identified by screening, which inhibited the metabolic activity of C. albicans by >50%. Of these, 26 compounds had fungistatic effects and 9 compounds had fungicidal effects on C. albicans. Five compounds, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate, ellipticine and CV-3988, had strong fungicidal effects and could inhibit the metabolic activity of Candida biofilms. However, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate and ellipticine were cytotoxic to hGF cells at low concentrations. CV-3988 showed no cytotoxicity at a fungicidal concentration.Four of the compounds identified, BAY11-7082, BAY11-7085, sanguinarine chloride hydrate and ellipticine, had toxic effects on Candida strains and hGF cells. In contrast, CV-3988 had fungicidal effects on Candida strains, but low cytotoxic effects on hGF cells. Therefore, this screening reveals agent, CV-3988 that was previously unknown to be antifungal agent, which could be a novel therapies for superficial mucosal

Protein dynamics and stability: The distribution of atomic fluctuations in thermophilic and mesophilic dihydrofolate reductase derived using elastic incoherent neutron scattering

International Nuclear Information System (INIS)

Meinhold, Lars; Clement, David; Tehei, M.; Daniel, R.M.; Finney, J.L.; Smith, Jeremy C.

2008-01-01

The temperature dependence of the dynamics of mesophilic and thermophilic dihydrofolate reductase is examined using elastic incoherent neutron scattering. It is demonstrated that the distribution of atomic displacement amplitudes can be derived from the elastic scattering data by assuming a (Weibull) functional form that resembles distributions seen in molecular dynamics simulations. The thermophilic enzyme has a significantly broader distribution than its mesophilic counterpart. Furthermore, although the rate of increase with temperature of the atomic mean-square displacements extracted from the dynamic structure factor is found to be comparable for both enzymes, the amplitudes are found to be slightly larger for the thermophilic enzyme. Therefore, these results imply that the thermophilic enzyme is the more flexible of the two

Genome-wide RNAi screening identifies genes inhibiting the migration of glioblastoma cells.

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Jian Yang

Full Text Available Glioblastoma Multiforme (GBM cells are highly invasive, infiltrating into the surrounding normal brain tissue, making it impossible to completely eradicate GBM tumors by surgery or radiation. Increasing evidence also shows that these migratory cells are highly resistant to cytotoxic reagents, but decreasing their migratory capability can re-sensitize them to chemotherapy. These evidences suggest that the migratory cell population may serve as a better therapeutic target for more effective treatment of GBM. In order to understand the regulatory mechanism underlying the motile phenotype, we carried out a genome-wide RNAi screen for genes inhibiting the migration of GBM cells. The screening identified a total of twenty-five primary hits; seven of them were confirmed by secondary screening. Further study showed that three of the genes, FLNA, KHSRP and HCFC1, also functioned in vivo, and knocking them down caused multifocal tumor in a mouse model. Interestingly, two genes, KHSRP and HCFC1, were also found to be correlated with the clinical outcome of GBM patients. These two genes have not been previously associated with cell migration.

Identifying factors to improve oral cancer screening uptake: a qualitative study.

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Fatemeh Vida Zohoori

Full Text Available To engage with high risk groups to identify knowledge and awareness of oral cancer signs and symptoms and the factors likely to contribute to improved screening uptake.Focus group discussions were undertaken with 18 males; 40+ years of age; smokers and/or drinkers (15+ cigarettes per day and/or 15+ units of alcohol per week, irregular dental attenders living in economically deprived areas of Teesside.There was a striking reported lack of knowledge and awareness of oral cancer and its signs and symptoms among the participants. When oral/mouth cancer leaflets produced by Cancer Research UK were presented to the participants, they claimed that they would seek help on noticing such a condition. There was a preference to seek help from their general practitioner rather than their dentist due to perceptions that a dentist is 'inaccessible' on a physical and psychological level, costly, a 'tooth specialist' not a 'mouth specialist', and also not able to prescribe medication and make referrals to specialists. Interestingly, none of the 18 participants who were offered a free oral cancer examination at a dental practice took up this offer.The uptake of oral cancer screening may be improved by increasing knowledge of the existence and signs and symptoms of oral cancer. Other factors that may increase uptake are increased awareness of the role of dentists in diagnosing oral cancer, promotion of oral cancer screening by health professionals during routine health checks, and the use of a "health" screening setting as opposed to a "dental" setting for such checks.

High-content phenotypic screening and triaging strategy to identify small molecules driving oligodendrocyte progenitor cell differentiation.

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Peppard, Jane V; Rugg, Catherine A; Smicker, Matthew A; Powers, Elaine; Harnish, Erica; Prisco, Joy; Cirovic, Dragan; Wright, Paul S; August, Paul R; Chandross, Karen J

2015-03-01

Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell-like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio- or cheminformatics studies, and their compelling biological activity merits further investigation. © 2014 Society for Laboratory Automation and Screening.

Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

International Nuclear Information System (INIS)

Chen, Li; Schmidt, Emmett V; Stuart, Lynda; Ohsumi, Toshiro K; Burgess, Shawn; Varshney, Gaurav K; Dastur, Anahita; Borowsky, Mark; Benes, Cyril; Lacy-Hulbert, Adam

2013-01-01

The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

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Deepika Kanojia

2017-11-01

Full Text Available Abstract Background Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. Methods Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. Results Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. Conclusions Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer’s Disease

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Philip W. Brownjohn

2017-04-01

Full Text Available Summary: Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP processing and the accumulation of APP-derived amyloid β (Aβ peptides are key processes in Alzheimer's disease (AD. We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation. : In this article, Livesey and colleagues perform a phenotypic drug screen in a human stem cell model of Alzheimer's disease. The anthelminthic avermectins are identified as a family of compounds that increase the production of short Aβ peptides over longer more toxic Aβ forms. The effect is analogous to existing γ-secretase modulators, but is independent of the core γ-secretase complex. Keywords: neural stem cells, Alzheimer's disease, phenotypic screening, iPSCs, human neurons, dementia, Down syndrome, amyloid beta, ivermectin, selamectin

Identifying a borderline personality disorder prodrome: Implications for community screening.

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Stepp, Stephanie D; Lazarus, Sophie A

2017-08-01

Elucidating early signs and symptoms of borderline personality disorder (BPD) has important implications for screening and identifying youth appropriate for early intervention. The purpose of this study was to identify dimensions of child temperament and psychopathology symptom severity that predict conversion to a positive screen for BPD over a 14-year follow-up period in a large, urban community sample of girls (n = 2 450). Parent and teacher reports of child temperament and psychopathology symptom severity assessed when girls were ages 5-8 years were examined as predictors of new-onset BPD cases when girls were ages 14-22 years. In the final model, parent and teacher ratings of emotionality remained significant predictors of new-onset BPD. Additionally, parent ratings of hyperactivity/impulsivity and depression severity, as well as teacher ratings of inattention severity, were also predictive. Results also revealed that elevations in these dimensions pose a notable increase in risk for conversion to BPD over the follow-up period. Supplementary analyses revealed that with the exception of parent-reported depression severity, these same predictors were associated with increases in BPD symptom severity over the follow-up period. These findings suggest BPD onset in adolescence and early adulthood can be detected from parent and teacher reports of temperament and symptom severity dimensions assessed in childhood. The identification of this prodrome holds promise for advancing early detection of children at risk prior to the development of the full-blown disorder. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

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Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

2011-01-01

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. PMID:22034911

Drug Repurposing Screening Identifies Novel Compounds That Effectively Inhibit Toxoplasma gondii Growth

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Dittmar, Ashley J.; Drozda, Allison A.

2016-01-01

ABSTRACT The urgent need to develop new antimicrobial therapies has spawned the development of repurposing screens in which well-studied drugs and other types of compounds are tested for potential off-label uses. As a proof-of-principle screen to identify compounds effective against Toxoplasma gondii, we screened a collection of 1,120 compounds for the ability to significantly reduce Toxoplasma replication. A total of 94 compounds blocked parasite replication with 50% inhibitory concentrations of parasite invasion and replication but did so independently of inhibition of dopamine or other neurotransmitter receptor signaling. Tamoxifen, which is an established inhibitor of the estrogen receptor, also reduced parasite invasion and replication. Even though Toxoplasma can activate the estrogen receptor, tamoxifen inhibits parasite growth independently of this transcription factor. Tamoxifen is also a potent inducer of autophagy, and we find that the drug stimulates recruitment of the autophagy marker light chain 3-green fluorescent protein onto the membrane of the vacuolar compartment in which the parasite resides and replicates. In contrast to other antiparasitic drugs, including pimozide, tamoxifen treatment of infected cells leads to a time-dependent elimination of intracellular parasites. Taken together, these data suggest that tamoxifen restricts Toxoplasma growth by inducing xenophagy or autophagic destruction of this obligate intracellular parasite. IMPORTANCE There is an urgent need to develop new therapies to treat microbial infections, and the repurposing of well-characterized compounds is emerging as one approach to achieving this goal. Using the protozoan parasite Toxoplasma gondii, we screened a library of 1,120 compounds and identified several compounds with significant antiparasitic activities. Among these were pimozide and tamoxifen, which are well-characterized drugs prescribed to treat patients with psychiatric disorders and breast cancer

A high throughput screening assay for identifying glycation inhibitors on MALDI-TOF target.

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Zhang, Qiuting; Tu, Zongcai; Wang, Hui; Fan, Liangliang; Huang, Xiaoqin; Xiao, Hui

2015-03-01

The Maillard reaction plays an important role in the food industry, however, the deleterious effects generated by the advanced glycation end-products (AGEs) have been well recognized. Many efforts have been made to seek new AGE inhibitors, in particular those natural ones without adverse effect. We have developed a rapid, mass spectrometry based, on-plate screening assay for novel AGE inhibitors. The glycation reaction, inhibition feedback as well as the subsequent MALDI mass spectrometric analysis occurred on one single MALDI plate. At 1:10 M ratio of peptide to sugar, as little as 4h incubation time allowed the screening test to be ready for analysis. DSP, inhibition and IC50 were calculated to evaluate selected inhibitors and resulting inhibition efficiencies were consistent with available references. We demonstrated that this method provide a potential high throughput screening assay to analyze and identify the anti-glycation agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genome-wide screen of Pseudomonas aeruginosa In Saccharomyces cerevisiae identifies new virulence factors

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Rafat eZrieq

2015-11-01

Full Text Available Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. 51 candidates were selected in a three-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec. By testing the cytotoxicity of wild type P. aeruginosa vs pec mutants towards macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

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Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai

2010-01-01

The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor...... radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B......) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA...

A genome-wide siRNA screen to identify modulators of insulin sensitivity and gluconeogenesis.

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Ruojing Yang

Full Text Available BACKGROUND: Hepatic insulin resistance impairs insulin's ability to suppress hepatic glucose production (HGP and contributes to the development of type 2 diabetes (T2D. Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC promoter (AH-G6PC cells. Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4 mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. CONCLUSIONS/SIGNIFICANCE: These results support the proposition that the proteins encoded by the genes identified in

Integrative screening approach identifies regulators of polyploidization and targets for acute megakaryocytic leukemia

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Wen, Qiang; Goldenson, Benjamin; Silver, Serena J.; Schenone, Monica; Dancik, Vladimir; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy; An, W. Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S.; Moore, Christopher B.; Bliss-Moreau, Meghan; VerPlank, Lynn; Tolliday, Nicola J.; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K.; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D. Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S.; Smith, Franklin O.; Woods, William G.; Taub, Jeffrey; Scherer, Christina A.; Bradner, James; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E.; Gould, Robert J.; Clemons, Paul A.; Carr, Steven A.; Root, David E.; Schreiber, Stuart L.; Stern, Andrew M.; Crispino, John D.

2012-01-01

Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy. PMID:22863010

Automated NMR fragment based screening identified a novel interface blocker to the LARG/RhoA complex.

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Jia Gao

Full Text Available The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to ¹⁵N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG.

Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities.

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Radic-Sarikas, Branka; Tsafou, Kalliopi P; Emdal, Kristina B; Papamarkou, Theodore; Huber, Kilian V M; Mutz, Cornelia; Toretsky, Jeffrey A; Bennett, Keiryn L; Olsen, Jesper V; Brunak, Søren; Kovar, Heinrich; Superti-Furga, Giulio

2017-01-01

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR. ©2016 American Association for Cancer Research.

Fitness trade-offs in the evolution of dihydrofolate reductase and drug resistance in Plasmodium falciparum.

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Marna S Costanzo

Full Text Available Patterns of emerging drug resistance reflect the underlying adaptive landscapes for specific drugs. In Plasmodium falciparum, the parasite that causes the most serious form of malaria, antifolate drugs inhibit the function of essential enzymes in the folate pathway. However, a handful of mutations in the gene coding for one such enzyme, dihydrofolate reductase, confer drug resistance. Understanding how evolution proceeds from drug susceptibility to drug resistance is critical if new antifolate treatments are to have sustained usefulness.We use a transgenic yeast expression system to build on previous studies that described the adaptive landscape for the antifolate drug pyrimethamine, and we describe the most likely evolutionary trajectories for the evolution of drug resistance to the antifolate chlorcycloguanil. We find that the adaptive landscape for chlorcycloguanil is multi-peaked, not all highly resistant alleles are equally accessible by evolution, and there are both commonalities and differences in adaptive landscapes for chlorcycloguanil and pyrimethamine.Our findings suggest that cross-resistance between drugs targeting the same enzyme reflect the fitness landscapes associated with each particular drug and the position of the genotype on both landscapes. The possible public health implications of these findings are discussed.

Guided genetic screen to identify genes essential in the regeneration of hair cells and other tissues.

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Pei, Wuhong; Xu, Lisha; Huang, Sunny C; Pettie, Kade; Idol, Jennifer; Rissone, Alberto; Jimenez, Erin; Sinclair, Jason W; Slevin, Claire; Varshney, Gaurav K; Jones, MaryPat; Carrington, Blake; Bishop, Kevin; Huang, Haigen; Sood, Raman; Lin, Shuo; Burgess, Shawn M

2018-01-01

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.

Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase.

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Luk, Louis Y P; Ruiz-Pernía, J Javier; Adesina, Aduragbemi S; Loveridge, E Joel; Tuñón, Iñaki; Moliner, Vincent; Allemann, Rudolf K

2015-07-27

Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes ((2) H, (13) C, (15) N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

RNAi-Mediated Reverse Genetic Screen Identified Drosophila Chaperones Regulating Eye and Neuromuscular Junction Morphology

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Sandeep Raut

2017-07-01

Full Text Available Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C-Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila. Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development.

A novel glutamine–RNA interaction identified by screening libraries in mammalian cells

OpenAIRE

Tan, Ruoying; Frankel, Alan D.

1998-01-01

The arginine-rich motif provides a versatile framework for RNA recognition in which few amino acids other than arginine are needed to mediate specific binding. Using a mammalian screening system based on transcriptional activation by HIV Tat, we identified novel arginine-rich peptides from combinatorial libraries that bind tightly to the Rev response element of HIV. Remarkably, a single glutamine, but not asparagine, within a stretch of polyarginine can mediate high-affinity binding. These re...

A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.

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Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man; He, Bin; Zhang, Liqing; Varmark, Hanne; Green, Michael R; Sheng, Zhi

2018-02-12

Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.

A genetic screen identifies interferon-α effector genes required to suppress hepatitis C virus replication.

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Fusco, Dahlene N; Brisac, Cynthia; John, Sinu P; Huang, Yi-Wen; Chin, Christopher R; Xie, Tiao; Zhao, Hong; Jilg, Nikolaus; Zhang, Leiliang; Chevaliez, Stephane; Wambua, Daniel; Lin, Wenyu; Peng, Lee; Chung, Raymond T; Brass, Abraham L

2013-06-01

Hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease. Interferon-α (IFNα) is an important component of anti-HCV therapy; it up-regulates transcription of IFN-stimulated genes, many of which have been investigated for their antiviral effects. However, all of the genes required for the antiviral function of IFNα (IFN effector genes [IEGs]) are not known. IEGs include not only IFN-stimulated genes, but other nontranscriptionally induced genes that are required for the antiviral effect of IFNα. In contrast to candidate approaches based on analyses of messenger RNA (mRNA) expression, identification of IEGs requires a broad functional approach. We performed an unbiased genome-wide small interfering RNA screen to identify IEGs that inhibit HCV. Huh7.5.1 hepatoma cells were transfected with small interfering RNAs incubated with IFNα and then infected with JFH1 HCV. Cells were stained using HCV core antibody, imaged, and analyzed to determine the percent infection. Candidate IEGs detected in the screen were validated and analyzed further. The screen identified 120 previously unreported IEGs. From these, we more fully evaluated the following: asparagine-linked glycosylation 10 homolog (yeast, α-1,2-glucosyltransferase); butyrylcholinesterase; dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2); glucokinase (hexokinase 4) regulator; guanylate cyclase 1, soluble, β 3; MYST histone acetyltransferase 1; protein phosphatase 3 (formerly 2B), catalytic subunit, β isoform; peroxisomal proliferator-activated receptor-γ-DBD-interacting protein 1; and solute carrier family 27 (fatty acid transporter), member 2; and demonstrated that they enabled IFNα-mediated suppression of HCV at multiple steps of its life cycle. Expression of these genes had more potent effects against flaviviridae because a subset was required for IFNα to suppress dengue virus but not influenza A virus. In addition, many of the host genes detected in this

HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

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Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

2016-11-01

A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

Novel inhibitors to Taenia solium Cu/Zn superoxide dismutase identified by virtual screening

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García-Gutiérrez, P.; Landa-Piedra, A.; Rodríguez-Romero, A.; Parra-Unda, R.; Rojo-Domínguez, A.

2011-12-01

We describe in this work a successful virtual screening and experimental testing aimed to the identification of novel inhibitors of superoxide dismutase of the worm Taenia solium ( TsCu/Zn-SOD), a human parasite. Conformers from LeadQuest® database of drug-like compounds were selected and then docked on the surface of TsCu/Zn-SOD. Results were screened looking for ligand contacts with receptor side-chains not conserved in the human homologue, with a subsequent development of a score optimization by a set of energy minimization steps, aimed to identify lead compounds for in vitro experiments. Six out of fifty experimentally tested compounds showed μM inhibitory activity toward TsCu/Zn-SOD. Two of them showed species selectivity since did not inhibit the homologous human enzyme when assayed in vitro.

A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target.

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Ito, Hideaki; Ura, Atsushi; Oyamada, Yoshihiro; Yoshida, Hiroaki; Yamagishi, Jun-Ichi; Narita, Shin-Ichiro; Matsuyama, Shin-Ichi; Tokuda, Hajime

2007-01-01

As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in-house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system.

Screening of 1331 Danish breast and/or ovarian cancer families identified 40 novel BRCA1 and BRCA2 mutations

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Hansen, Thomas V O; Jønson, Lars; Steffensen, Ane Y

2011-01-01

Germ-line mutations in the tumour suppressor genes BRCA1 and BRCA2 predispose to breast and ovarian cancer. Since 1999 we have performed mutational screening of breast and/or ovarian cancer patients in East Denmark. During this period we have identified 40 novel sequence variations in BRCA1...... and BRCA2 in high risk breast and/or ovarian cancer families. The mutations were detected via pre-screening using dHPLC or high-resolution melting and direct sequencing. We identified 16 variants in BRCA1, including 9 deleterious frame-shift mutations, 2 intronic variants, 4 missense mutations, and 1......, the presumed significance of the missense mutations was predicted in silico using the align GVGD algorithm. In conclusion, the mutation screening identified 40 novel variants in the BRCA1 and BRCA2 genes and thereby extends the knowledge of the BRCA1/BRCA2 mutation spectrum. Nineteen of the mutations were...

IspE inhibitors identified by a combination of in silico and in vitro high-throughput screening.

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Naomi Tidten-Luksch

Full Text Available CDP-ME kinase (IspE contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens.

Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening.

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Wagle, Neil; Xian, Jun; Shishova, Ekaterina Y; Wei, Jie; Glicksman, Marcie A; Cuny, Gregory D; Stein, Ross L; Cohen, David E

2008-12-01

Phosphatidylcholine transfer protein (PC-TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC-TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC-TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC-TP activity, with IC(50) values that ranged from 4.1 to 95.0muM under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC-TP. Because mice with homozygous disruption of the PC-TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.

Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

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Yuri Pevzner

2014-05-01

Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

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Yuri Pevzner

2015-08-01

Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

Identifying parents with risky alcohol consumption habits in a paediatric unit--are screening and brief intervention appropriate methods?

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Bjerregaard, Lene B L; Gerke, Oke; Rubak, Sune; Høst, Arne; Wagner, Lis

2011-06-01

There is no systematic identification of parents with excessive alcohol use who have a child admitted to hospital. Children in families with excessive alcohol issues form a high risk group as substantial alcohol consumption has a damaging influence on a child emotionally, cognitively, socially and physically. Alcohol consumption is a sensitive issue, and health staff needs knowledge, qualifications and adequate training in communicating with parents about this taboo. • To identify specific patterns in subgroups of parents by comparing results from screening and demographic variables • To identify systematic patterns in staff members by demographic variables to decide whether these factors influence the screening results. During 1 year, screening and brief intervention (SBI) was accomplished, including health staff conducting dialogues with parents of a hospitalized child using motivational interviewing (MI) and screening for risky alcohol behaviour by Cut down, Annoyance from others, feel Guilty, Early-morning Craving (CAGE)-C. Data were analysed by descriptive statistics, and relationships were tested with a statistical significance level of 0.05, using SPSS (version 16.0). Motivational dialogues with 779 parents were conducted by 43 staff members, and 11% of the parents were screened positive for risky alcohol behaviour. Drinking alcohol 4 days a week or more and drinking alcohol outside mealtimes were main risk factors. Parents' gender was the strongest predictor of screening positive and OR was 6.8 for men (CI 4.03-11.74) compared to women, pparents' age (CI 1.02-1.42) indicates the risk of screening positive increases with age, p=0.027. Brief intervention using CAGE-C and MI has proven successful in mapping parents' alcohol consumption patterns and in identifying parents with risky alcohol consumption habits. Health staff is able to manage health promotion and prevention when having the right competences and when being supervised. © 2010 The Authors

A genome scale RNAi screen identifies GLI1 as a novel gene regulating vorinostat sensitivity.

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Falkenberg, K J; Newbold, A; Gould, C M; Luu, J; Trapani, J A; Matthews, G M; Simpson, K J; Johnstone, R W

2016-07-01

Vorinostat is an FDA-approved histone deacetylase inhibitor (HDACi) that has proven clinical success in some patients; however, it remains unclear why certain patients remain unresponsive to this agent and other HDACis. Constitutive STAT (signal transducer and activator of transcription) activation, overexpression of prosurvival Bcl-2 proteins and loss of HR23B have been identified as potential biomarkers of HDACi resistance; however, none have yet been used to aid the clinical utility of HDACi. Herein, we aimed to further elucidate vorinostat-resistance mechanisms through a functional genomics screen to identify novel genes that when knocked down by RNA interference (RNAi) sensitized cells to vorinostat-induced apoptosis. A synthetic lethal functional screen using a whole-genome protein-coding RNAi library was used to identify genes that when knocked down cooperated with vorinostat to induce tumor cell apoptosis in otherwise resistant cells. Through iterative screening, we identified 10 vorinostat-resistance candidate genes that sensitized specifically to vorinostat. One of these vorinostat-resistance genes was GLI1, an oncogene not previously known to regulate the activity of HDACi. Treatment of vorinostat-resistant cells with the GLI1 small-molecule inhibitor, GANT61, phenocopied the effect of GLI1 knockdown. The mechanism by which GLI1 loss of function sensitized tumor cells to vorinostat-induced apoptosis is at least in part through interactions with vorinostat to alter gene expression in a manner that favored apoptosis. Upon GLI1 knockdown and vorinostat treatment, BCL2L1 expression was repressed and overexpression of BCL2L1 inhibited GLI1-knockdown-mediated vorinostat sensitization. Taken together, we present the identification and characterization of GLI1 as a new HDACi resistance gene, providing a strong rationale for development of GLI1 inhibitors for clinical use in combination with HDACi therapy.

Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.

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Zanotto-Filho, Alfeu; Dashnamoorthy, Ravi; Loranc, Eva; de Souza, Luis H T; Moreira, José C F; Suresh, Uthra; Chen, Yidong; Bishop, Alexander J R

2016-01-01

Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.

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Alfeu Zanotto-Filho

Full Text Available Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair, DNA-mRNA-protein metabolism (transcription/translation and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress/Unfolded Protein Responses (UPR in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.

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Oliver N F King

2010-11-01

Full Text Available Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors.High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4 family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II and to modulate demethylation at the H3K9 locus in a cell-based assay.These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication

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Julianna Han

2018-04-01

Full Text Available Summary: The emergence of influenza A viruses (IAVs from zoonotic reservoirs poses a great threat to human health. As seasonal vaccines are ineffective against zoonotic strains, and newly transmitted viruses can quickly acquire drug resistance, there remains a need for host-directed therapeutics against IAVs. Here, we performed a genome-scale CRISPR/Cas9 knockout screen in human lung epithelial cells with a human isolate of an avian H5N1 strain. Several genes involved in sialic acid biosynthesis and related glycosylation pathways were highly enriched post-H5N1 selection, including SLC35A1, a sialic acid transporter essential for IAV receptor expression and thus viral entry. Importantly, we have identified capicua (CIC as a negative regulator of cell-intrinsic immunity, as loss of CIC resulted in heightened antiviral responses and restricted replication of multiple viruses. Therefore, our study demonstrates that the CRISPR/Cas9 system can be utilized for the discovery of host factors critical for the replication of intracellular pathogens. : Using a genome-wide CRISPR/Cas9 screen, Han et al. demonstrate that the major hit, the sialic acid transporter SLC35A1, is an essential host factor for IAV entry. In addition, they identify the DNA-binding transcriptional repressor CIC as a negative regulator of cell-intrinsic immunity. Keywords: CRISPR/Cas9 screen, GeCKO, influenza virus, host factors, sialic acid pathway, SLC35A1, Capicua, CIC, cell-intrinsic immunity, H5N1

ENCoRE: an efficient software for CRISPR screens identifies new players in extrinsic apoptosis.

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Trümbach, Dietrich; Pfeiffer, Susanne; Poppe, Manuel; Scherb, Hagen; Doll, Sebastian; Wurst, Wolfgang; Schick, Joel A

2017-11-25

As CRISPR/Cas9 mediated screens with pooled guide libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged. We have developed a lightweight and efficient software to easily manipulate large raw next generation sequencing datasets derived from such screens into informative relational context with graphical support. The advantages of the software entitled ENCoRE (Easy NGS-to-Gene CRISPR REsults) include a simple graphical workflow, platform independence, local and fast multithreaded processing, data pre-processing and gene mapping with custom library import. We demonstrate the capabilities of ENCoRE to interrogate results from a pooled CRISPR cellular viability screen following Tumor Necrosis Factor-alpha challenge. The results not only identified stereotypical players in extrinsic apoptotic signaling but two as yet uncharacterized members of the extrinsic apoptotic cascade, Smg7 and Ces2a. We further validated and characterized cell lines containing mutations in these genes against a panel of cell death stimuli and involvement in p53 signaling. In summary, this software enables bench scientists with sensitive data or without access to informatic cores to rapidly interpret results from large scale experiments resulting from pooled CRISPR/Cas9 library screens.

Approaches to virtual screening and screening library selection.

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Wildman, Scott A

2013-01-01

The ease of access to virtual screening (VS) software in recent years has resulted in a large increase in literature reports. Over 300 publications in the last year report the use of virtual screening techniques to identify new chemical matter or present the development of new virtual screening techniques. The increased use is accompanied by a corresponding increase in misuse and misinterpretation of virtual screening results. This review aims to identify many of the common difficulties associated with virtual screening and allow researchers to better assess the reliability of their virtual screening effort.

Evaluation of an inpatient fall risk screening tool to identify the most critical fall risk factors in inpatients.

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Hou, Wen-Hsuan; Kang, Chun-Mei; Ho, Mu-Hsing; Kuo, Jessie Ming-Chuan; Chen, Hsiao-Lien; Chang, Wen-Yin

2017-03-01

To evaluate the accuracy of the inpatient fall risk screening tool and to identify the most critical fall risk factors in inpatients. Variations exist in several screening tools applied in acute care hospitals for examining risk factors for falls and identifying high-risk inpatients. Secondary data analysis. A subset of inpatient data for the period from June 2011-June 2014 was extracted from the nursing information system and adverse event reporting system of an 818-bed teaching medical centre in Taipei. Data were analysed using descriptive statistics, receiver operating characteristic curve analysis and logistic regression analysis. During the study period, 205 fallers and 37,232 nonfallers were identified. The results revealed that the inpatient fall risk screening tool (cut-off point of ≥3) had a low sensitivity level (60%), satisfactory specificity (87%), a positive predictive value of 2·0% and a negative predictive value of 99%. The receiver operating characteristic curve analysis revealed an area under the curve of 0·805 (sensitivity, 71·8%; specificity, 78%). To increase the sensitivity values, the Youden index suggests at least 1·5 points to be the most suitable cut-off point for the inpatient fall risk screening tool. Multivariate logistic regression analysis revealed a considerably increased fall risk in patients with impaired balance and impaired elimination. The fall risk factor was also significantly associated with days of hospital stay and with admission to surgical wards. The findings can raise awareness about the two most critical risk factors for falls among future clinical nurses and other healthcare professionals and thus facilitate the development of fall prevention interventions. This study highlights the needs for redefining the cut-off points of the inpatient fall risk screening tool to effectively identify inpatients at a high risk of falls. Furthermore, inpatients with impaired balance and impaired elimination should be closely

An update on the use of C. elegans for preclinical drug discovery: screening and identifying anti-infective drugs.

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Kim, Wooseong; Hendricks, Gabriel Lambert; Lee, Kiho; Mylonakis, Eleftherios

2017-06-01

The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds. Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules. Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.

A screen to identify drug resistant variants to target-directed anti-cancer agents

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Azam Mohammad

2003-01-01

Full Text Available The discovery of oncogenes and signal transduction pathways important for mitogenesis has triggered the development of target-specific small molecule anti-cancer compounds. As exemplified by imatinib (Gleevec, a specific inhibitor of the Chronic Myeloid Leukemia (CML-associated Bcr-Abl kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using imatinib and BCR/ABL as a paradigm for a drug-target pair, we recently reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair.

Identifying children at risk for language impairment: screening of communication at 18 months.

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Bruce, B; Kornfält, R; Radeborg, K; Hansson, K; Nettelbladt, U

2003-09-01

To investigate the possibility of identifying children at risk for language impairment based on a new screening instrument to assess communication and language skills at 18 mo of age. At 18 mo, 58 children were assessed with a screening instrument for communication and language consisting of a professional assessment and a parents' questionnaire. Students of speech and language pathology, well trained in child language assessment, carried out the professional assessment, which was based on observations of play behaviour, interaction and expressive and receptive language skills. Of the 58 children, 43 attended a follow-up assessment of language skills at 54 mo of age. Nine children were considered to be at risk for language impairment at 18 mo and 10 children were evaluated as being at risk at 54 mo. A significant positive correlation was found between the professional evaluations at 18 mo and the language tests at 54 mo. Verbal comprehension and pretend play correlated significantly with the results on the language tests. A professional screening of communication and language at 18 mo of age is worthwhile for predicting problems in language development. The results further show that language comprehension and pretend play rather than expressive skills should be emphasized.

Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications

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John Ong

2018-03-01

Full Text Available Summary: Use of hepatocytes derived from induced pluripotent stem cells (i-Heps is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications. : Rashid and colleagues demonstrate the utility of a high-throughput imaging platform for identification of physiologically relevant extracellular niche factors to advance i-Heps closer to their primary tissue counterparts. The extracellular matrix (ECM protein screen identified Laminin 411 as an important niche factor facilitating i-Hep-based disease modeling in vitro. Keywords: iPS hepatocytes, extracellular niche, image-based screening, disease modeling, laminin

SDOCT imaging to identify macular pathology in patients diagnosed with diabetic maculopathy by a digital photographic retinal screening programme.

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Sarah Mackenzie

Full Text Available INTRODUCTION: Diabetic macular edema (DME is an important cause of vision loss. England has a national systematic photographic retinal screening programme to identify patients with diabetic eye disease. Grading retinal photographs according to this national protocol identifies surrogate markers for DME. We audited a care pathway using a spectral-domain optical coherence tomography (SDOCT clinic to identify macular pathology in this subset of patients. METHODS: A prospective audit was performed of patients referred from screening with mild to moderate non-proliferative diabetic retinopathy (R1 and surrogate markers for diabetic macular edema (M1 attending an SDOCT clinic. The SDOCT images were graded by an ophthalmologist as SDOCT positive, borderline or negative. SDOCT positive patients were referred to the medical retina clinic. SDOCT negative and borderline patients were further reviewed in the SDOCT clinic in 6 months. RESULTS: From a registered screening population of 17 551 patients with diabetes mellitus, 311 patients met the inclusion criteria between (March 2008 and September 2009. We analyzed images from 311 patients' SDOCT clinic episodes. There were 131 SDOCT negative and 12 borderline patients booked for revisit in the OCT clinic. Twenty-four were referred back to photographic screening for a variety of reasons. A total of 144 were referred to ophthalmology with OCT evidence of definite macular pathology requiring review by an ophthalmologist. DISCUSSION: This analysis shows that patients with diabetes, mild to moderate non-proliferative diabetic retinopathy (R1 and evidence of diabetic maculopathy on non-stereoscopic retinal photographs (M1 have a 42.1% chance of having no macular edema on SDOCT imaging as defined by standard OCT definitions of DME when graded by a retinal specialist. SDOCT imaging is a useful adjunct to colour fundus photography in screening for referable diabetic maculopathy in our screening population.

Is Telephone Screening Feasible? Accuracy and Cost-Effectiveness of Identifying People Medically Eligible for Home- And Community-Based Services.

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Fries, Brant E.; James, Mary; Hammer, Susan S.; Shugarman, Lisa R.; Morris, John N.

2004-01-01

Purpose: To determine the accuracy of a telephone-screening system to identify persons eligible for home- and community-based long-term care. Design and Methods: Data from Michigan telephone screens were compared to data from in-person assessments using the Minimum Data Set for Home Care (MDS-HC). Weighted kappa statistics measured the level of…

Best practices in identifying, reporting and screening operating experience at nuclear power plants

International Nuclear Information System (INIS)

2008-03-01

IAEA Safety Standards Series No. SF-1 entitled Fundamental Safety Principles: Safety Fundamentals states the need for operating organizations to establish a programme for the collection and analysis of operating experience in nuclear power plants. Such a programme ensures that operating experience is analysed, events important to safety are reviewed in depth, lessons learned are disseminated to the staff of the organization and to relevant national and international organizations, and corrective actions are effectively implemented. This publication has been developed to provide advice and assistance to nuclear installations, and related institutions including contractors and support organizations to strengthen and enhance their own feedback process through the implementation of best practices in identifying, reporting and screening processes and to assess the effectiveness of the above areas. To support a proactive safety management approach the nuclear installations are enhancing the operating experience feedback (OEF) processes. For this purpose, the nuclear industry is striving to collect more information on occurrences that are useful to address the early signs of declining performance and improve operational safety performance. In this environment a strong reporting culture that motivates people to identify and report issues is an important attribute. As a consequence, the number and diversity of issues identified increases, and there is a need to set thresholds of screening for further treatment. Thus, the establishment of an effective identification, reporting and screening process is very beneficial to streamline the efforts, and ensure that major incidents and latent weaknesses are being addressed and that operating experience is treated according to its significance. This leads to improved safety and production. This publication was written to complement the publication IAEA Services Series No. 10 - PROSPER Guidelines - Guidelines for Peer Review and for

The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: Potential and limitations

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Stéphanie eCottier

2011-12-01

Full Text Available Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx. In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA binding domain (encoded in the yeast strain, and the bioactive molecule part binding to its potential protein target fused to a DNA activating domain (encoded on a cDNA expression vector. During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discussed the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.

Screening with an NMNAT2-MSD platform identifies small molecules that modulate NMNAT2 levels in cortical neurons.

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Ali, Yousuf O; Bradley, Gillian; Lu, Hui-Chen

2017-03-07

Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a key neuronal maintenance factor and provides potent neuroprotection in numerous preclinical models of neurological disorders. NMNAT2 is significantly reduced in Alzheimer's, Huntington's, Parkinson's diseases. Here we developed a Meso Scale Discovery (MSD)-based screening platform to quantify endogenous NMNAT2 in cortical neurons. The high sensitivity and large dynamic range of this NMNAT2-MSD platform allowed us to screen the Sigma LOPAC library consisting of 1280 compounds. This library had a 2.89% hit rate, with 24 NMNAT2 positive and 13 negative modulators identified. Western analysis was conducted to validate and determine the dose-dependency of identified modulators. Caffeine, one identified NMNAT2 positive-modulator, when systemically administered restored NMNAT2 expression in rTg4510 tauopathy mice to normal levels. We confirmed in a cell culture model that four selected positive-modulators exerted NMNAT2-specific neuroprotection against vincristine-induced cell death while four selected NMNAT2 negative modulators reduced neuronal viability in an NMNAT2-dependent manner. Many of the identified NMNAT2 positive modulators are predicted to increase cAMP concentration, suggesting that neuronal NMNAT2 levels are tightly regulated by cAMP signaling. Taken together, our findings indicate that the NMNAT2-MSD platform provides a sensitive phenotypic screen to detect NMNAT2 in neurons.

High-Throughput Screening of Small Molecules Identifies Hepcidin Antagonists

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Fung, Eileen; Sugianto, Priscilla; Hsu, Jason; Damoiseaux, Robert; Ganz, Tomas

2013-01-01

Anemia of inflammation (AI) is common in patients with infection, autoimmune diseases, cancer, and chronic kidney disease. Unless the underlying condition can be reversed, treatment options are limited to erythropoiesis-stimulating agents with or without intravenous iron therapy, modalities that are not always effective and can cause serious adverse effects. Hepcidin, the iron regulatory hormone, has been identified as a pathogenic factor in the development of AI. To explore new therapeutic options for AI and other iron-related disorders caused by hepcidin excess, we developed a cell-based screen to identify hepcidin antagonists. Of the 70,000 small molecules in the library, we identified 14 compounds that antagonized the hepcidin effect on ferroportin. One of these was fursultiamine, a Food and Drug Administration (FDA)–approved thiamine derivative. Fursultiamine directly interfered with hepcidin binding to its receptor, ferroportin, by blocking ferroportin C326 thiol residue essential for hepcidin binding. Consequently, fursultiamine prevented hepcidin-induced ferroportin ubiquitination, endocytosis, and degradation in vitro and allowed continuous cellular iron export despite the presence of hepcidin, with IC50 in the submicromolar range. Thiamine, the fursultiamine metabolite, and benfotiamine, another thiamine derivative, did not interfere with the effect of hepcidin on ferroportin. Other FDA-approved thiol-reactive compounds were at least 1000-fold less potent than fursultiamine in antagonizing hepcidin. In vivo, fursultiamine did not reproducibly antagonize the effect of hepcidin on serum iron, likely because of its rapid conversion to inactive metabolites. Fursultiamine is a unique antagonist of hepcidin in vitro that could serve as a template for the development of drug candidates that inhibit the hepcidin-ferroportin interaction. PMID:23292796

An Investigation to Validate the Grammar and Phonology Screening (GAPS) Test to Identify Children with Specific Language Impairment

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van der Lely, Heather K. J.; Payne, Elisabeth; McClelland, Alastair

2011-01-01

Background The extraordinarily high incidence of grammatical language impairments in developmental disorders suggests that this uniquely human cognitive function is “fragile”. Yet our understanding of the neurobiology of grammatical impairments is limited. Furthermore, there is no “gold-standard” to identify grammatical impairments and routine screening is not undertaken. An accurate screening test to identify grammatical abilities would serve the research, health and education communities, further our understanding of developmental disorders, and identify children who need remediation, many of whom are currently un-diagnosed. A potential realistic screening tool that could be widely administered is the Grammar and Phonology Screening (GAPS) test – a 10 minute test that can be administered by professionals and non-professionals alike. Here we provide a further step in evaluating the validity and accuracy (sensitivity and specificity) of the GAPS test in identifying children who have Specific Language Impairment (SLI). Methods and Findings We tested three groups of children; two groups aged 3;6–6:6, a typically developing (n = 30) group, and a group diagnosed with SLI: (n = 11) (Young (Y)-SLI), and a further group aged 6;9–8;11 with SLI (Older (O)-SLI) (n = 10) who were above the test age norms. We employed a battery of language assessments including the GAPS test to assess the children's language abilities. For Y-SLI children, analyses revealed a sensitivity and specificity at the 5th and 10th percentile of 1.00 and 0.98, respectively, and for O-SLI children at the 10th and 15th percentile .83 and .90, respectively. Conclusions The findings reveal that the GAPS is highly accurate in identifying impaired vs. non-impaired children up to 6;8 years, and has moderate-to-high accuracy up to 9 years. The results indicate that GAPS is a realistic tool for the early identification of grammatical abilities and impairment in young children. A larger

Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure

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Nath, Anjali K.; Roberts, Lee D.; Liu, Yan; Mahon, Sari B.; Kim, Sonia; Ryu, Justine H.; Werdich, Andreas; Januzzi, James L.; Boss, Gerry R.; Rockwood, Gary A.; MacRae, Calum A.; Brenner, Matthew; Gerszten, Robert E.; Peterson, Randall T.

2013-01-01

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.—Nath, A. K., Roberts, L. D., Liu, Y., Mahon, S. B., Kim, S., Ryu, J. H., Werdich, A., Januzzi, J. L., Boss, G. R., Rockwood, G. A., MacRae, C. A., Brenner, M., Gerszten, R. E., Peterson, R. T. Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure. PMID:23345455

Gametocytocidal screen identifies novel chemical classes with Plasmodium falciparum transmission blocking activity.

Directory of Open Access Journals (Sweden)

Natalie G Sanders

Full Text Available Discovery of transmission blocking compounds is an important intervention strategy necessary to eliminate and eradicate malaria. To date only a small number of drugs that inhibit gametocyte development and thereby transmission from the mosquito to the human host exist. This limitation is largely due to a lack of screening assays easily adaptable to high throughput because of multiple incubation steps or the requirement for high gametocytemia. Here we report the discovery of new compounds with gametocytocidal activity using a simple and robust SYBR Green I- based DNA assay. Our assay utilizes the exflagellation step in male gametocytes and a background suppressor, which masks the staining of dead cells to achieve healthy signal to noise ratio by increasing signal of viable parasites and subtracting signal from dead parasites. By determining the contribution of exflagellation to fluorescent signal and using appropriate cutoff values, we were able to screen for gametocytocidal compounds. After assay validation and optimization, we screened an FDA approved drug library of approximately 1500 compounds, as well as the 400 compound MMV malaria box and identified 44 gametocytocidal compounds with sub to low micromolar IC50s. Major classes of compounds with gametocytocidal activity included quaternary ammonium compounds with structural similarity to choline, acridine-like compounds similar to quinacrine and pyronaridine, as well as antidepressant, antineoplastic, and anthelminthic compounds. Top drug candidates showed near complete transmission blocking in membrane feeding assays. This assay is simple, reproducible and demonstrated robust Z-factor values at low gametocytemia levels, making it amenable to HTS for identification of novel and potent gametocytocidal compounds.

Yeast functional screen to identify genes conferring salt stress tolerance in Salicornia europaea.

Science.gov (United States)

Nakahara, Yoshiki; Sawabe, Shogo; Kainuma, Kenta; Katsuhara, Maki; Shibasaka, Mineo; Suzuki, Masanori; Yamamoto, Kosuke; Oguri, Suguru; Sakamoto, Hikaru

2015-01-01

Salinity is a critical environmental factor that adversely affects crop productivity. Halophytes have evolved various mechanisms to adapt to saline environments. Salicornia europaea L. is one of the most salt-tolerant plant species. It does not have special salt-secreting structures like a salt gland or salt bladder, and is therefore a good model for studying the common mechanisms underlying plant salt tolerance. To identify candidate genes encoding key proteins in the mediation of salt tolerance in S. europaea, we performed a functional screen of a cDNA library in yeast. The library was screened for genes that allowed the yeast to grow in the presence of 1.3 M NaCl. We obtained three full-length S. europaea genes that confer salt tolerance. The genes are predicted to encode (1) a novel protein highly homologous to thaumatin-like proteins, (2) a novel coiled-coil protein of unknown function, and (3) a novel short peptide of 32 residues. Exogenous application of a synthetic peptide corresponding to the 32 residues improved salt tolerance of Arabidopsis. The approach described in this report provides a rapid assay system for large-scale screening of S. europaea genes involved in salt stress tolerance and supports the identification of genes responsible for such mechanisms. These genes may be useful candidates for improving crop salt tolerance by genetic transformation.

Yeast functional screen to identify genes conferring salt stress tolerance in Salicornia europaea

Directory of Open Access Journals (Sweden)

Yoshiki eNakahara

2015-10-01

Full Text Available Salinity is a critical environmental factor that adversely affects crop productivity. Halophytes have evolved various mechanisms to adapt to saline environments. Salicornia europaea L. is one of the most salt-tolerant plant species. It does not have special salt-secreting structures like a salt gland or salt bladder, and is therefore a good model for studying the common mechanisms underlying plant salt tolerance. To identify candidate genes encoding key proteins in the mediation of salt tolerance in S. europaea, we performed a functional screen of a cDNA library in yeast. The library was screened for genes that allowed the yeast to grow in the presence of 1.3 M NaCl. We obtained three full-length S. europaea genes that confer salt tolerance. The genes are predicted to encode (1 a novel protein highly homologous to thaumatin-like proteins, (2 a novel coiled-coil protein of unknown function, and (3 a novel short peptide of 32 residues. Exogenous application of a synthetic peptide corresponding to the 32 residues improved salt tolerance of Arabidopsis. The approach described in this report provides a rapid assay system for large-scale screening of S. europaea genes involved in salt stress tolerance and supports the identification of genes responsible for such mechanisms. These genes may be useful candidates for improving crop salt tolerance by genetic transformation.

Nuclear magnetic resonance study of interaction of ligands with Streptococcus faecium dihydrofolate reductase labeled with [#betta#-13C]tryptophan

International Nuclear Information System (INIS)

London, R.E.; Groff, J.P.; Cocco, L.; Blakley, R.L.

1982-01-01

Dihydrofolate reductase from Streptococcus faecium has been labeled with [#betta#- 13 C]tryptophan. We have determined changes occurring in the chemical shifts and line widths of the four resonances of the 13 C NMR spectrum of the labeled enzyme, due to its interaction with various ligands. These include the coenzyme, NPDPH and related nucleotides, folate and its polyglutamate derivatives, and many inhibitors including methotrexate and trimethoprim. In addition, paramagnetic relaxation effects produced by a bound spin-labeled analogue of 2'-phosphoadenosine-5'-diphosphoribose on the tryptophan C/sup #betta#/ carbons have been measured. Distances calculated from the relaxation data have been compared with corresponding distances in the crystallographic model of the NADPH-methotrexate ternary complex of Lactobacillus casei reductase. The paramagnetic relaxation data indicate that the two downfield resonances (1 and 2) correspond to tryptophans (W/sub A/ and W/sub B/) that are more remote from the catalytic site, and from the crystallographic model these are seen to be Trp-115 and Trp-160. The upfield resonances (3 and 4) that show broadening due to chemical exchange correspond to closer residues (W/sub C/ and W/sub D/), and these are identified with Trp-6 and Trp-22. However, the relaxation data do not permit specific assignments within the nearer and farther pairs. Although resonance 3, which is split due to chemical exchange, was formerly assigned to Trp-6, data obtained for the enzyme in the presence of various ligands are better interpreted if resonance 3 is assigned to Trp-22, which is located on a loop that joins elements of secondary structure and forms one side of the ligand-binding cavity

A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein.

Directory of Open Access Journals (Sweden)

Michael S Rogers

Full Text Available Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA, a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2 protein and tumor endothelial marker 8 (TEM8. Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent

DEFF Research Database (Denmark)

Alifrangis, Michael; Enosse, Sonia; Pearce, Richard

2005-01-01

Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine....... However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA...

Permanent Childhood Hearing Impairment: Aetiological Evaluation of Infants identified through the Irish Newborn Hearing Screening Programme

LENUS (Irish Health Repository)

Smith, A

2017-11-01

The Newborn Hearing Screening Programme (NHSP) was established in Cork University Maternity Hospital (CUMH) in April 2011. Between April 2011 and July 2014, 42 infants were identified with a Permanent Childhood Hearing Impairment (PCHI). Following this diagnosis, infants underwent a paediatric assessment according to recognised guidelines with the intention of identifying the underlying aetiology of the PCHI. The aim of this study was to assess the findings of this aetiological workup via retrospective chart review. PCHI data was obtained from the eSP database. This is a web based information system (eSP) used to track each baby through the screening and referral process A retrospective chart review of these patients was performed. Sixteen (38%) infants were diagnosed with a bilateral sensorineural hearing loss. Two infants had congenital CMV infection. A Connexin 26 gene mutation was detected in one infant. Two infants were diagnosed with Waardenburg syndrome, One with Pendred syndrome and one with Pfeiffer syndrome. Five babies underwent cochlear implantation. Through adherence to the recommended protocol a possible cause of PCHI may be determined. This study has identified areas of future improvement for this service in Ireland.

High-Throughput Screening Using iPSC-Derived Neuronal Progenitors to Identify Compounds Counteracting Epigenetic Gene Silencing in Fragile X Syndrome.

Science.gov (United States)

Kaufmann, Markus; Schuffenhauer, Ansgar; Fruh, Isabelle; Klein, Jessica; Thiemeyer, Anke; Rigo, Pierre; Gomez-Mancilla, Baltazar; Heidinger-Millot, Valerie; Bouwmeester, Tewis; Schopfer, Ulrich; Mueller, Matthias; Fodor, Barna D; Cobos-Correa, Amanda

2015-10-01

Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused in most of cases by epigenetic silencing of the Fmr1 gene. Today, no specific therapy exists for FXS, and current treatments are only directed to improve behavioral symptoms. Neuronal progenitors derived from FXS patient induced pluripotent stem cells (iPSCs) represent a unique model to study the disease and develop assays for large-scale drug discovery screens since they conserve the Fmr1 gene silenced within the disease context. We have established a high-content imaging assay to run a large-scale phenotypic screen aimed to identify compounds that reactivate the silenced Fmr1 gene. A set of 50,000 compounds was tested, including modulators of several epigenetic targets. We describe an integrated drug discovery model comprising iPSC generation, culture scale-up, and quality control and screening with a very sensitive high-content imaging assay assisted by single-cell image analysis and multiparametric data analysis based on machine learning algorithms. The screening identified several compounds that induced a weak expression of fragile X mental retardation protein (FMRP) and thus sets the basis for further large-scale screens to find candidate drugs or targets tackling the underlying mechanism of FXS with potential for therapeutic intervention. © 2015 Society for Laboratory Automation and Screening.

A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis

Directory of Open Access Journals (Sweden)

Mala Misra

2016-08-01

Full Text Available Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating the asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types, and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system, to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins, and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling.

A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis

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Senthilkumar Deivasigamani

2014-10-01

Full Text Available Amyotrophic Lateral Sclerosis (ALS is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5–10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions. We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci – TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2 that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt and VAP(P58S, but in a contrasting manner. Reversal of VAP(P58S bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S expression. The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease.

A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis.

Science.gov (United States)

Deivasigamani, Senthilkumar; Verma, Hemant Kumar; Ueda, Ryu; Ratnaparkhi, Anuradha; Ratnaparkhi, Girish S

2014-10-31

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5-10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions. We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci - TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression. The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease. © 2014. Published by The Company of Biologists Ltd.

Development and validation of a screening procedure to identify speech-language delay in toddlers with cleft palate

DEFF Research Database (Denmark)

Jørgensen, Line Dahl; Willadsen, Elisabeth

2017-01-01

condition based on assessment of consonant inventory using a real-time listening procedure in combination with parent-reported expressive vocabulary. These measures allowed evaluation of early speech-language skills found to correlate significantly with later speech-language difficulties in longitudinal......The purpose of this study was to develop and validate a clinically useful speech-language screening procedure for young children with cleft palate +/- cleft lip (CP) to identify those in need of speech-language intervention. Twenty-two children with CP were assigned to a +/- need for intervention...... studies of children with CP. The external validity of this screening procedure was evaluated by comparing the +/- need for intervention assignment determined by the screening procedure to experienced speech-language pathologists’ (SLPs’) clinical judgment of whether or not a child needed early...

Effect of gamma rays at the dihydrofolate reductase locus: deletions and inversions

International Nuclear Information System (INIS)

Urlaub, G.; Mitchell, P.J.; Kas, E.; Chasin, L.A.; Funanage, V.L.; Myoda, T.T.; Hamlin, J.

1986-01-01

A series 11 gamma-ray-induced mutants at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells has been examined for the types of DNA sequence change brought about by this form of ionizing radiation. All 11 mutants were found to have suffered major structural changes affecting the dhfr gene. In eight of the mutants, all or part of the dhfr gene has been deleted. The extent of these deletions was examined in seven of these mutants and, for comparison, in two deletion mutants that were induced by UV irradiation. For this purpose, probes from an overlapping set of cosmids that span 210 kb of DNA in this region were used. Three of seven gamma-ray-induced mutants and one UV-induced mutant were shown to have deleted the entire 210-kb region. In the remaining mutants, endpoints ranging from within the dhfr gene to 100 kb downstream were observed. No upstream endpoints were detected, so that an upper limit on the size of these large deletions could not be assigned. Three of the 11 gamma-ray-induced mutants contained an interruption in the dhfr gene without any detectable loss of sequence. Restriction analysis of these interrupted mutants showed that at least 8-14 kb of foreign DNA sequence became joined to the gene at the point of disruption. Cytogenetic analysis of these mutants showed that in two cases an inversion of the banding pattern on chromosome Z-2 had taken place. The inverted dhfr mutants contain very low amounts of dhfr RNA sequences, and the 5' end of an inversion mutant gene exhibits the same pattern of DNA methylation and DNase I-hypersensitivity as the wild-type gene. Our results suggest that ionizing radiation causes primarily, if not exclusively, large deletions and inversions in mammalian cells

Drug discovery for schistosomiasis: hit and lead compounds identified in a library of known drugs by medium-throughput phenotypic screening.

Directory of Open Access Journals (Sweden)

Maha-Hamadien Abdulla

2009-07-01

Full Text Available Praziquantel (PZQ is the only widely available drug to treat schistosomiasis. Given the potential for drug resistance, it is prudent to search for novel therapeutics. Identification of anti-schistosomal chemicals has traditionally relied on phenotypic (whole organism screening with adult worms in vitro and/or animal models of disease-tools that limit automation and throughput with modern microtiter plate-formatted compound libraries.A partially automated, three-component phenotypic screen workflow is presented that utilizes at its apex the schistosomular stage of the parasite adapted to a 96-well plate format with a throughput of 640 compounds per month. Hits that arise are subsequently screened in vitro against adult parasites and finally for efficacy in a murine model of disease. Two GO/NO GO criteria filters in the workflow prioritize hit compounds for tests in the animal disease model in accordance with a target drug profile that demands short-course oral therapy. The screen workflow was inaugurated with 2,160 chemically diverse natural and synthetic compounds, of which 821 are drugs already approved for human use. This affords a unique starting point to 'reposition' (re-profile drugs as anti-schistosomals with potential savings in development timelines and costs.Multiple and dynamic phenotypes could be categorized for schistosomula and adults in vitro, and a diverse set of 'hit' drugs and chemistries were identified, including anti-schistosomals, anthelmintics, antibiotics, and neuromodulators. Of those hits prioritized for tests in the animal disease model, a number of leads were identified, one of which compares reasonably well with PZQ in significantly decreasing worm and egg burdens, and disease-associated pathology. Data arising from the three components of the screen are posted online as a community resource.To accelerate the identification of novel anti-schistosomals, we have developed a partially automated screen workflow that

Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate

Energy Technology Data Exchange (ETDEWEB)

Senkovich, Olga; Schormann, Norbert; Chattopadhyay, Debasish; (UAB)

2010-11-22

The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50 000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure-function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of

Description of 15 DNA-positive and antibody-negative "window-period" blood donations identified during prospective screening for Babesia microti.

Science.gov (United States)

Moritz, Erin D; Tonnetti, Laura; Hewins, Mary Ellen; Berardi, Victor P; Dodd, Roger Y; Stramer, Susan L

2017-07-01

Blood donation screening detecting only antibodies fails to identify donors in the earliest stage of infection, before a detectable immunologic response, that is, the "window period" (WP). We present data on WP donations identified during prospective screening for Babesia microti, a transfusion-transmissible parasite of increasing concern in the United States. Blood donations collected in Connecticut, Massachusetts, Minnesota, and Wisconsin were screened using polymerase chain reaction (PCR) and arrayed fluorescence immunoassay (AFIA) to detect B. microti DNA and antibodies, respectively. Parasite loads were estimated using quantitative PCR. Red blood cell (RBC) samples were inoculated into hamsters to assess infectivity. Donors screening reactive were indefinitely deferred, tested by supplemental methods, and followed to assess DNA and antibody clearance. Demographic data from WP donors (i.e., those screening PCR positive and AFIA negative) were compared to data from other positive donors. Of 220,479 donations screened from June 2012 to August 2016, a total of 700 were positive, of which 15 (2% of positive donations or 1 per 14,699 screened donations) were confirmed WP donations. The median estimated parasite load in WP donations was 350 parasites/mL, no different than AFIA-positive and PCR-positive donors. Parasite loads in RBC samples from WP units ranged from 14 to 11,022 parasites/mL; RBC samples from three of 10 (30%) WP donations infected hamsters. The mean age of WP donors was 48 years (range, 17-75 years); three (20%) were female. WP donor demographics did not differ significantly from demographics of other donors. We report one per 15,000 B. microti WP infections in blood donors in endemic areas, demonstrating the importance of nucleic acid testing to mitigate the risk of transfusion-transmitted babesiosis. © 2017 AABB.

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

Science.gov (United States)

Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B.; Kim, Jung-Hyun; Ang, J. Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P.; Andrews, Brenda; Boerkoel, Cornelius F.; Hieter, Philip

2016-01-01

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

A senescence rescue screen identifies BCL6 as an inhibitor of anti-proliferative p19ARF-p53 signaling

NARCIS (Netherlands)

Shvarts, A.; Brummelkamp, T.; Koh, E.; Daley, G.Q.; Bernards, R.A.

2002-01-01

Senescence limits the proliferative capacity of primary cells in culture. We describe here a genetic screen to identify genes that allow bypass of this checkpoint. Using retroviral cDNA expression libraries, we identify BCL6 as a potent inhibitor of senescence. BCL6 is frequently activated in

A senescence rescue screen identifies BCL6 as an inhibitor of anti-proliferative p19(ARF)-p53 signaling

NARCIS (Netherlands)

Shvarts, Avi; Brummelkamp, Thijn R.; Scheeren, Ferenc; Koh, Eugene; Daley, George Q.; Spits, Hergen; Bernards, René

2002-01-01

Senescence limits the proliferative capacity of primary cells in culture. We describe here a genetic screen to identify genes that allow bypass of this checkpoint. Using retroviral cDNA expression libraries, we identify BCL6 as a potent inhibitor of senescence. BCL6 is frequently activated in

Nickel-resistance determinants in Acidiphilium sp. PM identified by genome-wide functional screening.

Directory of Open Access Journals (Sweden)

Patxi San Martin-Uriz

Full Text Available Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metal-resistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY. This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Ni-resistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms.

Genes Important for Schizosaccharomyces pombe Meiosis Identified Through a Functional Genomics Screen

Science.gov (United States)

Blyth, Julie; Makrantoni, Vasso; Barton, Rachael E.; Spanos, Christos; Rappsilber, Juri; Marston, Adele L.

2018-01-01

Meiosis is a specialized cell division that generates gametes, such as eggs and sperm. Errors in meiosis result in miscarriages and are the leading cause of birth defects; however, the molecular origins of these defects remain unknown. Studies in model organisms are beginning to identify the genes and pathways important for meiosis, but the parts list is still poorly defined. Here we present a comprehensive catalog of genes important for meiosis in the fission yeast, Schizosaccharomyces pombe. Our genome-wide functional screen surveyed all nonessential genes for roles in chromosome segregation and spore formation. Novel genes important at distinct stages of the meiotic chromosome segregation and differentiation program were identified. Preliminary characterization implicated three of these genes in centrosome/spindle pole body, centromere, and cohesion function. Our findings represent a near-complete parts list of genes important for meiosis in fission yeast, providing a valuable resource to advance our molecular understanding of meiosis. PMID:29259000

An open-access endoscopy screen correctly and safely identifies patients for conscious sedation.

Science.gov (United States)

Kothari, Darshan; Feuerstein, Joseph D; Moss, Laureen; D'Souza, Julie; Montanaro, Kerri; Leffler, Daniel A; Sheth, Sunil G

2016-11-01

Open-access scheduling is highly utilized for facilitating generally low-risk endoscopies. Preprocedural screening addresses sedation requirements; however, procedural safety may be compromised if screening is inaccurate. We sought to determine the reliability of our open-access scheduling system for appropriate use of conscious sedation. We prospectively and consecutively enrolled outpatient procedures booked at an academic center by open-access using screening after in-office gastroenterology (GI) consultation. We collected the cases inappropriately booked for conscious sedation and compared the characteristics for significant differences. A total of 8063 outpatients were scheduled for procedures with conscious sedation, and 5959 were booked with open-access. Only 78 patients (0.97%, 78/8063) were identified as subsequently needing anesthesiologist-assisted sedation; 44 (56.4%, 44/78) were booked through open-access, of which chronic opioid (47.7%, 21/44) or benzodiazepine use (34.1%, 15/44) were the most common reasons for needing anesthesiologist-assisted sedation. Patients on chronic benzodiazepines required more midazolam than those not on chronic benzodiazepines (P = .03) of those patients who underwent conscious sedation. Similarly, patients with chronic opioid use required more fentanyl than those without chronic opioid use (P = .04). Advanced liver disease and alcohol use were common reasons for patients being booked after in-office consultation and were significantly higher than those booked with open-access (both P open-access scheduling. © The Author(s) 2016. Published by Oxford University Press and Sixth Affiliated Hospital of Sun Yat-Sen University.

Direct 19F NMR observation of the conformational selection of optically active rotamers of the antifolate compound fluoronitropyrimethamine bound to enzyme dihydrofolate reductase

International Nuclear Information System (INIS)

Tendler, S.J.B.; Birdsall, B.; Feeney, J.; Griffin, R.J.; Stevens, M.F.G.; Roberts, G.C.K.

1988-01-01

The molucular basis of the binding of the lipophilic antifolate compound fluoronitropyrimethamine to its target enzyme dihydrofolate reductase has been investigated using a combination of 19 F NMR spectroscopy and molecular mechanical calculations 19 F NMR reveals the presence of two different conformational states for the fluoronitropyrimethamine-Lactobacillus casei enzyme complex. MM2 molecular mechanical calculations predict restricted rotation about the C5-C1 bond of the ligand and this give rise to two slowly interconverting rotamers which are an enantiomeric pair. The results of 19 F NMR spectroscopy reveal that both these isomers bind to the enzyme, with different affinities. There is no detectable interconversion of the bound rotamers themselves on the NMR timescale. The effect of the addition of co-enzyme to the sample is to reverse the preference the enzyme has for each rotamer. (author). 11 refs.; 3 figs

Inter-rater reliability and stability of diagnoses of autism spectrum disorder in children identified through screening at a very young age

NARCIS (Netherlands)

van Daalen, Emma; Kemner, Chantal; Dietz, Claudine; Swinkels, Sophie H. N.; Buitelaar, Jan K.; van Engeland, Herman

2009-01-01

To examine the inter-rater reliability and stability of autism spectrum disorder (ASD) diagnoses made at a very early age in children identified through a screening procedure around 14 months of age. In a prospective design, preschoolers were recruited from a screening study for ASD. The inter-rater

Inter-rater reliability and stability of diagnoses of autism spectrum disorder in children identified through screening at a very young age.

NARCIS (Netherlands)

Daalen, E. van; Kemner, C.; Dietz, C.; Swinkels, S.H.N.; Buitelaar, J.K.; Engeland, H.M. van

2009-01-01

To examine the inter-rater reliability and stability of autism spectrum disorder (ASD) diagnoses made at a very early age in children identified through a screening procedure around 14 months of age. In a prospective design, preschoolers were recruited from a screening study for ASD. The inter-rater

CT screening for lung cancer: Frequency of enlarged adrenal glands identified in baseline and annual repeat rounds

Energy Technology Data Exchange (ETDEWEB)

Hu, Minxia [Icahn School of Medicine at Mount Sinai, Department of Radiology, New York, NY (United States); Capital Medical University, Department of Diagnostic Ultrasound, Beijing Tongren Hospital, Beijing (China); Yip, Rowena; Yankelevitz, David Y.; Henschke, Claudia I. [Icahn School of Medicine at Mount Sinai, Department of Radiology, New York, NY (United States)

2016-12-15

To determine the frequency of adrenal enlargement of participants in a CT-screening program for lung cancer and demonstrate the progression during follow-up, separately for baseline and annual repeat rounds. HIPAA-compliant informed consent was obtained in 4,776 participants. The adrenal gland was defined as enlarged if it measured ≥6 mm at its largest diameter. Logistic regression analyses were performed. At baseline, 202 (4 %) of 4,776 participants had adrenal enlargement. Significant factors were age (OR = 1.4, 95 % CI: 1.2-1.7) and current smoker (OR = 1.8, 95 % CI: 1.3-2.4). Follow-up 7-18 months after baseline for 133 cases with adrenal enlargement <40 mm showed it decreased or was stable in 85 (64 %), and increased by <10 mm in 48 (36 %). Five (0.04 %) cases of adrenal enlargement were newly identified, none increased beyond 40 mm on follow-up. Adrenal enlargement was a significant predictor of a subsequent diagnosis of lung cancer (OR = 2.0, 95 % CI: 1.2-3.4). Participants with adrenal enlargement <40 mm identified at baseline and on repeat screening could be reasonably assessed on subsequent annual screening. Adrenal enlargement increased with increasing pack-years of smoking. Adrenal enlargement was an independent predictor of a subsequent diagnosis of lung cancer. (orig.)

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

DEFF Research Database (Denmark)

Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.

2015-01-01

There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing...... interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV...... mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330...

A genome-scale RNA-interference screen identifies RRAS signaling as a pathologic feature of Huntington's disease.

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John P Miller

Full Text Available A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington's disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington's disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington's disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington's disease.

Novel immune-modulator identified by a rapid, functional screen of the parapoxvirus ovis (Orf virus genome

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McGuire Michael J

2012-01-01

Full Text Available Abstract Background The success of new sequencing technologies and informatic methods for identifying genes has made establishing gene product function a critical rate limiting step in progressing the molecular sciences. We present a method to functionally mine genomes for useful activities in vivo, using an unusual property of a member of the poxvirus family to demonstrate this screening approach. Results The genome of Parapoxvirus ovis (Orf virus was sequenced, annotated, and then used to PCR-amplify its open-reading-frames. Employing a cloning-independent protocol, a viral expression-library was rapidly built and arrayed into sub-library pools. These were directly delivered into mice as expressible cassettes and assayed for an immune-modulating activity associated with parapoxvirus infection. The product of the B2L gene, a homolog of vaccinia F13L, was identified as the factor eliciting immune cell accumulation at sites of skin inoculation. Administration of purified B2 protein also elicited immune cell accumulation activity, and additionally was found to serve as an adjuvant for antigen-specific responses. Co-delivery of the B2L gene with an influenza gene-vaccine significantly improved protection in mice. Furthermore, delivery of the B2L expression construct, without antigen, non-specifically reduced tumor growth in murine models of cancer. Conclusion A streamlined, functional approach to genome-wide screening of a biological activity in vivo is presented. Its application to screening in mice for an immune activity elicited by the pathogen genome of Parapoxvirus ovis yielded a novel immunomodulator. In this inverted discovery method, it was possible to identify the adjuvant responsible for a function of interest prior to a mechanistic study of the adjuvant. The non-specific immune activity of this modulator, B2, is similar to that associated with administration of inactivated particles to a host or to a live viral infection. Administration

Identifiability in N-mixture models: a large-scale screening test with bird data.

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Kéry, Marc

2018-02-01

Binomial N-mixture models have proven very useful in ecology, conservation, and monitoring: they allow estimation and modeling of abundance separately from detection probability using simple counts. Recently, doubts about parameter identifiability have been voiced. I conducted a large-scale screening test with 137 bird data sets from 2,037 sites. I found virtually no identifiability problems for Poisson and zero-inflated Poisson (ZIP) binomial N-mixture models, but negative-binomial (NB) models had problems in 25% of all data sets. The corresponding multinomial N-mixture models had no problems. Parameter estimates under Poisson and ZIP binomial and multinomial N-mixture models were extremely similar. Identifiability problems became a little more frequent with smaller sample sizes (267 and 50 sites), but were unaffected by whether the models did or did not include covariates. Hence, binomial N-mixture model parameters with Poisson and ZIP mixtures typically appeared identifiable. In contrast, NB mixtures were often unidentifiable, which is worrying since these were often selected by Akaike's information criterion. Identifiability of binomial N-mixture models should always be checked. If problems are found, simpler models, integrated models that combine different observation models or the use of external information via informative priors or penalized likelihoods, may help. © 2017 by the Ecological Society of America.

Molecular epidemiology of malaria in Cameroon. XXX. sequence analysis of Plasmodium falciparum ATPase 6, dihydrofolate reductase, and dihydropteroate synthase resistance markers in clinical isolates from children treated with an artesunate-sulfadoxine-pyrimethamine combination.

Science.gov (United States)

Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida

2011-07-01

Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance.

Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion.

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Oyen, David; Fenwick, R Bryn; Aoto, Phillip C; Stanfield, Robyn L; Wilson, Ian A; Dyson, H Jane; Wright, Peter E

2017-08-16

The rate-determining step in the catalytic cycle of E. coli dihydrofolate reductase is tetrahydrofolate (THF) product release, which can occur via an allosteric or an intrinsic pathway. The allosteric pathway, which becomes accessible when the reduced cofactor NADPH is bound, involves transient sampling of a higher energy conformational state, greatly increasing the product dissociation rate as compared to the intrinsic pathway that obtains when NADPH is absent. Although the kinetics of this process are known, the enzyme structure and the THF product conformation in the transiently formed excited state remain elusive. Here, we use side-chain proton NMR relaxation dispersion measurements, X-ray crystallography, and structure-based chemical shift predictions to explore the structural basis of allosteric product release. In the excited state of the E:THF:NADPH product release complex, the reduced nicotinamide ring of the cofactor transiently enters the active site where it displaces the pterin ring of the THF product. The p-aminobenzoyl-l-glutamate tail of THF remains weakly bound in a widened binding cleft. Thus, through transient entry of the nicotinamide ring into the active site, the NADPH cofactor remodels the enzyme structure and the conformation of the THF to form a weakly populated excited state that is poised for rapid product release.

Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour

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Fokkelman, Michiel; Balcıoğlu, Hayri E.; Klip, Janna E.; Yan, Kuan; Verbeek, Fons J.; Danen, Erik H. J.; van de Water, Bob

2016-01-01

Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour. PMID:27531518

A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

Science.gov (United States)

Bond, David; Foley, Edan

2009-01-01

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling. PMID:19893628

Mid-upper arm circumference as a screening tool for identifying children with obesity: a 12-country study.

Science.gov (United States)

Chaput, J-P; Katzmarzyk, P T; Barnes, J D; Fogelholm, M; Hu, G; Kuriyan, R; Kurpad, A; Lambert, E V; Maher, C; Maia, J; Matsudo, V; Olds, T; Onywera, V; Sarmiento, O L; Standage, M; Tudor-Locke, C; Zhao, P; Tremblay, M S

2017-12-01

No studies have examined if mid-upper arm circumference (MUAC) can be an alternative screening tool for obesity in an international sample of children differing widely in levels of human development. Our aim is to determine whether MUAC could be used to identify obesity in children from 12 countries in five major geographic regions of the world. This observational, multinational cross-sectional study included 7337 children aged 9-11 years. Anthropometric measurements were objectively assessed, and obesity was defined according to the World Health Organization reference data. In the total sample, MUAC was strongly correlated with adiposity indicators in both boys and girls (r > 0.86, p obesity was high in both sexes and across study sites (overall area under the curve of 0.97, sensitivity of 95% and specificity of 90%). The MUAC cut-off value to identify obesity was ~25 cm for both boys and girls. In country-specific analyses, the cut-off value to identify obesity ranged from 23.2 cm (boys in South Africa) to 26.2 cm (girls in the UK). Results from this 12-country study suggest that MUAC is a simple and accurate measurement that may be used to identify obesity in children aged 9-11 years. MUAC may be a promising screening tool for obesity in resource-limited settings. © 2016 World Obesity Federation.

A Morpholino-based screen to identify novel genes involved in craniofacial morphogenesis

Science.gov (United States)

Melvin, Vida Senkus; Feng, Weiguo; Hernandez-Lagunas, Laura; Artinger, Kristin Bruk; Williams, Trevor

2014-01-01

BACKGROUND The regulatory mechanisms underpinning facial development are conserved between diverse species. Therefore, results from model systems provide insight into the genetic causes of human craniofacial defects. Previously, we generated a comprehensive dataset examining gene expression during development and fusion of the mouse facial prominences. Here, we used this resource to identify genes that have dynamic expression patterns in the facial prominences, but for which only limited information exists concerning developmental function. RESULTS This set of ~80 genes was used for a high throughput functional analysis in the zebrafish system using Morpholino gene knockdown technology. This screen revealed three classes of cranial cartilage phenotypes depending upon whether knockdown of the gene affected the neurocranium, viscerocranium, or both. The targeted genes that produced consistent phenotypes encoded proteins linked to transcription (meis1, meis2a, tshz2, vgll4l), signaling (pkdcc, vlk, macc1, wu:fb16h09), and extracellular matrix function (smoc2). The majority of these phenotypes were not altered by reduction of p53 levels, demonstrating that both p53 dependent and independent mechanisms were involved in the craniofacial abnormalities. CONCLUSIONS This Morpholino-based screen highlights new genes involved in development of the zebrafish craniofacial skeleton with wider relevance to formation of the face in other species, particularly mouse and human. PMID:23559552

Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease

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Taddei Kevin

2010-01-01

Full Text Available Abstract Background The low-density lipoprotein receptor related protein 1 (LRP1 has been implicated in Alzheimer's disease (AD but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1. Results We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP, which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b. Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression. Conclusions These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered

A genetic screen in Myxococcus xanthus identifies mutants that uncouple outer membrane exchange from a downstream cellular response.

Science.gov (United States)

Dey, Arup; Wall, Daniel

2014-12-01

Upon physical contact with sibling cells, myxobacteria transiently fuse their outer membranes (OMs) and exchange OM proteins and lipids. From previous work, TraA and TraB were identified to be essential factors for OM exchange (OME) in donor and recipient cells. To define the genetic complexity of OME, we carried out a comprehensive forward genetic screen. The screen was based on the observation that Myxococcus xanthus nonmotile cells, by a Tra-dependent mechanism, block swarm expansion of motile cells when mixed. Thus, mutants defective in OME or a downstream responsive pathway were readily identified as escape flares from mixed inocula seeded on agar. This screen was surprisingly powerful, as we found >50 mutants defective in OME. Importantly, all of the mutations mapped to the traAB operon, suggesting that there may be few, if any, proteins besides TraA and TraB directly required for OME. We also found a second and phenotypically different class of mutants that exhibited wild-type OME but were defective in a responsive pathway. This pathway is postulated to control inner membrane homeostasis by covalently attaching amino acids to phospholipids. The identified proteins are homologous to the Staphylococcus aureus MprF protein, which is involved in membrane adaptation and antibiotic resistance. Interestingly, we also found that a small number of nonmotile cells were sufficient to block the swarming behavior of a large gliding-proficient population. This result suggests that an OME-derived signal could be amplified from a few nonmotile producers to act on many responder cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

Science.gov (United States)

Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

2015-07-28

West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis.

Science.gov (United States)

Meeske, Alexander J; Rodrigues, Christopher D A; Brady, Jacqueline; Lim, Hoong Chuin; Bernhardt, Thomas G; Rudner, David Z

2016-01-01

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell-cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis

Science.gov (United States)

Brady, Jacqueline; Lim, Hoong Chuin; Bernhardt, Thomas G.; Rudner, David Z.

2016-01-01

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell–cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes. PMID:26735940

Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

DEFF Research Database (Denmark)

Wittig-Blaich, Stephanie; Wittig, Rainer; Schmidt, Steffen

2017-01-01

Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technolo......Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few...... technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes......, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential...

Two crystal structures of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis reveal protein–ligand interactions including a structural basis for observed antifolate resistance

Energy Technology Data Exchange (ETDEWEB)

Anderson, Amy C., E-mail: aca@dartmouth.edu [Dartmouth College, Department of Chemistry, Burke Laboratories, Hanover, NH 03755 (United States)

2005-03-01

An analysis of the protein–ligand interactions in two crystal structures of DHFR-TS from C. hominis reveals a possible structural basis for observed antifolate resistance in C. hominis DHFR. A comparison with the structure of human DHFR reveals residue substitutions that may be exploited for the design of species-selective inhibitors. Cryptosporidium hominis is a protozoan parasite that causes acute gastrointestinal illness. There are no effective therapies for cryptosporidiosis, highlighting the need for new drug-lead discovery. An analysis of the protein–ligand interactions in two crystal structures of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from C. hominis, determined at 2.8 and 2.87 Å resolution, reveals that the interactions of residues Ile29, Thr58 and Cys113 in the active site of C. hominis DHFR provide a possible structural basis for the observed antifolate resistance. A comparison with the structure of human DHFR reveals active-site differences that may be exploited for the design of species-selective inhibitors.

A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance

DEFF Research Database (Denmark)

Menzel, Tobias; Nähse-Kumpf, Viola; Kousholt, Arne Nedergaard

2011-01-01

To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2......, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main...

Histology Verification Demonstrates That Biospectroscopy Analysis of Cervical Cytology Identifies Underlying Disease More Accurately than Conventional Screening: Removing the Confounder of Discordance

Science.gov (United States)

Gajjar, Ketan; Ahmadzai, Abdullah A.; Valasoulis, George; Trevisan, Júlio; Founta, Christina; Nasioutziki, Maria; Loufopoulos, Aristotelis; Kyrgiou, Maria; Stasinou, Sofia Melina; Karakitsos, Petros; Paraskevaidis, Evangelos; Da Gama-Rose, Bianca; Martin-Hirsch, Pierre L.; Martin, Francis L.

2014-01-01

Background Subjective visual assessment of cervical cytology is flawed, and this can manifest itself by inter- and intra-observer variability resulting ultimately in the degree of discordance in the grading categorisation of samples in screening vs. representative histology. Biospectroscopy methods have been suggested as sensor-based tools that can deliver objective assessments of cytology. However, studies to date have been apparently flawed by a corresponding lack of diagnostic efficiency when samples have previously been classed using cytology screening. This raises the question as to whether categorisation of cervical cytology based on imperfect conventional screening reduces the diagnostic accuracy of biospectroscopy approaches; are these latter methods more accurate and diagnose underlying disease? The purpose of this study was to compare the objective accuracy of infrared (IR) spectroscopy of cervical cytology samples using conventional cytology vs. histology-based categorisation. Methods Within a typical clinical setting, a total of n = 322 liquid-based cytology samples were collected immediately before biopsy. Of these, it was possible to acquire subsequent histology for n = 154. Cytology samples were categorised according to conventional screening methods and subsequently interrogated employing attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. IR spectra were pre-processed and analysed using linear discriminant analysis. Dunn’s test was applied to identify the differences in spectra. Within the diagnostic categories, histology allowed us to determine the comparative efficiency of conventional screening vs. biospectroscopy to correctly identify either true atypia or underlying disease. Results Conventional cytology-based screening results in poor sensitivity and specificity. IR spectra derived from cervical cytology do not appear to discriminate in a diagnostic fashion when categories were based on conventional screening

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

Directory of Open Access Journals (Sweden)

Anna C Llewellyn

Full Text Available Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI, validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and

The usefulness and feasibility of a screening instrument to identify psychosocial problems in patients receiving curative radiotherapy: a process evaluation

International Nuclear Information System (INIS)

Braeken, Anna PBM; Kempen, Gertrudis IJM; Eekers, Daniëlle; Gils, Francis CJM van; Houben, Ruud MA; Lechner, Lilian

2011-01-01

Psychosocial problems in cancer patients are often unrecognized and untreated due to the low awareness of the existence of these problems or pressures of time. The awareness of the need to identify psychosocial problems in cancer patients is growing and has affected the development of screening instruments. This study explored the usefulness and feasibility of using a screening instrument (SIPP: Screening Inventory of Psychosocial Problems) to identify psychosocial problems in cancer patients receiving curative radiotherapy treatment (RT). The study was conducted in a radiation oncology department in the Netherlands. Several methods were used to document the usefulness and feasibility of the SIPP. Data were collected using self-report questionnaires completed by seven radiotherapists and 268 cancer patients. Regarding the screening procedure 33 patients were offered to consult a psychosocial care provider (e.g. social worker, psychologist) during the first consultation with their radiotherapist. Of these patients, 31 patients suffered from at least sub-clinical symptoms and two patients hardly suffered from any symptoms. Patients' acceptance rate 63.6% (21/33) was high. Patients were positive about the content of the SIPP (mean scores vary from 8.00 to 8.88, out of a range between 0 and 10) and about the importance of discussing items of the SIPP with their radiotherapist (mean score = 7.42). Radiotherapists' perspectives about the contribution of the SIPP to discuss the different psychosocial problems were mixed (mean scores varied from 3.17 to 4.67). Patients were more positive about discussing items of the SIPP if the radiotherapists had positive attitudes towards screening and discussing psychosocial problems. The screening procedure appeared to be feasible in a radiotherapy department. In general, patients' perspectives were at least moderate. Radiotherapists considered the usefulness and feasibility of the SIPP generally to be lower, but their

cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-telomeric Roles of Arabidopsis Telomerase

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Ladislav eDokládal

2015-11-01

Full Text Available Telomerase-reverse transcriptase (TERT plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE TERT domain and identified a nuclear-localized protein that contains a RNA recognition motif (RRM. This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

Combinatorial drug screening identifies Ewing sarcoma-specific sensitivities

OpenAIRE

Radic-Sarikas, Branka; Tsafou, Kalliopi P.; Emdal, Kristina B.; Papamarkou, Theodore; Huber, Kilian V.M.; Mutz, Cornelia; Toretsky, Jeffrey A.; Bennett, Keiryn L.; Olsen, Jesper V.; Brunak, Søren; Kovar, Heinrich; Superti-Furga, Giulio

2017-01-01

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three p...

Identifying harmful drinking using a single screening question in a psychiatric consultation-liaison population.

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Massey, Suena H; Norris, Lorenzo; Lausin, Melissa; Nwaneri, Chinyere; Lieberman, Daniel Z

2011-01-01

Harmful drinking is common in medical inpatients, yet commonly missed due in part to time pressures. A screening question about past year heavy drinking recommended by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) has been validated in primary care and emergency room settings. We tested the psychometric properties of a modified single screening question (SSQ) in hospitalized patients referred to a consultation-liaison service. A psychiatry attending (n = 40), a psychiatry resident (n = 30) and a medical student (n = 30) administered the SSQ, followed by a self-report 10-item Alcohol Use Disorders Identification Test (AUDIT) to a sample of 100 consultation-liaison patients who were able to give informed consent for participation. Using the AUDIT as a reference, the sensitivity and specificity of the SSQ to detect harmful drinking in this sample were .96 and .82, respectively. Gender differences in specificity were not found. The single question also had a strong correlation with dependence (r(b) = .457, p past year heavy drinking can rapidly identify harmful drinking in alert nonpsychotic consultation-liaison patients. Copyright © 2011 The Academy of Psychosomatic Medicine. Published by Elsevier Inc. All rights reserved.

Screening for type 2 diabetes in a multiethnic setting using known risk factors to identify those at high risk

DEFF Research Database (Denmark)

Gray, Laura J.; Tringham, Jennifer R.; Davies, Melanie J.

2010-01-01

population to identify those with abnormal glucose tolerance. ethods: A sample of individuals aged 25-75 years (40-75 white European) with at least one risk factor for T2DM were invited for screening from 17 Leicestershire (UK) general practices or through a health awareness campaign. All participants...... received a 75 g oral glucose tolerance test, cardiovascular risk assessment, detailed medical and family histories and anthropometric measurements. Results: In the 3,225 participants who were screened. 640 (20%) were found to have some form of abnormal glucose tolerance of whom 4% had T2DM, 3% impaired...

Novel curcumin- and emodin-related compounds identified by in silico 2D/3D conformer screening induce apoptosis in tumor cells

International Nuclear Information System (INIS)

Füllbeck, Melanie; Huang, Xiaohua; Dumdey, Renate; Frommel, Cornelius; Dubiel, Wolfgang; Preissner, Robert

2005-01-01

Inhibition of the COP9 signalosome (CSN) associated kinases CK2 and PKD by curcumin causes stabilization of the tumor suppressor p53. It has been shown that curcumin induces tumor cell death and apoptosis. Curcumin and emodin block the CSN-directed c-Jun signaling pathway, which results in diminished c-Jun steady state levels in HeLa cells. The aim of this work was to search for new CSN kinase inhibitors analogue to curcumin and emodin by means of an in silico screening method. Here we present a novel method to identify efficient inhibitors of CSN-associated kinases. Using curcumin and emodin as lead structures an in silico screening with our in-house database containing more than 10 6 structures was carried out. Thirty-five compounds were identified and further evaluated by the Lipinski's rule-of-five. Two groups of compounds can be clearly discriminated according to their structures: the curcumin-group and the emodin-group. The compounds were evaluated in in vitro kinase assays and in cell culture experiments. The data revealed 3 compounds of the curcumin-group (e.g. piceatannol) and 4 of the emodin-group (e.g. anthrachinone) as potent inhibitors of CSN-associated kinases. Identified agents increased p53 levels and induced apoptosis in tumor cells as determined by annexin V-FITC binding, DNA fragmentation and caspase activity assays. Our data demonstrate that the new in silico screening method is highly efficient for identifying potential anti-tumor drugs

Potent Plasmodium falciparum gametocytocidal activity of diaminonaphthoquinones, lead antimalarial chemotypes identified in an antimalarial compound screen.

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Tanaka, Takeshi Q; Guiguemde, W Armand; Barnett, David S; Maron, Maxim I; Min, Jaeki; Connelly, Michele C; Suryadevara, Praveen Kumar; Guy, R Kiplin; Williamson, Kim C

2015-03-01

Forty percent of the world's population is threatened by malaria, which is caused by Plasmodium parasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. Drug resistance has been reported for all commonly used antimalarials and has prompted screens to identify new drug candidates. However, many of these new candidates have not been evaluated against the parasite stage responsible for transmission, gametocytes. If Plasmodium falciparum gametocytes are not eliminated, patients continue to spread malaria for weeks after asexual parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for transmission, and a safe, effective gametocytocidal agent could also be used in community-wide malaria control programs. Here, we identify 15 small molecules with nanomolar activity against late-stage gametocytes. Fourteen are diaminonaphthoquinones (DANQs), and one is a 2-imino-benzo[d]imidazole (IBI). One of the DANQs identified, SJ000030570, is a lead antimalarial candidate. In contrast, 94% of the 650 compounds tested are inactive against late-stage gametocytes. Consistent with the ineffectiveness of most approved antimalarials against gametocytes, of the 19 novel compounds with activity against known anti-asexual-stage targets, only 3 had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the transmission stages and emphasize the importance of screening for gametocytocidal activity. The potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual parasites provides leads for the development of antimalarials with the potential to prevent both the symptoms and the spread of malaria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues.

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Parsons, Linda M; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

2017-08-07

In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 ( SIK2 and 3 ) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development. Copyright © 2017 Parsons et al.

Metabolomic screening using ESI-FT MS identifies potential radiation-responsive molecules in mouse urine

International Nuclear Information System (INIS)

Iizuka, Daisuke; Yoshioka, Susumu; Kawai, Hidehiko; Izumi, Shunsuke; Suzuki, Fumio; Kamiya, Kenji

2017-01-01

The demand for establishment of high-throughput biodosimetric methods is increasing. Our aim in this study was to identify low-molecular-weight urinary radiation-responsive molecules using electrospray ionization Fourier transform mass spectrometry (ESI-FT MS), and our final goal was to develop a sensitive biodosimetry technique that can be applied in the early triage of a radiation emergency medical system. We identified nine metabolites by statistical comparison of mouse urine before and 8 h after irradiation. Time-course analysis showed that, of these metabolites, thymidine and either thymine or imidazoleacetic acid were significantly increased dose-dependently 8 h after radiation exposure; these molecules have already been reported as potential radiation biomarkers. Phenyl glucuronide was significantly decreased 8 h after radiation exposure, irrespective of the dose. Histamine and 1-methylhistamine were newly identified by MS/MS and showed significant, dose-dependent increases 72 h after irradiation. Quantification of 1-methylhistamine by enzyme-linked immunosorbent assay (ELISA) analysis also showed a significant increase 72 h after 4 Gy irradiation. These results suggest that urinary metabolomics screening using ESI-FT MS can be a powerful tool for identifying promising radiation-responsive molecules, and that urinary 1-methylhistamine is a potential radiation-responsive molecule for acute, high-dose exposure.

Use of a single alcohol screening question to identify other drug use.

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Smith, Peter C; Cheng, Debbie M; Allensworth-Davies, Donald; Winter, Michael R; Saitz, Richard

2014-06-01

People who consume unhealthy amounts of alcohol are more likely to use illicit drugs. We tested the ability of a screening test for unhealthy alcohol use to simultaneously detect drug use. Adult English speaking patients (n=286) were enrolled from a primary care waiting room. They were asked the screening question for unhealthy alcohol use "How many times in the past year have you had X or more drinks in a day?", where X is 5 for men and 4 for women, and a response of one or more is considered positive. A standard diagnostic interview was used to determine current (past year) drug use or a drug use disorder (abuse or dependence). Oral fluid testing was also used to detect recent use of common drugs of abuse. The single screening question for unhealthy alcohol use was 67.6% sensitive (95% confidence interval [CI], 50.2-82.0%) and 64.7% specific (95% CI, 58.4-70.6%) for the detection of a drug use disorder. It was similarly insensitive for drug use detected by oral fluid testing and/or self-report. Although a patient with a drug use disorder has twice the odds of screening positive for unhealthy alcohol use compared to one without a drug use disorder, suggesting patients who screen positive for alcohol should be asked about drug use, a single screening question for unhealthy alcohol use was not sensitive or specific for the detection of other drug use or drug use disorders in a sample of primary care patients. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

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Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

2015-12-15

Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments

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Juan J. González-Plaza

2018-01-01

Full Text Available Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs, which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments

Science.gov (United States)

González-Plaza, Juan J.; Šimatović, Ana; Milaković, Milena; Bielen, Ana; Wichmann, Fabienne; Udiković-Kolić, Nikolina

2018-01-01

Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs

A Large-Scale RNAi Screen Identifies SGK1 as a Key Survival Kinase for GBM Stem Cells.

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Kulkarni, Shreya; Goel-Bhattacharya, Surbhi; Sengupta, Sejuti; Cochran, Brent H

2018-01-01

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain cancer and has a very poor prognosis. A subpopulation of cells known as GBM stem-like cells (GBM-SC) have the capacity to initiate and sustain tumor growth and possess molecular characteristics similar to the parental tumor. GBM-SCs are known to be enriched in hypoxic niches and may contribute to therapeutic resistance. Therefore, to identify genetic determinants important for the proliferation and survival of GBM stem cells, an unbiased pooled shRNA screen of 10,000 genes was conducted under normoxic as well as hypoxic conditions. A number of essential genes were identified that are required for GBM-SC growth, under either or both oxygen conditions, in two different GBM-SC lines. Interestingly, only about a third of the essential genes were common to both cell lines. The oxygen environment significantly impacts the cellular genetic dependencies as 30% of the genes required under hypoxia were not required under normoxic conditions. In addition to identifying essential genes already implicated in GBM such as CDK4, KIF11 , and RAN , the screen also identified new genes that have not been previously implicated in GBM stem cell biology. The importance of the serum and glucocorticoid-regulated kinase 1 (SGK1) for cellular survival was validated in multiple patient-derived GBM stem cell lines using shRNA, CRISPR, and pharmacologic inhibitors. However, SGK1 depletion and inhibition has little effect on traditional serum grown glioma lines and on differentiated GBM-SCs indicating its specific importance in GBM stem cell survival. Implications: This study identifies genes required for the growth and survival of GBM stem cells under both normoxic and hypoxic conditions and finds SGK1 as a novel potential drug target for GBM. Mol Cancer Res; 16(1); 103-14. ©2017 AACR . ©2017 American Association for Cancer Research.

A Proteomic Screen Identified Stress-Induced Chaperone Proteins as Targets of Akt Phosphorylation in Mesangial Cells

OpenAIRE

Barati, Michelle T.; Rane, Madhavi J.; Klein, Jon B.; McLeish, Kenneth R.

2006-01-01

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90α, Hsp90β, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphoryl...

Genome-wide RNAi Screen Identifies Networks Involved in Intestinal Stem Cell Regulation in Drosophila

Directory of Open Access Journals (Sweden)

Xiankun Zeng

2015-02-01

Full Text Available The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC lineage differentiation, and ISC-to-enteroendocrine (EE lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.

Trade-offs with stability modulate innate and mutationally acquired drug-resistance in bacterial dihydrofolate reductase enzymes.

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Matange, Nishad; Bodkhe, Swapnil; Patel, Maitri; Shah, Pooja

2018-06-05

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In this study, we show that trimethoprim-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harbouring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild type making them prone to aggregation and proteolysis. This destabilization is associated with lower expression level resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis we show that perturbation of critical stabilizing hydrophobic interactions in wild type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial DHFRs. Mutational and computational analyses in EcDHFR as well as in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues, modulates TMP-resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to trimethoprim. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme. ©2018 The Author(s).

A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene.

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Gobeil, Stephane; Zhu, Xiaochun; Doillon, Charles J; Green, Michael R

2008-11-01

Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.

Hearing Screening Follow-Up: Completing the Process to Identify Hearing Health Needs

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Eiserman, William; Shisler, Lenore; Hoffman, Jeff

2015-01-01

Hearing is at the heart of language development and school readiness; increasing numbers of Early Head Start programs have come to rely on otoacoustic emissions (OAE) technology to screen all infants and toddlers for hearing loss. Successful identification of hearing health needs is dependent not only on an appropriate screening method, but also…

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

Science.gov (United States)

Westrick, Randal J; Tomberg, Kärt; Siebert, Amy E; Zhu, Guojing; Winn, Mary E; Dobies, Sarah L; Manning, Sara L; Brake, Marisa A; Cleuren, Audrey C; Hobbs, Linzi M; Mishack, Lena M; Johnston, Alexander J; Kotnik, Emilee; Siemieniak, David R; Xu, Jishu; Li, Jun Z; Saunders, Thomas L; Ginsburg, David

2017-09-05

Factor V Leiden ( F5 L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N -ethyl- N -nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5 L ( F5 L/L ) and haploinsufficient for tissue factor pathway inhibitor ( Tfpi +/- ). F8 deficiency enhanced the survival of F5 L/L Tfpi +/- mice, demonstrating that F5 L/L Tfpi +/- lethality is genetically suppressible. ENU-mutagenized F5 L/L males and F5 L/+ Tfpi +/- females were crossed to generate 6,729 progeny, with 98 F5 L/L Tfpi +/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden ( MF5L1-16 )," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene ( F3 ). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 ( F3 +/- ) suppressed F5 L/L Tfpi +/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5 L/L Tfpi +/- lethality ( P = 1.7 × 10 -6 ), suggesting that Actr2 p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2 p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5 L and also suggest a role for Actr2 in this process.

Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX

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Dong, Lili; Tan, Qiwen; Ye, Wei; Liu, Dongli; Chen, Haifeng; Hu, Hongwei; Wen, Duo; Liu, Yang; Cao, Ya; Kang, Jingwu; Fan, Jia; Guo, Wei; Wu, Weizhong

2015-01-01

Alpha-fetoprotein (AFP) is a liver cancer associated protein and has long been utilized as a serum tumor biomarker of disease progression. AFP is usually detected in HCC patients by an antibody based system. Recently, however, aptamers generated from systematic evolution of ligands by exponential enrichment (SELEX) were reported to have an alternative potential in targeted imaging, diagnosis and therapy. In this study, AFP-bound ssDNA aptamers were screened and identified using capillary electrophoresis (CE) SELEX technology. After cloning, sequencing and motif analysis, we successfully confirmed an aptamer, named AP273, specifically targeting AFP. The aptamer could be used as a probe in AFP immunofluorescence imaging in HepG2, one AFP positive cancer cell line, but not in A549, an AFP negative cancer cell line. More interesting, the aptamer efficiently inhibited the migration and invasion of HCC cells after in vivo transfection. Motif analysis revealed that AP273 had several stable secondary motifs in its structure. Our results indicate that CE-SELEX technology is an efficient method to screen specific protein-bound ssDNA, and AP273 could be used as an agent in AFP-based staining, diagnosis and therapy, although more works are still needed. PMID:26497223

Virtual screening approach to identifying influenza virus neuraminidase inhibitors using molecular docking combined with machine-learning-based scoring function.

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Zhang, Li; Ai, Hai-Xin; Li, Shi-Meng; Qi, Meng-Yuan; Zhao, Jian; Zhao, Qi; Liu, Hong-Sheng

2017-10-10

In recent years, an epidemic of the highly pathogenic avian influenza H7N9 virus has persisted in China, with a high mortality rate. To develop novel anti-influenza therapies, we have constructed a machine-learning-based scoring function (RF-NA-Score) for the effective virtual screening of lead compounds targeting the viral neuraminidase (NA) protein. RF-NA-Score is more accurate than RF-Score, with a root-mean-square error of 1.46, Pearson's correlation coefficient of 0.707, and Spearman's rank correlation coefficient of 0.707 in a 5-fold cross-validation study. The performance of RF-NA-Score in a docking-based virtual screening of NA inhibitors was evaluated with a dataset containing 281 NA inhibitors and 322 noninhibitors. Compared with other docking-rescoring virtual screening strategies, rescoring with RF-NA-Score significantly improved the efficiency of virtual screening, and a strategy that averaged the scores given by RF-NA-Score, based on the binding conformations predicted with AutoDock, AutoDock Vina, and LeDock, was shown to be the best strategy. This strategy was then applied to the virtual screening of NA inhibitors in the SPECS database. The 100 selected compounds were tested in an in vitro H7N9 NA inhibition assay, and two compounds with novel scaffolds showed moderate inhibitory activities. These results indicate that RF-NA-Score improves the efficiency of virtual screening for NA inhibitors, and can be used successfully to identify new NA inhibitor scaffolds. Scoring functions specific for other drug targets could also be established with the same method.

Large-scale functional RNAi screen in C. elegans identifies genes that regulate the dysfunction of mutant polyglutamine neurons.

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Lejeune, François-Xavier; Mesrob, Lilia; Parmentier, Frédéric; Bicep, Cedric; Vazquez-Manrique, Rafael P; Parker, J Alex; Vert, Jean-Philippe; Tourette, Cendrine; Neri, Christian

2012-03-13

A central goal in Huntington's disease (HD) research is to identify and prioritize candidate targets for neuroprotective intervention, which requires genome-scale information on the modifiers of early-stage neuron injury in HD. Here, we performed a large-scale RNA interference screen in C. elegans strains that express N-terminal huntingtin (htt) in touch receptor neurons. These neurons control the response to light touch. Their function is strongly impaired by expanded polyglutamines (128Q) as shown by the nearly complete loss of touch response in adult animals, providing an in vivo model in which to manipulate the early phases of expanded-polyQ neurotoxicity. In total, 6034 genes were examined, revealing 662 gene inactivations that either reduce or aggravate defective touch response in 128Q animals. Several genes were previously implicated in HD or neurodegenerative disease, suggesting that this screen has effectively identified candidate targets for HD. Network-based analysis emphasized a subset of high-confidence modifier genes in pathways of interest in HD including metabolic, neurodevelopmental and pro-survival pathways. Finally, 49 modifiers of 128Q-neuron dysfunction that are dysregulated in the striatum of either R/2 or CHL2 HD mice, or both, were identified. Collectively, these results highlight the relevance to HD pathogenesis, providing novel information on the potential therapeutic targets for neuroprotection in HD. © 2012 Lejeune et al; licensee BioMed Central Ltd.

Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

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Richard T Timms

Full Text Available 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.

Development of the Aboriginal Communication Assessment After Brain Injury (ACAABI): A screening tool for identifying acquired communication disorders in Aboriginal Australians.

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Armstrong, Elizabeth M; Ciccone, Natalie; Hersh, Deborah; Katzenellebogen, Judith; Coffin, Juli; Thompson, Sandra; Flicker, Leon; Hayward, Colleen; Woods, Deborah; McAllister, Meaghan

2017-06-01

Acquired communication disorders (ACD), following stroke and traumatic brain injury, may not be correctly identified in Aboriginal Australians due to a lack of linguistically and culturally appropriate assessment tools. Within this paper we explore key issues that were considered in the development of the Aboriginal Communication Assessment After Brain Injury (ACAABI) - a screening tool designed to assess the presence of ACD in Aboriginal populations. A literature review and consultation with key stakeholders were undertaken to explore directions needed to develop a new tool, based on existing tools and recommendations for future developments. The literature searches revealed no existing screening tool for ACD in these populations, but identified tools in the areas of cognition and social-emotional wellbeing. Articles retrieved described details of the content and style of these tools, with recommendations for the development and administration of a new tool. The findings from the interview and focus group views were consistent with the approach recommended in the literature. There is a need for a screening tool for ACD to be developed but any tool must be informed by knowledge of Aboriginal language, culture and community input in order to be acceptable and valid.

A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen

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Ashlee J. Conway

2017-08-01

Full Text Available A genome-wide ethyl-N-nitrosourea (ENU mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX. A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53.

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Bechill, John; Zhong, Rong; Zhang, Chen; Solomaha, Elena; Spiotto, Michael T

2016-01-01

p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways.

Assessment of Folic Acid Supplementation in Pregnant Women by Estimation of Serum Levels of Tetrahydrofolic Acid, Dihydrofolate Reductase, and Homocysteine

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Manisha Naithani

2016-01-01

Full Text Available Background. Status of folic acid use in pregnant women of the hilly regions in North India was little known. This study was carried out to assess the folic acid use and estimate folate metabolites in pregnant women of this region. Materials and Methods. This cross-sectional study is comprised of 76 pregnant women, whose folic acid supplementation was assessed by a questionnaire and serum levels of homocysteine, tetrahydrofolic acid (THFA, and dihydrofolate reductase (DHFR were estimated using Enzyme Linked Immunoassays. Results. The study data revealed awareness of folic acid use during pregnancy was present in 46.1% and 23.7% were taking folic acid supplements. The study depicted that there was no statistically significant difference between serum levels of THFA and DHFR in pregnant women with and without folic acid supplements (p=0.790. Hyperhomocysteinemia was present in 15.78% of the participants. Conclusion. Less awareness about folic acid supplementation and low use of folic acid by pregnant women were observed in this region. Sufficient dietary ingestion may suffice for the escalated requirements in pregnancy, but since this cannot be ensured, hence folic acid supplementation should be made as an integral part of education and reproductive health programs for its better metabolic use, growth, and development of fetus.

Screening of Natural Product Derivatives Identifies Two Structurally Related Flavonoids as Potent Quorum Sensing Inhibitors against Gram-Negative Bacteria

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Suvi Manner

2018-05-01

Full Text Available Owing to the failure of conventional antibiotics in biofilm control, alternative approaches are urgently needed. Inhibition of quorum sensing (QS represents an attractive target since it is involved in several processes essential for biofilm formation. In this study, a compound library of natural product derivatives (n = 3040 was screened for anti-quorum sensing activity using Chromobacterium violaceum as reporter bacteria. Screening assays, based on QS-mediated violacein production and viability, were performed in parallel to identify non-bactericidal QS inhibitors (QSIs. Nine highly active QSIs were identified, while 328 compounds were classified as moderately actives and 2062 compounds as inactives. Re-testing of the highly actives at a lower concentration against C. violaceum, complemented by a literature search, led to the identification of two flavonoid derivatives as the most potent QSIs, and their impact on biofilm maturation in Escherichia coli and Pseudomonas aeruginosa was further investigated. Finally, effects of these leads on swimming and swarming motility of P. aeruginosa were quantified. The identified flavonoids affected all the studied QS-related functions at micromolar concentrations. These compounds can serve as starting points for further optimization and development of more potent QSIs as adjunctive agents used with antibiotics in the treatment of biofilms.

Chemical biology drug sensitivity screen identifies sunitinib as synergistic agent with disulfiram in prostate cancer cells.

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Kirsi Ketola

Full Text Available Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects.In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression.Taken together, our results propose novel combinatorial approaches to inhibit prostate cancer cell growth. Disulfiram

Identifying the trauma recovery needs of maltreated children: An examination of child welfare workers' effectiveness in screening for traumatic stress.

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Whitt-Woosley, Adrienne; Sprang, Ginny; Royse, David G

2018-07-01

Children in the child welfare system comprise a group characterized by exposure to trauma via experiences of maltreatment, under circumstances presenting multiple risk factors for traumatic stress. High rates of posttraumatic stress have been observed in this population. However, there is currently no standard for the universal screening of children in child welfare for trauma exposure and traumatic stress. This study examined the trauma experiences of a sample of maltreated children and whether their child welfare workers were effective screeners of traumatic stress symptoms. Descriptive and correlational analyses were conducted regarding a sample of children (N = 131) with trauma screenings completed by their child welfare workers and clinical measures of traumatic stress symptoms. Four hierarchical regression models were also examined to determine whether workers' screening information regarding child age, trauma exposure history and symptoms of traumatic stress were predictive of outcomes on clinical measures. The analyses revealed complex trauma exposure histories and high rates of traumatic stress symptoms among this generally younger sample of maltreated children. Additionally, the models supported workers' efficacy in screening for symptoms of total posttraumatic stress and specific trauma symptoms of intrusion and avoidance. Workers were less effective in screening for the symptoms of arousal. These findings support the importance of identifying the trauma recovery needs of maltreated children and the utility of child protection workers in assisting with the trauma screening process. Implications are provided for related practice, policy and training efforts in child welfare. Published by Elsevier Ltd.

A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion

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Toret, Christopher P.; D’Ambrosio, Michael V.; Vale, Ronald D.; Simon, Michael A.

2014-01-01

Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (∼14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca2+-dependent adhesion in DE-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling. PMID:24446484

A Clinical Drug Library Screen Identifies Tosufloxacin as Being Highly Active against Staphylococcus aureus Persisters

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Hongxia Niu

2015-07-01

Full Text Available To identify effective compounds that are active against Staphylococcus aureus (S. aureus persisters, we screened a clinical drug library consisting of 1524 compounds and identified six drug candidates that had anti-persister activity: tosufloxacin, clinafloxacin, sarafloxacin, doxycycline, thiostrepton, and chlorosalicylanilide. Among them, tosufloxacin had the highest anti-persister activity, which could completely eradicate S. aureus persisters within 2 days in vitro. Clinafloxacin ranked the second with very few persisters surviving the drug exposure. Interestingly, we found that both tosufloxacin and trovafloxacin that had high activity against persisters contained at the N-1 position the 2,4-difluorophenyl group, which is absent in other less active quinolones and may be associated with the high anti-persister activity. Further studies are needed to evaluate tosufloxacin in animal models and to explain its unique activity against bacterial persisters. Our findings may have implications for improved treatment of persistent bacterial infections.

Target based screening of small molecule library identifies pregnelonene, a Nrf2 agonist, as a potential radioprotector in zebrafish

International Nuclear Information System (INIS)

Joshi, Jayadev; Ghosh, Subhajit; Dimri, Manali; Shrivastava, Nitisha; Indracanti, Prem Kumar; Ray, Jharna

2014-01-01

Reactive oxygen species, cellular oxidative stress, tissue inflammation and cell death are the downstream consequences of radiation exposures which ultimately could lead to organism death. Present study aims at identifying potential targets and screening of small molecule compound library for identifying novel and effective radioprotectors. In-silco analysis of known radioprotectors revealed three main function, antioxidant, anti-inflammation and antiapoptosis. In this study, a collection of small molecules (John Hopkins Clinical Compound Library, JHCCL) were screened for these different functions using the biological activity database of NCBI with the help of in-house developed python script. Further, filtering of the JHCCL was done by searching for molecules which are known to be active against target of radiobiological significance, Nrf-2. Close observation of potential hits identified, pregnenolone, as an Nrf-2 agonist which was further evaluated for radioprotection in zebrafish model. Pregnenolone rendered significant protection (at 40 μM; added 1 hour prior to 20 Gy gamma radiation) in terms of damage manifestations (pericardial edema, microcephaly, micropthalmia, yolk sac resorption, curvature of spine, blood flow, body length, heart-beat, blood clot, roughness of skin) and survival advantage (60%) when compared to irradiated control. Further, the ability of pregnenolone to act as a neuroprotectant was also carried out using in-house developed software for assessing neuromotor functions. In comparison to radiation alone group, pregnenolone was found to possess significant neuroactive functions and diminished radiation induced neuronal impairment. Over all these results suggests that pregnenolone is an effective radioprotector which warrants further investigation for validation of its radioprotective action in higher vertebrates. Apart from that the utility of approach to screen out bioactivity data base of various chemical compound libraries for possible

Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells

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Thomas D. B. MacVicar

2015-06-01

Full Text Available Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy, mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca2+ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1, as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT and the IP3-receptors (IP3Rs as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca2+ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca2+ uniporter (MCU primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca2+ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca2+ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.

Implementing a screening tool for identifying patients at risk for hereditary breast and ovarian cancer: a statewide initiative.

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Brannon Traxler, L; Martin, Monique L; Kerber, Alice S; Bellcross, Cecelia A; Crane, Barbara E; Green, Victoria; Matthews, Roland; Paris, Nancy M; Gabram, Sheryl G A

2014-10-01

The Georgia Breast Cancer Genomic Health Consortium is a partnership created with funding from the Centers for Disease Control and Prevention (CDC) to the Georgia Department of Public Health to reduce cancer disparities among high-risk minority women. The project addresses young women at increased risk for hereditary breast and ovarian cancer (HBOC) syndrome through outreach efforts. The consortium provides education and collects surveillance data using the breast cancer genetics referral screening tool (B-RST) available at www.BreastCancerGeneScreen.org . The HBOC educational protocol was presented to 73 staff in 6 public health centers. Staff used the tool during the collection of medical history. Further family history assessments and testing for mutations in the BRCA1/2 genes were facilitated if appropriate. Data was collected from November 2012 through December 2013, including 2,159 screened women. The majority of patients identified as black/African American and were 18-49 years old. Also, 6.0 % (n = 130) had positive screens, and 60.9 % (n = 67) of the 110 patients who agreed to be contacted provided a detailed family history. A total of 47 patients (42.7 %) met National Comprehensive Cancer Network guidelines when family history was clarified. Fourteen (12.7 %) underwent genetic testing; 1 patient was positive for a BRCA2 mutation, and 1 patient was found to carry a variant of uncertain significance. The introduction of genomics practice within public health departments has provided access to comprehensive cancer care for uninsured individuals. The successful implementation of the B-RST into public health centers demonstrates the opportunity for integration of HBOC screening into primary care practices.

Quantitative and mixed analyses to identify factors that affect cervical cancer screening uptake among lesbian and bisexual women and transgender men.

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Johnson, Michael J; Mueller, Martina; Eliason, Michele J; Stuart, Gail; Nemeth, Lynne S

2016-12-01

The purposes of this study were to measure the prevalence of, and identify factors associated with, cervical cancer screening among a sample of lesbian, bisexual and queer women, and transgender men. Past research has found that lesbian, bisexual and queer women underuse cervical screening service. Because deficient screening remains the most significant risk factor for cervical cancer, it is essential to understand the differences between routine and nonroutine screeners. A convergent-parallel mixed methods design. A convenience sample of 21- to 65-year-old lesbian and bisexual women and transgender men were recruited in the USA from August-December 2014. Quantitative data were collected via a 48-item Internet questionnaire (N = 226), and qualitative data were collected through in-depth telephone interviews (N = 20) and open-ended questions on the Internet questionnaire. Seventy-three per cent of the sample was routine cervical screeners. The results showed that a constellation of factors influence the use of cervical cancer screening among lesbian, bisexual and queer women. Some of those factors overlap with the general female population, whereas others are specific to the lesbian, bisexual or queer identity. Routine screeners reported feeling more welcome in the health care setting, while nonroutine screeners reported more discrimination related to their sexual orientation and gender expression. Routine screeners were also more likely to 'out' to their provider. The quantitative and qualitative factors were also compared and contrasted. Many of the factors identified in this study to influence cervical cancer screening relate to the health care environment and to interactions between the patient and provider. Nurses should be involved with creating welcoming environments for lesbian, bisexual and queer women and their partners. Moreover, nurses play a large role in patient education and should promote self-care behaviours among lesbian women and transgender

An approximate but efficient method to calculate free energy trends by computer simulation: Application to dihydrofolate reductase-inhibitor complexes

Science.gov (United States)

Gerber, Paul R.; Mark, Alan E.; van Gunsteren, Wilfred F.

1993-06-01

Derivatives of free energy differences have been calculated by molecular dynamics techniques. The systems under study were ternary complexes of Trimethoprim (TMP) with dihydrofolate reductases of E. coli and chicken liver, containing the cofactor NADPH. Derivatives are taken with respect to modification of TMP, with emphasis on altering the 3-, 4- and 5-substituents of the phenyl ring. A linear approximation allows the encompassing of a whole set of modifications in a single simulation, as opposed to a full perturbation calculation, which requires a separate simulation for each modification. In the case considered here, the proposed technique requires a factor of 1000 less computing effort than a full free energy perturbation calculation. For the linear approximation to yield a significant result, one has to find ways of choosing the perturbation evolution, such that the initial trend mirrors the full calculation. The generation of new atoms requires a careful treatment of the singular terms in the non-bonded interaction. The result can be represented by maps of the changed molecule, which indicate whether complex formation is favoured under movement of partial charges and change in atom polarizabilities. Comparison with experimental measurements of inhibition constants reveals fair agreement in the range of values covered. However, detailed comparison fails to show a significant correlation. Possible reasons for the most pronounced deviations are given.

Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen*

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Kozlov, Sergei V.; Waardenberg, Ashley J.; Engholm-Keller, Kasper; Arthur, Jonathan W.; Graham, Mark E.; Lavin, Martin

2016-01-01

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

shRNA screening identifies JMJD1C as being required for leukemia maintenance

DEFF Research Database (Denmark)

Sroczynska, Patrycja; Cruickshank, V Adam; Bukowski, John-Paul

2014-01-01

Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid and lymphoid leukemia (AML and ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic...... candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro, as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines......, with the strongest effect observed in the MLL-rearranged ALL cell line, SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia....

Hypersexual Disorder According to the Hypersexual Disorder Screening Inventory in Help-Seeking Swedish Men and Women With Self-Identified Hypersexual Behavior

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Katarina Görts Öberg, PhD

2017-12-01

Öberg KG, Hallberg J, Kaldo V, et al. Hypersexual Disorder According to the Hypersexual Disorder Screening Inventory in Help-Seeking Swedish Men and Women With Self-Identified Hypersexual Behavior. Sex Med 2017;5:e229–e236.

Cancer Screening Among Patients Who Self-Identify as Muslim: Combining Self-Reported Data with Medical Records in a Family Practice Setting.

Science.gov (United States)

Lofters, A K; Slater, M; Vahabi, M

2018-02-01

Cancer screening is a core component of family medicine but screening inequalities are well documented in Canada for foreign-born persons. Although people of Muslim faith and culture are the fastest growing immigrant population in Canada, there is little information in the literature about their cancer screening practices. Determining screening gaps could inform practice-based quality improvement initiatives. We conducted a retrospective chart review combining patient-level medical record data with self-reported religious affiliation to examine the relationship between religion and cancer screening in a large multi-site urban family practice. Religious affiliation was classified as Muslim, other affiliation, or atheist/no religious affiliation. 5311 patients were included in the study sample. Muslim patients were significantly less likely to prefer English for spoken communication than the other two groups, less likely to be Canadian-born, more likely to have a female family physician, and were over-represented in the lowest income quintile. Muslim women were most likely to be up-to-date on breast cancer screening (85.2 vs. 77.5 % for those with other religions vs. 69.5 % for those with no religious affiliation). There were no significant differences in cancer screening by physician sex. In this pilot study conducted within a primary care practice, we used self-reported data on religious affiliation to examine possible inequities in cancer screening and observed intriguing variations in screening by self-identified religious affiliation. Future efforts to collect and use similar patient-level data should incorporate non-official languages and intensively outreach to patients with less health system contact. Regardless, the family medicine context may be the ideal setting to collect and act on patient-level sociodemographic data such as religious affiliation.

The HLJ1-targeting drug screening identified Chinese herb andrographolide that can suppress tumour growth and invasion in non-small-cell lung cancer.

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Lai, Yi-Hua; Yu, Sung-Liang; Chen, Hsuan-Yu; Wang, Chi-Chung; Chen, Huei-Wen; Chen, Jeremy J W

2013-05-01

HLJ1 is a novel tumour suppressor and is a potential druggable target for non-small-cell lung cancer (NSCLC). In this report, using a promoter-containing enhancer region as the HLJ1-targeting drug-screening platform, we identified several herbal compounds from a Chinese herbal bank with the capacity to enhance HLJ1 promoter activity and suppress tumour growth and invasion of NSCLC. Among the herbal drugs identified, the andrographolide (from Andrographis paniculata [Burm. f.] Nees.) most significantly induced HLJ1 expression and suppressed tumorigenesis both in vitro and in vivo. The andrographolide upregulates HLJ1 via JunB activation, which modulates AP-2α binding at the MMP-2 promoter and represses the expression of MMP-2. In addition, silencing of HLJ1 partially reverses the inhibition of cancer-cell invasion by andrographolide. Microarray transcriptomic analysis was performed to comprehensively depict the andrographolide-regulated signalling pathways. We showed that andrographolide can affect 939 genes (analysis of variance, false discovery rate andrographolide on anticancer invasion and proliferation. In conclusion, the HLJ1-targeting drug-screening platform is useful for screening of novel anticancer compounds. Using this platform, we identified andrographolide is a promising new anticancer agent that could suppress tumour growth and invasion in NSCLC.

Food Insecurity Screening in Pediatric Primary Care: Can Offering Referrals Help Identify Families in Need?

Science.gov (United States)

Bottino, Clement J; Rhodes, Erinn T; Kreatsoulas, Catherine; Cox, Joanne E; Fleegler, Eric W

2017-07-01

To describe a clinical approach for food insecurity screening incorporating a menu offering food-assistance referrals, and to examine relationships between food insecurity and referral selection. Caregivers of 3- to 10-year-old children presenting for well-child care completed a self-administered questionnaire on a laptop computer. Items included the US Household Food Security Survey Module: 6-Item Short Form (food insecurity screen) and a referral menu offering assistance with: 1) finding a food pantry, 2) getting hot meals, 3) applying for Supplemental Nutrition Assistance Program (SNAP), and 4) applying for Special Supplemental Nutrition Program for Women, Infants, and Children (WIC). Referrals were offered independent of food insecurity status or eligibility. We examined associations between food insecurity and referral selection using multiple logistic regression while adjusting for covariates. A total of 340 caregivers participated; 106 (31.2%) reported food insecurity, and 107 (31.5%) selected one or more referrals. Forty-nine caregivers (14.4%) reported food insecurity but selected no referrals; 50 caregivers (14.7%) selected one or more referrals but did not report food insecurity; and 57 caregivers (16.8%) both reported food insecurity and selected one or more referrals. After adjustment, caregivers who selected one or more referrals had greater odds of food insecurity compared to caregivers who selected no referrals (adjusted odds ratio 4.0; 95% confidence interval 2.4-7.0). In this sample, there was incomplete overlap between food insecurity and referral selection. Offering referrals may be a helpful adjunct to standard screening for eliciting family preferences and identifying unmet social needs. Copyright © 2016 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

International Nuclear Information System (INIS)

Birdsall, B.; Andrews, J.; Ostler, G.; Tendler, S.J.B.; Feeney, J.; Roberts, G.C.K.; Davies, R.W.; Cheung, H.T.A.

1989-01-01

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21 → Leu and Asp 26 → Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1 H, 13 C, and 31 P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1 H NMR studies of the Trp 21 → Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 angstrom away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21 → Leu mutant indicate that the delicately poised equilibria can be perturbed. Some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim

Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

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Marjanovic, Jasmina; Chalupska, Dominika; Patenode, Caroline; Coster, Adam; Arnold, Evan; Ye, Alice; Anesi, George; Lu, Ying; Okun, Ilya; Tkachenko, Sergey; Haselkorn, Robert; Gornicki, Piotr

2010-01-01

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In “proof of concept” experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 μM IC50 and having no effect on human ACC1 at 100 μM. PMID:20439761

An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers.

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Tang, H M; Kuay, K T; Koh, P F; Asad, M; Tan, T Z; Chung, V Y; Lee, S C; Thiery, J P; Huang, Ry-J

2016-01-01

Epithelial-mesenchymal transition (EMT), a crucial mechanism in development, mediates aggressiveness during carcinoma progression and therapeutic refractoriness. The reversibility of EMT makes it an attractive strategy in designing novel therapeutic approaches. Therefore, drug discovery pipelines for EMT reversal are in need to discover emerging classes of compounds. Here, we outline a pre-clinical drug screening platform for EMT reversal that consists of three phases of drug discovery and validation. From the Phase 1 epithelial marker promoter induction (EpI) screen on a library consisting of compounds being approved by Food and Drug Administration (FDA), Vorinostat (SAHA), a histone deacetylase inhibitor (HDACi), is identified to exert EMT reversal effects by restoring the expression of an epithelial marker, E-cadherin. An expanded screen on 41 HDACi further identifies 28 compounds, such as class I-specific HDACi Mocetinosat, Entinostat and CI994, to restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells. Mocetinostat is the most potent HDACi to restore epithelial differentiation with the lowest concentration required for 50% induction of epithelial promoter activity (EpIC-50).The HDACi exerts paradoxical effects on EMT transcriptional factors such as SNAI and ZEB family and the effects are context-dependent in epithelial- and mesenchymal-like cells. In vitro functional studies further show that HDACi induced significant increase in anoikis and decrease in spheroid formation in ovarian and bladder carcinoma cells with mesenchymal features. This study demonstrates a robust drug screening pipeline for the discovery of compounds capable of restoring epithelial differentiation that lead to significant functional lethality.

Optimal screening of children with acute malnutrition requires a change in current WHO guidelines as MUAC and WHZ identify different patient groups

DEFF Research Database (Denmark)

Laillou, Arnaud; Prak, Sophonneary; de Groot, Richard

2014-01-01

BACKGROUND: Timely treatment of acute malnutrition in children 500,000 deaths annually. Screening at community level is essential to identify children with malnutrition. Current WHO guidelines for community screening for malnutrition recommend a Mid Upper Arm...... Circumference (MUAC) of malnutrition (SAM). However, it is currently unclear how MUAC relates to the other indicator used to define acute malnutrition: weight-for-height Z-score (WHZ). METHODS: Secondary data from >11,000 Cambodian children, obtained by different surveys between...... 2010 and 2012, was used to calculate sensitivity and ROC curves for MUAC and WHZ. FINDINGS: The secondary analysis showed that using the current WHO cut-off of 115 mm for screening for severe acute malnutrition over 90% of children with a weight-for-height z-score (WHZ)

Newborn blood spot screening for sickle cell disease by using tandem mass spectrometry: implementation of a protocol to identify only the disease states of sickle cell disease.

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Moat, Stuart J; Rees, Derek; King, Lawrence; Ifederu, Adeboye; Harvey, Katie; Hall, Kate; Lloyd, Geoff; Morrell, Christine; Hillier, Sharon

2014-02-01

The currently recommended technologies of HPLC and isoelectric focusing for newborn blood spot screening for sickle cell disease (SCD) identify both the disease and carrier states, resulting in large numbers of infants being followed up unnecessarily. Analysis of blood spot tryptic peptides performed by using tandem mass spectrometry (MS/MS) is an alternative technology to detect hemoglobin (Hb) variant disorders. We analyzed 2154 residual newborn blood spots and 675 newborn blood spots from infants with Hb variants by using MS/MS after trypsin digestion. Screening cutoffs were developed by using the ratio between the variant peptide-to-wild-type peptide abundance for HbS, C, D(Punjab), O(Arab), Lepore, and E peptides. A postanalytical data analysis protocol was developed using these cutoffs to detect only the disease states of SCD and not to identify carrier states. A parallel study of 13 249 newborn blood spots from a high-prevalence SCD area were analyzed by both MS/MS and HPLC. Screening cutoffs developed distinguished the infants with the disease states of SCD, infants who were carriers of SCD, and infants with normal Hb. In the parallel study no false-negative results were identified, and all clinically relevant cases were correctly identified using the MS/MS protocol. Unblinding the data revealed a total of 328 carrier infants that were successfully excluded by the protocol. The screening protocol developed correctly identified infants with the disease states of SCD. Furthermore, large numbers of sickle cell carrier infants were successfully not identified, thereby avoiding unnecessary follow-up testing and referral for genetic counseling.

A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis

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Moy Richard L

2008-07-01

Full Text Available Abstract Background Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis. Results We constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis. Conclusion Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

Optical pre-screening in breast screening programs: Can we identify women who benefit most from limited mammography resources?

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Walter, Jane; Loshchenov, Maxim; Zhilkin, Vladimir; Peake, Rachel; Stone, Jennifer; Lilge, Lothar

2017-04-01

Background: In excess of 60% of all cancers are detected in low and middle-income countries, with breast cancer (BC) the dominant malignancy for women. Incidence rates continue to climb, most noticeably in the less than 50-year-old population. Expansion of mammography infrastructure and resources is lacking, resulting in over 60% of women diagnosed with stage III/IV BC in the majority of these countries. Optical Breast Spectroscopy (OBS) was shown to correlate well with mammographic breast density (MBD). OBS could aid breast screening programs in low- and middle-income countries by lowering the number of mammographs required for complete population coverage. However, its performance needs to be tested in large population trails to ensure high sensitivity and acceptable specificity. Methods: For the planned studies in low- and middle-income countries in different continents, online methods need to be implemented to monitor the performance and data collection by these devices, operated by trained nurses. Based on existing datasets, procedures were developed to validate an individual woman's data integrity and to identify operator errors versus system malfunctions. Results: Using a dataset comprising spectra from 360 women collected by 2 instruments in different locations and with 3 different trained operators, automated methods were developed to identify 100% of the source or photodetector malfunctions as well as incorrect calibrations and 96% of instances of insufficient tissue contact. Conclusions: Implementing the dataset validation locally in each instrument and tethered to a cloud database will allow the planned clinical trials to proceed.

Rational approach to identify newer caspase-1 inhibitors using pharmacophore based virtual screening, docking and molecular dynamic simulation studies.

Science.gov (United States)

Patel, Shivani; Modi, Palmi; Chhabria, Mahesh

2018-05-01

Caspase-1 is a key endoprotease responsible for the post-translational processing of pro-inflammatory cytokines IL-1β, 18 & 33. Excessive secretion of IL-1β leads to numerous inflammatory and autoimmune diseases. Thus caspase-1 inhibition would be considered as an important therapeutic strategy for development of newer anti-inflammatory agents. Here we have employed an integrated virtual screening by combining pharmacophore mapping and docking to identify small molecules as caspase-1 inhibitors. The ligand based 3D pharmacophore model was generated having the essential structural features of (HBA, HY & RA) using a data set of 27 compounds. A validated pharmacophore hypothesis (Hypo 1) was used to screen ZINC and Minimaybridge chemical databases. The retrieved virtual hits were filtered by ADMET properties and molecular docking analysis. Subsequently, the cross-docking study was also carried out using crystal structure of caspase-1, 3, 7 and 8 to identify the key residual interaction for specific caspase-1 inhibition. Finally, the best mapped and top scored (ZINC00885612, ZINC72003647, BTB04175 and BTB04410) molecules were subjected to molecular dynamics simulation for accessing the dynamic structure of protein after ligand binding. This study identifies the most promising hits, which can be leads for the development of novel caspase-1 inhibitors as anti-inflammatory agents. Copyright © 2018 Elsevier Inc. All rights reserved.

Multiplexed screening of natural humoral immunity identifies antibodies at fine specificity for complex and dynamic viral targets.

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McCutcheon, Krista M; Gray, Julia; Chen, Natalie Y; Liu, Keyi; Park, Minha; Ellsworth, Stote; Tripp, Ralph A; Tompkins, S Mark; Johnson, Scott K; Samet, Shelly; Pereira, Lenore; Kauvar, Lawrence M

2014-01-01

Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

Barriers and strategies for identifying and managing risk factors of cardiovascular diseases in levels of preventing, screening, and treating

Directory of Open Access Journals (Sweden)

Saber Azami Aghdash

2015-11-01

Full Text Available Introduction: Cardiovascular diseases (CVD are of the main causes of mortality in the world and impose a heavy economic, social, and health burden on society. Therefore, the objective of this study was to determine the barriers and strategies for identifying and managing risk factors of CVD in levels of preventing, screening, and treating. Methods: During present qualitative study with phenomenological approach, 60 subjects of cardiologists, nurses, patients, and their relatives were selected based on purposive sampling from educational-medical cardiothoracic subspecialty centers. Data were collected using an open-ended questionnaire and was extracted and analyzed with content analysis method. Results: Barriers were divided into three groups of individual barriers (low awareness, delay in referring for treatment and screening, incorrect beliefs, and not caring about health, socio-economic barriers (high costs, lack of resources, mental and psychological pressures, and health care barriers (non-alignment of doctors, being therapy-oriented, managerial and planning weaknesses, and lack of health care facilities. The most important presenting strategies are: providing public educations, improving family physician program, reduction of costs, cooperation of patients, and using functional indices to evaluate and improve the quality of services. Conclusion: Low awareness of people, high costs of services, lack of health care facilities, socio-cultural problems of people, and delay in referring of people, for treatment and screening are of the most important barriers of proper identifying and managing risk factors of CVD. Strategies provided in this study to overcome these barriers could be used.

Screening and Identifying Antioxidative Components in Ginkgo biloba Pollen by DPPH-HPLC-PAD Coupled with HPLC-ESI-MS2

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Netrusov, A. I.; Zhou, Qingxin; Guo, Danyang; Liu, Xiaoyong; He, Hailun; Xin, Xue; Wang, Yifen; Chen, Leilei

2017-01-01

The Ginkgo biloba is one of ancient trees that exists from billions of years ago, its leaf and nut are used as herbs and foods in China, while so far its pollen does not have any application except pollination. In order to evaluate the antioxidant activity of Ginkgo biloba pollen, and rapidly screen its antioxidative components, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability, total flavonoid, total phenol, and proanthocyanidin of Ginkgo biloba pollen were determined and compared with those of Ginkgo biloba leaf and nut, and the off-line DPPH-HPLC-PAD and HPLC-ESI-MS2 were applied for screening and identifying the antioxidant flavonoids in Ginkgo biloba pollen. The results showed that the DPPH scavenging ability of Ginkgo biloba pollen was much higher than Ginkgo biloba nut, but lower than Ginkgo biloba leaf, while the total content of flavonoid in Ginkgo biloba pollen was approximately 4.37 times higher than in Ginkgo biloba leaf. Further studies found that the major flavonol aglycone in Ginkgo biloba pollen was kaempferol, which accounted for 96.71% of the total aglycones (includes quercetin, kaempferol and isorhamnetin), and the main flavonoid components in Ginkgo biloba pollen were flavonoid glycosides. Finally, ten antioxidant peaks were screened and identified to be flavonoids (including kaempferol and nine flavonoid glycosides), so flavonoids were likely to be the main antioxidant components in GP, and among them, three novel kaempferol glycosides (peaks 1, 2, and 3) were found in Ginkgo biloba pollen for the first time, which had never been found in Ginkgo biloba. PMID:28095510

Screening for Intimate Partner Violence in Orthopedic Patients: A Comparison of Three Screening Tools

Science.gov (United States)

Sprague, Sheila; Madden, Kim; Dosanjh, Sonia; Petrisor, Brad; Schemitsch, Emil H.; Bhandari, Mohit

2012-01-01

Accurately identifying victims of intimate partner violence (IPV) can be a challenge for clinicians and clinical researchers. Multiple instruments have been developed and validated to identify IPV in patients presenting to health care practitioners, including the Woman Abuse Screening Tool (WAST) and the Partner Violence Screen (PVS). The purpose…

A novel anti-tumor inhibitor identified by virtual screen with PLK1 structure and zebrafish assay.

Directory of Open Access Journals (Sweden)

Jing Lu

Full Text Available Polo-like kinase 1 (PLK1, one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC50 values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.

HCS-Neurons: identifying phenotypic changes in multi-neuron images upon drug treatments of high-content screening.

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Charoenkwan, Phasit; Hwang, Eric; Cutler, Robert W; Lee, Hua-Chin; Ko, Li-Wei; Huang, Hui-Ling; Ho, Shinn-Ying

2013-01-01

High-content screening (HCS) has become a powerful tool for drug discovery. However, the discovery of drugs targeting neurons is still hampered by the inability to accurately identify and quantify the phenotypic changes of multiple neurons in a single image (named multi-neuron image) of a high-content screen. Therefore, it is desirable to develop an automated image analysis method for analyzing multi-neuron images. We propose an automated analysis method with novel descriptors of neuromorphology features for analyzing HCS-based multi-neuron images, called HCS-neurons. To observe multiple phenotypic changes of neurons, we propose two kinds of descriptors which are neuron feature descriptor (NFD) of 13 neuromorphology features, e.g., neurite length, and generic feature descriptors (GFDs), e.g., Haralick texture. HCS-neurons can 1) automatically extract all quantitative phenotype features in both NFD and GFDs, 2) identify statistically significant phenotypic changes upon drug treatments using ANOVA and regression analysis, and 3) generate an accurate classifier to group neurons treated by different drug concentrations using support vector machine and an intelligent feature selection method. To evaluate HCS-neurons, we treated P19 neurons with nocodazole (a microtubule depolymerizing drug which has been shown to impair neurite development) at six concentrations ranging from 0 to 1000 ng/mL. The experimental results show that all the 13 features of NFD have statistically significant difference with respect to changes in various levels of nocodazole drug concentrations (NDC) and the phenotypic changes of neurites were consistent to the known effect of nocodazole in promoting neurite retraction. Three identified features, total neurite length, average neurite length, and average neurite area were able to achieve an independent test accuracy of 90.28% for the six-dosage classification problem. This NFD module and neuron image datasets are provided as a freely downloadable

A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila.

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Reid Aikin

Full Text Available Hedgehog (Hh proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia.

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Zuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R; Herrmann, Harald; Sison, Edward A; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J; Johns, Christopher; Chicas, Agustin; Mulloy, James C; Kogan, Scott C; Brown, Patrick; Valent, Peter; Bradner, James E; Lowe, Scott W; Vakoc, Christopher R

2011-08-03

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

High throughput Screening to Identify Natural Human Monoamine Oxidase B Inhibitors

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Mazzio, E; Deiab, S; Park, K; Soliman, KFA

2012-01-01

Age-related increase in monoamine oxidase B (MAO-B) may contribute to CNS neurodegenerative diseases. Moreover, MAO-B inhibitors are used in the treatment of idiopathic Parkinson disease as preliminary monotherapy or adjunct therapy with L-dopa. To date, meager natural sources of MAO-B inhibitors have been identified, and the relative strength, potency and rank of many plants relative to standard drugs such as Selegiline (L-deprenyl, Eldepryl) are not known. In this work, we developed and utilized a high throughput enzyme microarray format to screen and evaluate 905 natural product extracts (0.025–.7 mg/ml) to inhibit human MAO-B derived from BTI-TN-5B1-4 cells infected with recombinant baculovirus. The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis-matrix assisted laser desorption ionization-time-of-flight-tandem mass spectroscopy, and enzyme activity was confirmed by [1] substrate conversion (3-mM benzylamine) to H202 and [2] benzaldehyde. Of the 905 natural extracts tested, the lowest IC50s [Comfrey, Bringraj, Skullcap, Kava-kava, Wild Indigo, Gentian and Green Tea. In conclusion, the data reflect relative potency information by rank of commonly used herbs and plants that contain human MAO-B inhibitory properties in their natural form. PMID:22887993

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

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Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A comparative genomics screen identifies a Sinorhizobium meliloti 1021 sodM-like gene strongly expressed within host plant nodules

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Queiroux Clothilde

2012-05-01

Full Text Available Abstract Background We have used the genomic data in the Integrated Microbial Genomes system of the Department of Energy’s Joint Genome Institute to make predictions about rhizobial open reading frames that play a role in nodulation of host plants. The genomic data was screened by searching for ORFs conserved in α-proteobacterial rhizobia, but not conserved in closely-related non-nitrogen-fixing α-proteobacteria. Results Using this approach, we identified many genes known to be involved in nodulation or nitrogen fixation, as well as several new candidate genes. We knocked out selected new genes and assayed for the presence of nodulation phenotypes and/or nodule-specific expression. One of these genes, SMc00911, is strongly expressed by bacterial cells within host plant nodules, but is expressed minimally by free-living bacterial cells. A strain carrying an insertion mutation in SMc00911 is not defective in the symbiosis with host plants, but in contrast to expectations, this mutant strain is able to out-compete the S. meliloti 1021 wild type strain for nodule occupancy in co-inoculation experiments. The SMc00911 ORF is predicted to encode a “SodM-like” (superoxide dismutase-like protein containing a rhodanese sulfurtransferase domain at the N-terminus and a chromate-resistance superfamily domain at the C-terminus. Several other ORFs (SMb20360, SMc01562, SMc01266, SMc03964, and the SMc01424-22 operon identified in the screen are expressed at a moderate level by bacteria within nodules, but not by free-living bacteria. Conclusions Based on the analysis of ORFs identified in this study, we conclude that this comparative genomics approach can identify rhizobial genes involved in the nitrogen-fixing symbiosis with host plants, although none of the newly identified genes were found to be essential for this process.

A Targeted Approach for Congenital Cytomegalovirus Screening Within Newborn Hearing Screening.

Science.gov (United States)

Fowler, Karen B; McCollister, Faye P; Sabo, Diane L; Shoup, Angela G; Owen, Kris E; Woodruff, Julie L; Cox, Edith; Mohamed, Lisa S; Choo, Daniel I; Boppana, Suresh B

2017-02-01

Congenital cytomegalovirus (cCMV) infection remains a leading cause of childhood hearing loss. Currently universal CMV screening at birth does not exist in the United States. An alternative approach could be testing infants who do not pass their newborn hearing screening (NHS) for cCMV. This study was undertaken to evaluate whether a targeted approach will identify infants with CMV-related sensorineural hearing loss (SNHL). Infants born at 7 US medical centers received NHS and were also screened for cCMV while in the newborn nursery. Infants who tested positive for CMV received further diagnostic audiologic evaluations to identify or confirm hearing loss. Between 2007 and 2012, 99 945 newborns were screened for both hearing impairment and cCMV. Overall, 7.0% of CMV-positive infants did not pass NHS compared with 0.9% of CMV-negative infants (P CMV-infected infants who passed their NHS had SNHL confirmed by further evaluation during early infancy. NHS in this cohort identified 57% of all CMV-related SNHL that occurred in the neonatal period. A targeted CMV approach that tests newborns who fail their NHS identified the majority of infants with CMV-related SNHL at birth. However, 43% of the infants with CMV-related SNHL in the neonatal period and cCMV infants who are at risk for late onset SNHL were not identified by NHS. Copyright © 2017 by the American Academy of Pediatrics.

A Screen Identifies the Oncogenic Micro-RNA miR-378a-5p as a Negative Regulator of Oncogene-Induced Senescence

DEFF Research Database (Denmark)

Kooistra, Susanne Marije; Rudkjær, Lise Christine; Lees, Michael James

2014-01-01

Oncogene-induced senescence (OIS) can occur in response to hyperactive oncogenic signals and is believed to be a fail-safe mechanism protecting against tumorigenesis. To identify new factors involved in OIS, we performed a screen for microRNAs that can overcome or inhibit OIS in human diploid fib...

Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.

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Liu, C Tony; Layfield, Joshua P; Stewart, Robert J; French, Jarrod B; Hanoian, Philip; Asbury, John B; Hammes-Schiffer, Sharon; Benkovic, Stephen J

2014-07-23

Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

Science.gov (United States)

Miyashita, Yurina; Ohmae, Eiji; Ikura, Teikichi; Nakasone, Kaoru; Katayanagi, Katsuo

2017-05-01

Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500 mM NaCl at pH 6.0, fourfold by 250 mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000 mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.

Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

Science.gov (United States)

Sherman, Sean P; Pyle, April D

2013-01-01

Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

Using lessons from breast, cervical, and colorectal cancer screening to inform the development of lung cancer screening programs.

Science.gov (United States)

Armstrong, Katrina; Kim, Jane J; Halm, Ethan A; Ballard, Rachel M; Schnall, Mitchell D

2016-05-01

Multiple advisory groups now recommend that high-risk smokers be screened for lung cancer by low-dose computed tomography. Given that the development of lung cancer screening programs will face many of the same issues that have challenged other cancer screening programs, the National Cancer Institute-funded Population-based Research Optimizing Screening through Personalized Regimens (PROSPR) consortium was used to identify lessons learned from the implementation of breast, cervical, and colorectal cancer screening that should inform the introduction of lung cancer screening. These lessons include the importance of developing systems for identifying and recruiting eligible individuals in primary care, ensuring that screening centers are qualified and performance is monitored, creating clear communication standards for reporting screening results to referring physicians and patients, ensuring follow-up is available for individuals with abnormal test results, avoiding overscreening, remembering primary prevention, and leveraging advances in cancer genetics and immunology. Overall, this experience emphasizes that effective cancer screening is a multistep activity that requires robust strategies to initiate, report, follow up, and track each step as well as a dynamic and ongoing oversight process to revise current screening practices as new evidence regarding screening is created, new screening technologies are developed, new biological markers are identified, and new approaches to health care delivery are disseminated. Cancer 2016;122:1338-1342. © 2016 American Cancer Society. © 2016 American Cancer Society.

Vision Screening

Science.gov (United States)

... an efficient and cost-effective method to identify children with visual impairment or eye conditions that are likely to lead ... main goal of vision screening is to identify children who have or are at ... visual impairment unless treated in early childhood. Other problems that ...

Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry.

Directory of Open Access Journals (Sweden)

Yaw Shin Ooi

Full Text Available The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ, a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

Prognostic factors for specific lower extremity and spinal musculoskeletal injuries identified through medical screening and training load monitoring in professional football (soccer): a systematic review

Science.gov (United States)

Sergeant, Jamie C; Parkes, Matthew J; Callaghan, Michael J

2017-01-01

Background Medical screening and load monitoring procedures are commonly used in professional football to assess factors perceived to be associated with injury. Objectives To identify prognostic factors (PFs) and models for lower extremity and spinal musculoskeletal injuries in professional/elite football players from medical screening and training load monitoring processes. Methods The MEDLINE, AMED, EMBASE, CINAHL Plus, SPORTDiscus and PubMed electronic bibliographic databases were searched (from inception to January 2017). Prospective and retrospective cohort studies of lower extremity and spinal musculoskeletal injury incidence in professional/elite football players aged between 16 and 40 years were included. The Quality in Prognostic Studies appraisal tool and the modified Grading of Recommendations Assessment, Development and Evaluation synthesis approach was used to assess the quality of the evidence. Results Fourteen studies were included. 16 specific lower extremity injury outcomes were identified. No spinal injury outcomes were identified. Meta-analysis was not possible due to heterogeneity and study quality. All evidence related to PFs and specific lower extremity injury outcomes was of very low to low quality. On the few occasions where multiple studies could be used to compare PFs and outcomes, only two factors demonstrated consensus. A history of previous hamstring injuries (HSI) and increasing age may be prognostic for future HSI in male players. Conclusions The assumed ability of medical screening tests to predict specific musculoskeletal injuries is not supported by the current evidence. Screening procedures should currently be considered as benchmarks of function or performance only. The prognostic value of load monitoring modalities is unknown. PMID:29177074

Breast cancer screening in older women.

Science.gov (United States)

Caplan, L S; Haynes, S G

1996-01-01

There is currently an epidemic of breast cancer in women 65 years of age and older. The purposes of this paper are to explore the breast cancer screening behaviors of older women and to identify some of the determinants of screening in these women. Data were analyzed from the 1987 National Health Interview Survey, a continuous nationwide household interview survey of the U.S. civilian, noninstitutionalized population. As in other studies, the utilization of breast cancer screening by older women was less in older women than in younger women. This was true for both mammography and clinical breast examination. A number of determinants of screening in older women were identified here. Women with a usual source of care and/or no activity limitation, as well as high school graduates, were the ones most likely to have received a screening mammogram and/or a screening clinical breast exam during the past year. The failure of older women to receive adequate breast cancer screening is an important concern which should be reevaluated, given the breast cancer epidemic in this population. This study identified a number of determinants of breast cancer screening in older women. For the most part, these determinants point to the primary care physician as the key to breast cancer screening in these women. Therefore, the primary care physician must be informed of, and encouraged to follow, the recommendations for periodic breast cancer screening in older women.

Random small interfering RNA library screen identifies siRNAs that induce human erythroleukemia cell differentiation.

Science.gov (United States)

Fan, Cuiqing; Xiong, Yuan; Zhu, Ning; Lu, Yabin; Zhang, Jiewen; Wang, Song; Liang, Zicai; Shen, Yan; Chen, Meihong

2011-03-01

Cancers are characterized by poor differentiation. Differentiation therapy is a strategy to alleviate malignant phenotypes by inducing cancer cell differentiation. Here we carried out a combinatorial high-throughput screen with a random siRNA library on human erythroleukemia K-562 cell differentiation. Two siRNAs screened from the library were validated to be able to induce erythroid differentiation to varying degrees, determined by CD235 and globin up-regulation, GATA-2 down-regulation, and cell growth inhibition. The screen we performed here is the first trial of screening cancer differentiation-inducing agents from a random siRNA library, demonstrating that a random siRNA library can be considered as a new resource in efforts to seek new therapeutic agents for cancers. As a random siRNA library has a broad coverage for the entire genome, including known/unknown genes and protein coding/non-coding sequences, screening using a random siRNA library can be expected to greatly augment the repertoire of therapeutic siRNAs for cancers.

Frequencies distribution of dihydrofolate reductase and dihydropteroate synthetase mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum population from Hadhramout Governorate, Yemen.

Science.gov (United States)

Bamaga, Omar A A; Mahdy, Mohammed A K; Lim, Yvonne A L

2015-12-22

Malaria in Yemen is mainly caused by Plasmodium falciparum and 25% of the population is at high risk. Sulfadoxine-pyrimethamine (SP) had been used as monotherapy against P. falciparum. Emergence of chloroquine resistance led to the shift in anti-malarial treatment policy in Yemen to artemisinin-based combination therapy, that is artesunate (AS) plus SP as first-line therapy for uncomplicated malaria and artemether-lumefantrine as second-line treatment. This study aimed to screen mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes associated with SP resistance among P. falciparum population in Hadhramout governorate, Yemen. Genomic DNA was extracted from dried blood spots of 137 P. falciparum isolates collected from a community-based study. DNA was amplified using nested polymerase chain reaction (PCR) and subsequently sequenced for Pfdhfr and Pfdhps genes. Sequences were analysed for mutations in Pfdhfr gene codons 51, 59, 108, and 164 and in Pfdhps gene codons 436, 437, and 540. A total of 128 and 114 P. falciparum isolates were successfully sequenced for Pfdhfr and Pfdhps genes, respectively. Each Pfdhfr mutant allele (I51 and N108) in P. falciparum population had a frequency of 84%. Pfdhfr R59 mutant allele was detected in one isolate. Mutation at codon 437 (G437) in the Pfdhps gene was detected in 44.7% of falciparum malaria isolates. Frequencies of Pfdhfr double mutant genotype (I51C59N108I164) and Pfdhfr/Pfdhps triple mutant genotype (I51C59N108I164-S436G437K540) were 82.8 and 39.3%, respectively. One isolate harboured Pfdhfr triple mutant genotype (I51, R59, N108, I164) and Pfdhfr/Pfdhps quadruple mutant genotype (I51R59N108I164-S436G437K540). High frequencies of Pfdhfr and Pfdhps mutant alleles and genotypes in P. falciparum population in Hadhramout, Yemen, highlight the risk of developing resistance for SP, the partner drug of AS, which subsequently will expose the parasite to AS monotherapy increasing then the

Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design.

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Thomas S Peat

Full Text Available A fragment-based screen against human immunodeficiency virus type 1 (HIV integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.

Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

Directory of Open Access Journals (Sweden)

Sean P Sherman

Full Text Available Differentiated cells from human embryonic stem cells (hESCs provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

Inhibitors of MyD88-dependent proinflammatory cytokine production identified utilizing a novel RNA interference screening approach.

Directory of Open Access Journals (Sweden)

John S Cho

2009-09-01

Full Text Available The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha. How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.

Nutrition screening: science behind simplicity

African Journals Online (AJOL)

Nutrition screening triggers entry into the nutrition care process.1 Screening has informally been described as simple, quick or low-intensity proxy for more complex procedures. More formal definitions for the nutrition setting have been proposed, describing nutrition screening as a process of identifying patients, clients, or ...

In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

Science.gov (United States)

Miller, Peter G.; Al-Shahrour, Fatima; Hartwell, Kimberly A.; Chu, Lisa P.; Järås, Marcus; Puram, Rishi V.; Puissant, Alexandre; Callahan, Kevin P.; Ashton, John; McConkey, Marie E.; Poveromo, Luke P.; Cowley, Glenn S.; Kharas, Michael G.; Labelle, Myriam; Shterental, Sebastian; Fujisaki, Joji; Silberstein, Lev; Alexe, Gabriela; Al-Hajj, Muhammad A.; Shelton, Christopher A.; Armstrong, Scott A.; Root, David E.; Scadden, David T.; Hynes, Richard O.; Mukherjee, Siddhartha; Stegmaier, Kimberly; Jordan, Craig T.; Ebert, Benjamin L.

2013-01-01

SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML. PMID:23770013

RNA interference screen to identify pathways that enhance or reduce nonviral gene transfer during lipofection.

Science.gov (United States)

Barker, Gregory A; Diamond, Scott L

2008-09-01

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

MSBIS: A Multi-Step Biomedical Informatics Screening Approach for Identifying Medications that Mitigate the Risks of Metoclopramide-Induced Tardive Dyskinesia

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Dong Xu

2017-12-01

Full Text Available In 2009 the U.S. Food and Drug Administration (FDA placed a black box warning on metoclopramide (MCP due to the increased risks and prevalence of tardive dyskinesia (TD. In this study, we developed a multi-step biomedical informatics screening (MSBIS approach leveraging publicly available bioactivity and drug safety data to identify concomitant drugs that mitigate the risks of MCP-induced TD. MSBIS includes (1 TargetSearch (http://dxulab.org/software bioinformatics scoring for drug anticholinergic activity using CHEMBL bioactivity data; (2 unadjusted odds ratio (UOR scoring for indications of TD-mitigating effects using the FDA Adverse Event Reporting System (FAERS; (3 adjusted odds ratio (AOR re-scoring by removing the effect of cofounding factors (age, gender, reporting year; (4 logistic regression (LR coefficient scoring for confirming the best TD-mitigating drug candidates. Drugs with increasing TD protective potential and statistical significance were obtained at each screening step. Fentanyl is identified as the most promising drug against MCP-induced TD (coefficient: −2.68; p-value < 0.01. The discovery is supported by clinical reports that patients fully recovered from MCP-induced TD after fentanyl-induced general anesthesia. Loperamide is identified as a potent mitigating drug against a broader range of drug-induced movement disorders through pharmacokinetic modifications. Using drug-induced TD as an example, we demonstrated that MSBIS is an efficient in silico tool for unknown drug-drug interaction detection, drug repurposing, and combination therapy design.

MSBIS: A Multi-Step Biomedical Informatics Screening Approach for Identifying Medications that Mitigate the Risks of Metoclopramide-Induced Tardive Dyskinesia.

Science.gov (United States)

Xu, Dong; Ham, Alexandrea G; Tivis, Rickey D; Caylor, Matthew L; Tao, Aoxiang; Flynn, Steve T; Economen, Peter J; Dang, Hung K; Johnson, Royal W; Culbertson, Vaughn L

2017-12-01

In 2009 the U.S. Food and Drug Administration (FDA) placed a black box warning on metoclopramide (MCP) due to the increased risks and prevalence of tardive dyskinesia (TD). In this study, we developed a multi-step biomedical informatics screening (MSBIS) approach leveraging publicly available bioactivity and drug safety data to identify concomitant drugs that mitigate the risks of MCP-induced TD. MSBIS includes (1) TargetSearch (http://dxulab.org/software) bioinformatics scoring for drug anticholinergic activity using CHEMBL bioactivity data; (2) unadjusted odds ratio (UOR) scoring for indications of TD-mitigating effects using the FDA Adverse Event Reporting System (FAERS); (3) adjusted odds ratio (AOR) re-scoring by removing the effect of cofounding factors (age, gender, reporting year); (4) logistic regression (LR) coefficient scoring for confirming the best TD-mitigating drug candidates. Drugs with increasing TD protective potential and statistical significance were obtained at each screening step. Fentanyl is identified as the most promising drug against MCP-induced TD (coefficient: -2.68; p-valueTD after fentanyl-induced general anesthesia. Loperamide is identified as a potent mitigating drug against a broader range of drug-induced movement disorders through pharmacokinetic modifications. Using drug-induced TD as an example, we demonstrated that MSBIS is an efficient in silico tool for unknown drug-drug interaction detection, drug repurposing, and combination therapy design. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I-catalyzed azide-alkyne cycloaddition with retained activity.

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Sung In Lim

Full Text Available Cu(I-catalyzed azide-alkyne cycloaddition (CuAAC is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR, in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.

Design, Synthesis, and X-ray Crystal Structures of 2,4-Diaminofuro[2,3-d]pyrimidines as Multireceptor Tyrosine Kinase and Dihydrofolate Reductase Inhibitors

Science.gov (United States)

Gangjee, Aleem; Li, Wei; Lin, Lu; Zeng, Yibin; Ihnat, Michael; Warnke, Linda A.; Green, Dixy W.; Cody, Vivian; Pace, Jim; Queener, Sherry F.

2009-01-01

To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and 1b) were separated by HPLC and the X-ray crystal structures (2.0 Å and 1.4 Å respectively) with mouse DHFR and NADPH as well as 1b with human DHFR (1.5 Å) were determined. The E- and Z-isomers adopt different binding modes when bound to mouse DHFR. A series of 2,4-diaminofuro[2,3-d]pyrimidines 2–13 were designed and synthesized using the X-ray crystal structures of 1a and 1b with DHFR to increase their DHFR inhibitory activity. Wittig reactions of appropriate 2-methoxyphenyl ketones with 2,4-diamino-6-chloromethyl furo[2,3-d]pyrimidine afforded the C8–C9 unsaturated compounds 2–7 and catalytic reduction gave the saturated 8–13. Homologation of the C9-methyl analog maintains DHFR inhibitory activity. In addition, inhibition of EGFR and PDGFR-β were discovered for saturated C9-homologated analogs 9 and 10 that were absent in the saturated C9-methyl analogs. PMID:19748785

Parallel screening of FDA-approved antineoplastic drugs for identifying sensitizers of TRAIL-induced apoptosis in cancer cells

International Nuclear Information System (INIS)

Taylor, David J; Parsons, Christine E; Han, Haiyong; Jayaraman, Arul; Rege, Kaushal

2011-01-01

Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to death receptor 4 and 5 are promising candidates for cancer therapy due to their ability to induce apoptosis selectively in a variety of human cancer cells, while demonstrating little cytotoxicity in normal cells. Although TRAIL and agonistic antibodies to DR4 and DR5 are considered safe and promising candidates in cancer therapy, many malignant cells are resistant to DR-mediated, TRAIL-induced apoptosis. In the current work, we screened a small library of fifty-five FDA and foreign-approved anti-neoplastic drugs in order to identify candidates that sensitized resistant prostate and pancreatic cancer cells to TRAIL-induced apoptosis. FDA-approved drugs were screened for their ability to sensitize TRAIL resistant prostate cancer cells to TRAIL using an MTT assay for cell viability. Analysis of variance was used to identify drugs that exhibited synergy with TRAIL. Drugs demonstrating the highest synergy were selected as leads and tested in different prostate and pancreatic cancer cell lines, and one immortalized human pancreatic epithelial cell line. Sequential and simultaneous dosing modalities were investigated and the annexin V/propidium iodide assay, in concert with fluorescence microscopy, was employed to visualize cells undergoing apoptosis. Fourteen drugs were identified as having synergy with TRAIL, including those whose TRAIL sensitization activities were previously unknown in either prostate or pancreatic cancer cells or both. Five leads were tested in additional cancer cell lines of which, doxorubicin, mitoxantrone, and mithramycin demonstrated synergy in all lines. In particular, mitoxantrone and mithramycin demonstrated significant synergy with TRAIL and led to reduction of cancer cell viability at concentrations lower than 1 μM. At these low concentrations, mitoxantrone demonstrated selectivity toward malignant cells over normal pancreatic epithelial cells

Parallel screening of FDA-approved antineoplastic drugs for identifying sensitizers of TRAIL-induced apoptosis in cancer cells

Directory of Open Access Journals (Sweden)

Taylor David J

2011-11-01

Full Text Available Abstract Background Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL and agonistic antibodies to death receptor 4 and 5 are promising candidates for cancer therapy due to their ability to induce apoptosis selectively in a variety of human cancer cells, while demonstrating little cytotoxicity in normal cells. Although TRAIL and agonistic antibodies to DR4 and DR5 are considered safe and promising candidates in cancer therapy, many malignant cells are resistant to DR-mediated, TRAIL-induced apoptosis. In the current work, we screened a small library of fifty-five FDA and foreign-approved anti-neoplastic drugs in order to identify candidates that sensitized resistant prostate and pancreatic cancer cells to TRAIL-induced apoptosis. Methods FDA-approved drugs were screened for their ability to sensitize TRAIL resistant prostate cancer cells to TRAIL using an MTT assay for cell viability. Analysis of variance was used to identify drugs that exhibited synergy with TRAIL. Drugs demonstrating the highest synergy were selected as leads and tested in different prostate and pancreatic cancer cell lines, and one immortalized human pancreatic epithelial cell line. Sequential and simultaneous dosing modalities were investigated and the annexin V/propidium iodide assay, in concert with fluorescence microscopy, was employed to visualize cells undergoing apoptosis. Results Fourteen drugs were identified as having synergy with TRAIL, including those whose TRAIL sensitization activities were previously unknown in either prostate or pancreatic cancer cells or both. Five leads were tested in additional cancer cell lines of which, doxorubicin, mitoxantrone, and mithramycin demonstrated synergy in all lines. In particular, mitoxantrone and mithramycin demonstrated significant synergy with TRAIL and led to reduction of cancer cell viability at concentrations lower than 1 μM. At these low concentrations, mitoxantrone demonstrated selectivity toward

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ORF Alignment: NC_002755 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available hain A, Dihydrofolate ... Reductase Of Mycobacterium Tuberculosis Complexed With ... Nadph And... Methotrexate pdb|1DG8|A Chain A, Dihydrofolate ... Reductase Of Mycobacterium Tuberculosis... Complexed With ... Nadph pdb|1DG7|A Chain A, Dihydrofolate Reductase Of ... Mycobacterium Tuberculosi...late ... Reductase Of Mycobacterium Tuberculosis Complexed With ... Nadph And Trimethoprim emb

A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics

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Kirsten Tschapalda

2016-06-01

Full Text Available Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1, a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.

Symptom screening rules to identify active pulmonary tuberculosis: Findings from the Zambian South African Tuberculosis and HIV/AIDS Reduction (ZAMSTAR trial prevalence surveys.

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M M Claassens

Full Text Available High tuberculosis (TB burden countries should consider systematic screening among adults in the general population. We identified symptom screening rules to be used in addition to cough ≥2 weeks, in a context where X-ray screening is not feasible, aiming to increase the sensitivity of screening while achieving a specificity of ≥85%.We used 2010 Zambia South Africa Tuberculosis and HIV/AIDS Reduction (ZAMSTAR survey data: a South African (SA training dataset, a SA testing dataset for internal validation and a Zambian dataset for external validation. Regression analyses investigated relationships between symptoms or combinations of symptoms and active disease. Sensitivity and specificity were calculated for candidate rules.Among all participants, the sensitivity of using only cough ≥2 weeks as a screening rule was less than 25% in both SA and Zambia. The addition of any three of six TB symptoms (cough <2 weeks, night sweats, weight loss, fever, chest pain, shortness of breath, or 2 or more of cough <2 weeks, night sweats, and weight loss, increased the sensitivity to ~38%, while reducing specificity from ~95% to ~85% in SA and ~97% to ~92% in Zambia. Among HIV-negative adults, findings were similar in SA, whereas in Zambia the increase in sensitivity was relatively small (15% to 22%.High TB burden countries should investigate cost-effective strategies for systematic screening: one such strategy could be to use our rule in addition to cough ≥2 weeks.

Universal screening for alcohol misuse in acute medical admissions is feasible and identifies patients at high risk of liver disease.

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Westwood, Greta; Meredith, Paul; Atkins, Susan; Greengross, Peter; Schmidt, Paul E; Aspinall, Richard J

2017-09-01

Many people who die from alcohol related liver disease (ARLD) have a history of recurrent admissions to hospital, representing potential missed opportunities for intervention. Universal screening for alcohol misuse has been advocated but it is not known if this is achievable or effective at detecting individuals at high risk of ARLD. We systematically screened all admissions to the Acute Medical Unit (AMU) of a large acute hospital using an electronic data capture system in real time. Patients at an increasing risk of alcohol harm were referred for either brief intervention (BI) or further assessment by an Alcohol Specialist Nursing Service (ASNS). Additional data were recorded on admission diagnoses, alcohol unit consumption, previous attendances, previous admissions, length of stay and mortality. Between July 2011 and March 2014, there were 53,165 admissions and 48,211 (90.68%) completed screening. Of these, 1,122 (2.3%) were classified as "increasing", and 1,921 (4.0%) as "high" risk of alcohol harm. High risk patients had more hospital admissions in the three previous years (average 4.74) than the low (3.00) and increasing (2.92) risk groups (prisk patients also had more frequent emergency department (ED) attendances (7.68) than the lower (2.64) and increasing (3.81) groups (prisk group were seen by the ASNS and 1,135 (81.2%) had an Alcohol Use Disorders Identification Test (AUDIT) score over 20 with 527 (37.8%) recording the maximum value of 40. Compared to the other groups, high risk patients had a distinct profile of admissions with the most common diagnoses being mental health disorders, gastro-intestinal bleeding, poisoning and liver disease. Universal screening of admissions for alcohol misuse is feasible and identifies a cohort with frequent ED attendances, recurrent admissions and an elevated risk of ARLD. An additional group of patients at an increasing risk of alcohol harm can be identified in a range of common presentations. These patients can be

Diagnostic accuracy of an integrated respiratory guideline in identifying patients with respiratory symptoms requiring screening for pulmonary tuberculosis: a cross-sectional study

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Majara Bosielo P

2006-08-01

Full Text Available Abstract Background To evaluate the diagnostic accuracy of the integrated Practical Approach to Lung Health in South Africa (PALSA guideline in identifying patients requiring bacteriological screening for tuberculosis (TB, and to determine which clinical features best predict suspected and bacteriologically-confirmed tuberculosis among patients with respiratory symptoms. Methods A prospective, cross-sectional study in which 1392 adult patients with cough and/or difficult breathing, attending a primary care facility in Cape Town, South Africa, were evaluated by a nurse using the guideline. The accuracy of a nurse using the guideline to identify TB suspects was compared to that of primary care physicians' diagnoses of (1 suspected TB, and (2 proven TB supported by clinical information and chest radiographs. Results The nurse using the guideline identified 516 patients as TB suspects compared with 365 by the primary care physicians, representing a sensitivity of 76% (95% confidence interval (CI 71%–79%, specificity of 77% (95% CI 74%–79%, positive predictive value of 53% (95% CI 49%–58%, negative predictive value of 90% (95% CI 88%–92%, and area under the receiver operating characteristic curve (ARUC of 0.76 (95% CI 0.74–0.79. Sputum results were obtained in 320 of the 365 primary care physicians TB suspects (88%; 40 (13% of these were positive for TB. Only 4 cases were not identified by the nurse using the guideline. The primary care physicians diagnostic accuracy in diagnosing bacteriologically-confirmed TB (n = 320 was as follows: sensitivity 90% (95% CI 76%–97%, specificity 65% (95% CI 63%–68%, negative predictive value 7% (95% CI 5%–10%, positive predictive value 99.5% (95% CI 98.8%–99.8%, and ARUC 0.78 (95% CI 0.73–0.82. Weight loss, pleuritic pain, and night sweats were independently associated with the diagnosis of bacteriologically-confirmed tuberculosis (positive likelihood ratio if all three present = 16.7, 95% CI 5

Nutritional Risk Screening 2002, Short Nutritional Assessment Questionnaire, Malnutrition Screening Tool, and Malnutrition Universal Screening Tool Are Good Predictors of Nutrition Risk in an Emergency Service.

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Rabito, Estela Iraci; Marcadenti, Aline; da Silva Fink, Jaqueline; Figueira, Luciane; Silva, Flávia Moraes

2017-08-01

There is an international consensus that nutrition screening be performed at the hospital; however, there is no "best tool" for screening of malnutrition risk in hospitalized patients. To evaluate (1) the accuracy of the MUST (Malnutrition Universal Screening Tool), MST (Malnutrition Screening Tool), and SNAQ (Short Nutritional Assessment Questionnaire) in comparison with the NRS-2002 (Nutritional Risk Screening 2002) to identify patients at risk of malnutrition and (2) the ability of these nutrition screening tools to predict morbidity and mortality. A specific questionnaire was administered to complete the 4 screening tools. Outcomes measures included length of hospital stay, transfer to the intensive care unit, presence of infection, and incidence of death. A total of 752 patients were included. The nutrition risk was 29.3%, 37.1%, 33.6%, and 31.3% according to the NRS-2002, MUST, MST, and SNAQ, respectively. All screening tools showed satisfactory performance to identify patients at nutrition risk (area under the receiver operating characteristic curve between 0.765-0.808). Patients at nutrition risk showed higher risk of very long length of hospital stay as compared with those not at nutrition risk, independent of the tool applied (relative risk, 1.35-1.78). Increased risk of mortality (2.34 times) was detected by the MUST. The MUST, MST, and SNAQ share similar accuracy to the NRS-2002 in identifying risk of malnutrition, and all instruments were positively associated with very long hospital stay. In clinical practice, the 4 tools could be applied, and the choice for one of them should be made per the particularities of the service.

Chemical screening identifies ROCK as a target for recovering mitochondrial function in Hutchinson-Gilford progeria syndrome.

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Kang, Hyun Tae; Park, Joon Tae; Choi, Kobong; Choi, Hyo Jei Claudia; Jung, Chul Won; Kim, Gyu Ree; Lee, Young-Sam; Park, Sang Chul

2017-06-01

Hutchinson-Gilford progeria syndrome (HGPS) constitutes a genetic disease wherein an aging phenotype manifests in childhood. Recent studies indicate that reactive oxygen species (ROS) play important roles in HGPS phenotype progression. Thus, pharmacological reduction in ROS levels has been proposed as a potentially effective treatment for patient with this disorder. In this study, we performed high-throughput screening to find compounds that could reduce ROS levels in HGPS fibroblasts and identified rho-associated protein kinase (ROCK) inhibitor (Y-27632) as an effective agent. To elucidate the underlying mechanism of ROCK in regulating ROS levels, we performed a yeast two-hybrid screen and discovered that ROCK1 interacts with Rac1b. ROCK activation phosphorylated Rac1b at Ser71 and increased ROS levels by facilitating the interaction between Rac1b and cytochrome c. Conversely, ROCK inactivation with Y-27632 abolished their interaction, concomitant with ROS reduction. Additionally, ROCK activation resulted in mitochondrial dysfunction, whereas ROCK inactivation with Y-27632 induced the recovery of mitochondrial function. Furthermore, a reduction in the frequency of abnormal nuclear morphology and DNA double-strand breaks was observed along with decreased ROS levels. Thus, our study reveals a novel mechanism through which alleviation of the HGPS phenotype is mediated by the recovery of mitochondrial function upon ROCK inactivation. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Optimal screening of children with acute malnutrition requires a change in current WHO guidelines as MUAC and WHZ identify different patient groups.

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Laillou, Arnaud; Prak, Sophonneary; de Groot, Richard; Whitney, Sophie; Conkle, Joel; Horton, Lindsey; Un, Sam Oeurn; Dijkhuizen, Marjoleine A; Wieringa, Frank T

2014-01-01

Timely treatment of acute malnutrition in children 500,000 deaths annually. Screening at community level is essential to identify children with malnutrition. Current WHO guidelines for community screening for malnutrition recommend a Mid Upper Arm Circumference (MUAC) of malnutrition (SAM). However, it is currently unclear how MUAC relates to the other indicator used to define acute malnutrition: weight-for-height Z-score (WHZ). Secondary data from >11,000 Cambodian children, obtained by different surveys between 2010 and 2012, was used to calculate sensitivity and ROC curves for MUAC and WHZ. The secondary analysis showed that using the current WHO cut-off of 115 mm for screening for severe acute malnutrition over 90% of children with a weight-for-height z-score (WHZ) children with a MUAC65% of children with a WHZchildren with acute malnutrition, therefore these 2 indicators should be regarded as independent from each other. We suggest a 2-step model with MUAC used a screening at community level, followed by MUAC and WHZ measured at a primary health care unit, with both indicators used independently to diagnose severe acute malnutrition. Current guidelines should be changed to reflect this, with treatment initiated when either MUAC <115 mm or WHZ<-3.

Optimal Screening of Children with Acute Malnutrition Requires a Change in Current WHO Guidelines as MUAC and WHZ Identify Different Patient Groups

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Laillou, Arnaud; Prak, Sophonneary; de Groot, Richard; Whitney, Sophie; Conkle, Joel; Horton, Lindsey; Un, Sam Oeurn; Dijkhuizen, Marjoleine A.; Wieringa, Frank T.

2014-01-01

Background Timely treatment of acute malnutrition in children 500,000 deaths annually. Screening at community level is essential to identify children with malnutrition. Current WHO guidelines for community screening for malnutrition recommend a Mid Upper Arm Circumference (MUAC) of malnutrition (SAM). However, it is currently unclear how MUAC relates to the other indicator used to define acute malnutrition: weight-for-height Z-score (WHZ). Methods Secondary data from >11,000 Cambodian children, obtained by different surveys between 2010 and 2012, was used to calculate sensitivity and ROC curves for MUAC and WHZ. Findings The secondary analysis showed that using the current WHO cut-off of 115 mm for screening for severe acute malnutrition over 90% of children with a weight-for-height z-score (WHZ) 65% of children with a WHZmalnutrition, therefore these 2 indicators should be regarded as independent from each other. We suggest a 2-step model with MUAC used a screening at community level, followed by MUAC and WHZ measured at a primary health care unit, with both indicators used independently to diagnose severe acute malnutrition. Current guidelines should be changed to reflect this, with treatment initiated when either MUAC <115 mm or WHZ<−3. PMID:24983995

WISDOM-II: Screening against multiple targets implicated in malaria using computational grid infrastructures

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Kenyon Colin

2009-05-01

Full Text Available Abstract Background Despite continuous efforts of the international community to reduce the impact of malaria on developing countries, no significant progress has been made in the recent years and the discovery of new drugs is more than ever needed. Out of the many proteins involved in the metabolic activities of the Plasmodium parasite, some are promising targets to carry out rational drug discovery. Motivation Recent years have witnessed the emergence of grids, which are highly distributed computing infrastructures particularly well fitted for embarrassingly parallel computations like docking. In 2005, a first attempt at using grids for large-scale virtual screening focused on plasmepsins and ended up in the identification of previously unknown scaffolds, which were confirmed in vitro to be active plasmepsin inhibitors. Following this success, a second deployment took place in the fall of 2006 focussing on one well known target, dihydrofolate reductase (DHFR, and on a new promising one, glutathione-S-transferase. Methods In silico drug design, especially vHTS is a widely and well-accepted technology in lead identification and lead optimization. This approach, therefore builds, upon the progress made in computational chemistry to achieve more accurate in silico docking and in information technology to design and operate large scale grid infrastructures. Results On the computational side, a sustained infrastructure has been developed: docking at large scale, using different strategies in result analysis, storing of the results on the fly into MySQL databases and application of molecular dynamics refinement are MM-PBSA and MM-GBSA rescoring. The modeling results obtained are very promising. Based on the modeling results, In vitro results are underway for all the targets against which screening is performed. Conclusion The current paper describes the rational drug discovery activity at large scale, especially molecular docking using FlexX software

Structure Based Virtual Screening Studies to Identify Novel Potential Compounds for GPR142 and Their Relative Dynamic Analysis for Study of Type 2 Diabetes

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Aman C. Kaushik

2018-02-01

Full Text Available GPR142 (G protein receptor 142 is a novel orphan GPCR (G protein coupled receptor belonging to “Class A” of GPCR family and expressed in β cells of pancreas. In this study, we reported the structure based virtual screening to identify the hit compounds which can be developed as leads for potential agonists. The results were validated through induced fit docking, pharmacophore modeling, and system biology approaches. Since, there is no solved crystal structure of GPR142, we attempted to predict the 3D structure followed by validation and then identification of active site using threading and ab initio methods. Also, structure based virtual screening was performed against a total of 1171519 compounds from different libraries and only top 20 best hit compounds were screened and analyzed. Moreover, the biochemical pathway of GPR142 complex with screened compound2 was also designed and compared with experimental data. Interestingly, compound2 showed an increase in insulin production via Gq mediated signaling pathway suggesting the possible role of novel GPR142 agonists in therapy against type 2 diabetes.

Structure Based Virtual Screening Studies to Identify Novel Potential Compounds for GPR142 and Their Relative Dynamic Analysis for Study of Type 2 Diabetes

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Kaushik, Aman C.; Kumar, Sanjay; Wei, Dong Q.; Sahi, Shakti

2018-02-01

GPR142 (G protein receptor 142) is a novel orphan GPCR (G protein coupled receptor) belonging to ‘Class A’ of GPCR family and expressed in beta cells of pancreas. In this study, we reported the structure based virtual screening to identify the hit compounds which can be developed as leads for potential agonists. The results were validated through induced fit docking, pharmacophore modeling and system biology approaches. Since, there is no solved crystal structure of GPR142, we attempted to predict the 3D structure followed by validation and then identification of active site using threading and ab initio methods. Also, structure based virtual screening was performed against a total of 1171519 compounds from different libraries and only top 20 best hit compounds were screened and analyzed. Moreover, the biochemical pathway of GPR142 complex with screened compound2 was also designed and compared with experimental data. Interestingly, compound2 showed an increase in insulin production via Gq mediated signaling pathway suggesting the possible role of novel GPR142 agonists in therapy against type 2 diabetes.

Domestic violence screening in a military setting: provider screening and attitudes.

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Lutgendorf, Monica; Busch, Jeanne; Magann, Everett F; Morrison, John C

2010-06-01

Domestic violence is an important healthcare problem, and it appears more prevalent in military patient populations although no one has demonstrated the cause behind this phenomenon. The purpose of this observational study was to assess data regarding domestic violence screening from practitioners at one military training center. This study used an anonymous questionnaire for physicians, nurses and nurse midwives, which surveyed current methods, attitudes toward screening, and barriers for such assessment. Fifty-seven surveys were distributed, and 26 were returned for a response rate of 45.6%. Only about a third (38.5%) of the practitioners screened all obstetric patients while the remainder screened selected patients for domestic violence. Even less (19%) screened gynecology patients routinely, whereas 69% reported they screened selected women with chronic or somatic complaints. A history of prior abuse in the respondents led practitioners to try to identify such patients within their practice. Lack of education or training was the most common barrier to universal screening followed by time constraints and frustration about not being able to address adequately the problem when noted. These results emphasized the importance of an educational program to increase domestic violence awareness and routine screening.

Screening children for elevated blood lead - Learnings from the literature

International Nuclear Information System (INIS)

Boreland, Frances; Lyle, David

2008-01-01

Introduction: Although it is important that children at risk of developing elevated blood lead receive regular screening, attendance at screening programs is variable. A literature review was undertaken to better understand the factors that affect carers' decisions about whether or not to take their children for blood lead screening. Method: Electronic databases (Medline, EMBASE, CINAHL, Psychinfo) were searched to identify relevant publications, supported by reviewing reference lists of identified articles and searching with internet-based search engines. Results: Thirty-four published studies dealing with blood lead screening rates were identified, of which only seven papers focused specifically on parent's attitudes to blood lead screening. The barriers to and enablers of screening for elevated blood lead levels appear to be similar to those identified for other screening programs. Discussion: It is recommended that attendance at screening be routinely monitored, and that where participation is suboptimal further research be undertaken, in close co-operation with affected communities or sub-groups, to determine how best to encourage screening and to protect children from lead. It is important to minimize stigma and to ensure, as far as possible, that practical barriers such as lack of transport do not restrict access to screening programs

Selective anti-proliferation of HER2-positive breast cancer cells by anthocyanins identified by high-throughput screening.

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Weihua Liu

Full Text Available Overexpressed Human epidermal growth factor receptor 2 (HER2 drives the biology of 20% breast cancer and is a prediction of a poor prognosis for patients. HER2-targeted therapies significantly improve outcomes for HER2-positive patients. Traditional Chinese herbs/medicines have been used to treat breast cancer patients including HER2-positive patients in Asia for decades. Although the traditional medicines demonstrate efficacy in clinics for HER2-positive patients, the mechanism is largely unknown. In this article, we screened a 10,000 natural product library in 6 different cell lines representing breast cancer, and assessed the ability of each drug to cause cytotoxicity through a high-throughput screening approach. We have identified eight natural compounds that selectively inhibit the proliferation of HER2-positive cells. Two of the hit compounds, peonidin-3-glucoside and cyaniding-3-glucoside, are both extracts from black rice. They inhibit the phospho-HER2 and phospho-AKT and were confirmed to induce HER2-psotive breast cancer cells apoptosis both in vitro and in vivo. Peonidin-3-glucoside and cyaniding-3-glucoside treatments significantly reduced the tumor size and volume in vivo compared to the control group. There is no significant difference of antitumorgenic effects between peonidin-3-glucoside and cyaniding-3-glucoside treatments.

The comparison of the performance of two screening strategies identifying newly-diagnosed HIV during pregnancy

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Boer, K.; Smit, C.; Flier, M. van der; Wolf, F. de; Koopmans †, P.P.; Crevel, R. van; Eggink, A.J.; Groot, R. de; Keuter, M.; Post, F.; Ven, A.J.A.M. van der; Warris, A.; et al.,

2011-01-01

BACKGROUND: In the Netherlands, a non-selective opt-out instead of a selective opt-in antenatal HIV screening strategy was implemented in 2004. In case of infection, screening was followed by prevention of mother-to-child-transmission (PMTCT). We compared the performance of the two strategies in

Transcriptional Profiling of Biofilm Regulators Identified by an Overexpression Screen in Saccharomyces cerevisiae

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Cromie, Gareth A.; Tan, Zhihao; Hays, Michelle; Sirr, Amy; Jeffery, Eric W.; Dudley, Aimée M.

2017-01-01

Biofilm formation by microorganisms is a major cause of recurring infections and removal of biofilms has proven to be extremely difficult given their inherent drug resistance . Understanding the biological processes that underlie biofilm formation is thus extremely important and could lead to the development of more effective drug therapies, resulting in better infection outcomes. Using the yeast Saccharomyces cerevisiae as a biofilm model, overexpression screens identified DIG1, SFL1, HEK2, TOS8, SAN1, and ROF1/YHR177W as regulators of biofilm formation. Subsequent RNA-seq analysis of biofilm and nonbiofilm-forming strains revealed that all of the overexpression strains, other than DIG1 and TOS8, were adopting a single differential expression profile, although induced to varying degrees. TOS8 adopted a separate profile, while the expression profile of DIG1 reflected the common pattern seen in most of the strains, plus substantial DIG1-specific expression changes. We interpret the existence of the common transcriptional pattern seen across multiple, unrelated overexpression strains as reflecting a transcriptional state, that the yeast cell can access through regulatory signaling mechanisms, allowing an adaptive morphological change between biofilm-forming and nonbiofilm states. PMID:28673928

Identifying obstructive sleep apnea after stroke/TIA: evaluating four simple screening tools.

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Boulos, Mark I; Wan, Anthony; Im, James; Elias, Sara; Frankul, Fadi; Atalla, Mina; Black, Sandra E; Basile, Vincenzo S; Sundaram, Arun; Hopyan, Julia J; Boyle, Karl; Gladstone, David J; Murray, Brian J; Swartz, Richard H

2016-05-01

Despite its high prevalence and unfavorable clinical consequences, obstructive sleep apnea (OSA) often remains underappreciated after cerebrovascular events. The purpose of our study was to evaluate the clinical utility of four simple paper-based screening tools for excluding OSA after stroke or transient ischemic attack (TIA). Sixty-nine inpatients and outpatients with stroke or TIA during the past 180 days completed the 4-Variable screening tool (4V), STOP-BAG questionnaire (ie, STOP-BANG questionnaire without the neck circumference measurement), Berlin questionnaire, and the Sleep Obstructive apnea score optimized for Stroke (SOS). They subsequently underwent objective testing using a portable sleep monitoring device. Cutoffs were selected to maximize sensitivity and exclude OSA (AHI ≥ 10) in ≥10% of the cohort. The mean age was 68.3 ± 14.2 years and 47.8% were male. Thirty-two patients (46.4%) were found to have OSA. Male sex, body mass index (BMI), and atrial fibrillation were independent predictors of OSA. Among the screening tools, the 4V had the greatest area under the curve (AUC) of 0.688 (p = 0.007); the sensitivity was 96.9% for a cutoff of stroke/TIA. Due to the atypical presentation of poststroke/TIA OSA, these tools are only moderately predictive; objective testing should still be used for OSA diagnosis in this population. Copyright © 2016 Elsevier B.V. All rights reserved.

Screening and prioritisation of chemical risks from metal mining operations, identifying exposure media of concern.

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Pan, Jilang; Oates, Christopher J; Ihlenfeld, Christian; Plant, Jane A; Voulvoulis, Nikolaos

2010-04-01

Metals have been central to the development of human civilisation from the Bronze Age to modern times, although in the past, metal mining and smelting have been the cause of serious environmental pollution with the potential to harm human health. Despite problems from artisanal mining in some developing countries, modern mining to Western standards now uses the best available mining technology combined with environmental monitoring, mitigation and remediation measures to limit emissions to the environment. This paper develops risk screening and prioritisation methods previously used for contaminated land on military and civilian sites and engineering systems for the analysis and prioritisation of chemical risks from modern metal mining operations. It uses hierarchical holographic modelling and multi-criteria decision making to analyse and prioritise the risks from potentially hazardous inorganic chemical substances released by mining operations. A case study of an active platinum group metals mine in South Africa is used to demonstrate the potential of the method. This risk-based methodology for identifying, filtering and ranking mining-related environmental and human health risks can be used to identify exposure media of greatest concern to inform risk management. It also provides a practical decision-making tool for mine acquisition and helps to communicate risk to all members of mining operation teams.

Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

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Felgueiras, Juliana; Silva, Joana Vieira; Fardilha, Margarida

2018-01-16

"A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of

The Clinical Validation of the Athlete Sleep Screening Questionnaire: an Instrument to Identify Athletes that Need Further Sleep Assessment.

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Bender, Amy M; Lawson, Doug; Werthner, Penny; Samuels, Charles H

2018-06-04

Previous research has established that general sleep screening questionnaires are not valid and reliable in an athlete population. The Athlete Sleep Screening Questionnaire (ASSQ) was developed to address this need. While the initial validation of the ASSQ has been established, the clinical validity of the ASSQ has yet to be determined. The main objective of the current study was to evaluate the clinical validity of the ASSQ. Canadian National Team athletes (N = 199; mean age 24.0 ± 4.2 years, 62% females; from 23 sports) completed the ASSQ. A subset of athletes (N = 46) were randomized to the clinical validation sub-study which required subjects to complete an ASSQ at times 2 and 3 and to have a clinical sleep interview by a sleep medicine physician (SMP) who rated each subjects' category of clinical sleep problem and provided recommendations to improve sleep. To assess clinical validity, the SMP category of clinical sleep problem was compared to the ASSQ. The internal consistency (Cronbach's alpha = 0.74) and test-retest reliability (r = 0.86) of the ASSQ were acceptable. The ASSQ demonstrated good agreement with the SMP (Cohen's kappa = 0.84) which yielded a diagnostic sensitivity of 81%, specificity of 93%, positive predictive value of 87%, and negative predictive value of 90%. There were 25.1% of athletes identified to have clinically relevant sleep disturbances that required further clinical sleep assessment. Sleep improved from time 1 at baseline to after the recommendations at time 3. Sleep screening athletes with the ASSQ provides a method of accurately determining which athletes would benefit from preventative measures and which athletes suffer from clinically significant sleep problems. The process of sleep screening athletes and providing recommendations improves sleep and offers a clinical intervention output that is simple and efficient for teams and athletes to implement.

Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

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Craig B Bennett

2008-01-01

Full Text Available BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1 to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34 and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1. Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII carboxy terminal domain (P-CTD, phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1

The comparison of the performance of two screening strategies identifying newly-diagnosed HIV during pregnancy

NARCIS (Netherlands)

Boer, Kees; Smit, Colette; van der Flier, Michiel; de Wolf, Frank; Bezemer, D. O.; Gras, L. A. J.; Kesselring, A. M.; van Sighem, A. I.; Smit, C.; Zhang, S.; Zaheri, S.; Bronsveld, W.; Hillebrand-Haverkort, M. E.; Prins, J. M.; Branger, J.; Eeftinck Schattenkerk, J. K. M.; Gisolf, J.; Godfried, M. H.; Lange, J. M. A.; Lettinga, K. D.; van der Meer, J. T. M.; Nellen, F. J. B.; van der Poll, T.; Reiss, P.; Ruys, Th A.; Steingrover, R.; van Twillert, G.; Vermeulen, J. N.; Vrouenraets, S. M. E.; van Vugt, M.; Wit, F. W. M. N.; Kuijpers, T. W.; Pajkrt, D.; Scherpbier, H. J.; van Eeden, A.; Brinkman, K.; van den Berk, G. E. L.; Blok, W. L.; Frissen, P. H. J.; Roos, J. C.; Schouten, W. E. M.; Bekendam, D. J.; Weigel, H. M.; Mulder, J. W.; van Gorp, E. C. M.; Wagenaar, J.; Veenstra, J.; Danner, S. A.; van Agtmael, M. A.; Claessen, F. A. P.

2011-01-01

In the Netherlands, a non-selective opt-out instead of a selective opt-in antenatal HIV screening strategy was implemented in 2004. In case of infection, screening was followed by prevention of mother-to-child-transmission (PMTCT). We compared the performance of the two strategies in terms of

MSBIS: A Multi-Step Biomedical Informatics Screening Approach for Identifying Medications that Mitigate the Risks of Metoclopramide-Induced Tardive Dyskinesia

OpenAIRE

Dong Xu; Alexandrea G. Ham; Rickey D. Tivis; Matthew L. Caylor; Aoxiang Tao; Steve T. Flynn; Peter J. Economen; Hung K. Dang; Royal W. Johnson; Vaughn L. Culbertson

2017-01-01

In 2009 the U.S. Food and Drug Administration (FDA) placed a black box warning on metoclopramide (MCP) due to the increased risks and prevalence of tardive dyskinesia (TD). In this study, we developed a multi-step biomedical informatics screening (MSBIS) approach leveraging publicly available bioactivity and drug safety data to identify concomitant drugs that mitigate the risks of MCP-induced TD. MSBIS includes (1) TargetSearch (http://dxulab.org/software) bioinformatics scoring for drug anti...

Screening for diabetes in unconventional locations: resource implications and economics of screening in optometry practices.

Science.gov (United States)

Howse, Jennifer H; Jones, Steve; Hungin, A Pali S

2011-10-01

Unconventional locations outwith general medical practice may prove opportunities for screening. The aim was to determine the resource implications and economics of a screening service using random capillary blood glucose (rCBG) tests to detect raised blood glucose levels in the "at risk" population attending high street optometry practices. A screening service was implemented in optometry practices in North East England: the cost of the service and the implication of different screening strategies was estimated. The cost of a screening test was £5.53-£11.20, depending on the screening strategy employed and who carried out the testing. Refining the screening strategy to target those ≥40 years with BMI of ≥25 kg/m(2) and/or family history of diabetes resulted in a cost per case referred to the GP of £14.38-£26.36. Implementing this strategy in half of optometric practices in England would have the potential to identify up to 150,000 new cases of diabetes and prediabetes a year. Optometry practices provide an effective way of identifying people who would benefit from further investigation for diabetes. Effectiveness could be improved further by improving cooperation and communication between optometrists and medical practitioners. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis.

Science.gov (United States)

Fazly, Ahmed; Jain, Charu; Dehner, Amie C; Issi, Luca; Lilly, Elizabeth A; Ali, Akbar; Cao, Hong; Fidel, Paul L; Rao, Reeta P; Kaufman, Paul D

2013-08-13

Infection by pathogenic fungi, such as Candida albicans, begins with adhesion to host cells or implanted medical devices followed by biofilm formation. By high-throughput phenotypic screening of small molecules, we identified compounds that inhibit adhesion of C. albicans to polystyrene. Our lead candidate compound also inhibits binding of C. albicans to cultured human epithelial cells, the yeast-to-hyphal morphological transition, induction of the hyphal-specific HWP1 promoter, biofilm formation on silicone elastomers, and pathogenesis in a nematode infection model as well as alters fungal morphology in a mouse mucosal infection assay. We term this compound filastatin based on its strong inhibition of filamentation, and we use chemical genetic experiments to show that it acts downstream of multiple signaling pathways. These studies show that high-throughput functional assays targeting fungal adhesion can provide chemical probes for study of multiple aspects of fungal pathogenesis.

Application of ToxCast High-Throughput Screening and ...

Science.gov (United States)

Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenesis Distruptors Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenssis Distruptors

Social Health Insurance-Based Simultaneous Screening for 154 Mutations in 19 Deafness Genes Efficiently Identified Causative Mutations in Japanese Hearing Loss Patients.

Directory of Open Access Journals (Sweden)

Kentaro Mori

Full Text Available Sensorineural hearing loss is one of the most common neurosensory disorders in humans. The incidence of SNHL is estimated to be 1 in 500-1000 newborns. In more than half of these patients, the hearing loss is associated with genetic causes. In Japan, genetic testing for the patients with SNHL using the Invader assay to screen for 46 mutations in 13 deafness genes was approved by the Ministry of Health, Labour and Welfare for inclusion in social health insurance coverage in 2012. Furthermore, from August 2015, this genetic testing has been expanded to screen for 154 mutations in 19 deafness genes using targeted genomic enrichment with massively parallel DNA sequencing combined with the Invader assay and TaqMan genotyping. For this study we analyzed 717 unrelated Japanese hearing loss patients. The total allele frequency of 154 mutations in 19 deafness genes was 32.64% (468/1434 and the total numbers of cases associated with at least one mutation was 44.07% (316/717. Among these, we were able to diagnose 212 (30% patients, indicating that the present screening could efficiently identify causative mutations in hearing loss patients. It is noteworthy that 27 patients (3.8% had coexistent multiple mutations in different genes. Five of these 27 patients (0.7%, 5/717 overall were diagnosed with genetic hearing loss affected by concomitant with responsible mutations in more than two different genes. For patients identified with multiple mutations in different genes, it is necessary to consider that several genes might have an impact on their phenotypes.

A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

NARCIS (Netherlands)

Damveld, R.A.; Franken, A.; Arentshorst, M.; Punt, P.J.; Klis, F.M.; van den Hondel, C.A.M.J.J.; Ram, A.F.J.

2008-01-01

To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative

Validation of the TAPS-1: A Four-Item Screening Tool to Identify Unhealthy Substance Use in Primary Care.

Science.gov (United States)

Gryczynski, Jan; McNeely, Jennifer; Wu, Li-Tzy; Subramaniam, Geetha A; Svikis, Dace S; Cathers, Lauretta A; Sharma, Gaurav; King, Jacqueline; Jelstrom, Eve; Nordeck, Courtney D; Sharma, Anjalee; Mitchell, Shannon G; O'Grady, Kevin E; Schwartz, Robert P

2017-09-01

The Tobacco, Alcohol, Prescription Medication, and Other Substance use (TAPS) tool is a combined two-part screening and brief assessment developed for adult primary care patients. The tool's first-stage screening component (TAPS-1) consists of four items asking about past 12-month use for four substance categories, with response options of never, less than monthly, monthly, weekly, and daily or almost daily. To validate the TAPS-1 in primary care patients. Participants completed the TAPS tool in self- and interviewer-administered formats, in random order. In this secondary analysis, the TAPS-1 was evaluated against DSM-5 substance use disorder (SUD) criteria to determine optimal cut-points for identifying unhealthy substance use at three severity levels (problem use, mild SUD, and moderate-to-severe SUD). Two thousand adult patients at five primary care sites. DSM-5 SUD criteria were determined via the modified Composite International Diagnostic Interview. Oral fluid was used as a biomarker of recent drug use. Optimal frequency-of-use cut-points on the self-administered TAPS-1 for identifying SUDs were ≥ monthly use for tobacco and alcohol (sensitivity = 0.92 and 0.71, specificity = 0.80 and 0.85, AUC = 0.86 and 0.78, respectively) and any reported use for illicit drugs and prescription medication misuse (sensitivity = 0.93 and 0.89, specificity = 0.85 and 0.91, AUC = 0.89 and 0.90, respectively). The performance of the interviewer-administered format was similar. When administered first, the self-administered format yielded higher disclosure rates for past 12-month alcohol use, illicit drug use, and prescription medication misuse. Frequency of use alone did not provide sufficient information to discriminate between gradations of substance use problem severity. Among those who denied drug use on the TAPS-1, less than 4% had a drug-positive biomarker. The TAPS-1 can identify unhealthy substance use in primary care patients with a high level of accuracy

RNAi Screen in Drosophila melanogastor Identifies Regulators of Steroidogenesis and Developmental Maturation

DEFF Research Database (Denmark)

Danielsen, Erik Thomas

and duration required for juvenile-adult transition. This PhD project demonstrates the power of Drosophila genetics by taking an in vivo genome-wide RNAi screening approach to uncover genes required for the function of steroid producing tissue and developmental maturation. In total, 1909 genes were found...... to be required for the prothoracic gland function and affected the developmental timing for the juvenile-adult transition. Among the screen hits, we focused on an uncharacterized gene, sit (CG5278), which is highly expressed in the gland and is required for ecdysone production. Sit is a homolog of mammalian very...... flux of cholesterol uptake in the gland cells and affected the endosomal trafficking. Therefore this gene was suggested to be named stuck in traffic (sit). Sit’s role in cholesterol uptake was also supported by the observation that the developmental delayed phenotype from loss of sit expression...

Screening children for elevated blood lead - Learnings from the literature

Energy Technology Data Exchange (ETDEWEB)

Boreland, Frances [Broken Hill Centre for Remote Health Research, Broken Hill University Department of Rural Health, University of Sydney, Corrindah Court, PO Box 457, Broken Hill, NSW 2880 (Australia)], E-mail: fboreland@gwahs.health.nsw.gov.au; Lyle, David [Broken Hill Centre for Remote Health Research, Broken Hill University Department of Rural Health, University of Sydney, Corrindah Court, PO Box 457, Broken Hill, NSW 2880 (Australia)], E-mail: dlyle@gwahs.health.nsw.gov.au

2008-02-01

Introduction: Although it is important that children at risk of developing elevated blood lead receive regular screening, attendance at screening programs is variable. A literature review was undertaken to better understand the factors that affect carers' decisions about whether or not to take their children for blood lead screening. Method: Electronic databases (Medline, EMBASE, CINAHL, Psychinfo) were searched to identify relevant publications, supported by reviewing reference lists of identified articles and searching with internet-based search engines. Results: Thirty-four published studies dealing with blood lead screening rates were identified, of which only seven papers focused specifically on parent's attitudes to blood lead screening. The barriers to and enablers of screening for elevated blood lead levels appear to be similar to those identified for other screening programs. Discussion: It is recommended that attendance at screening be routinely monitored, and that where participation is suboptimal further research be undertaken, in close co-operation with affected communities or sub-groups, to determine how best to encourage screening and to protect children from lead. It is important to minimize stigma and to ensure, as far as possible, that practical barriers such as lack of transport do not restrict access to screening programs.

Newborn screening for galactosaemia.

Science.gov (United States)

Lak, Rohollah; Yazdizadeh, Bahareh; Davari, Majid; Nouhi, Mojtaba; Kelishadi, Roya

2017-12-23

Classical galactosaemia is an autosomal recessive inborn error of metabolism caused by a deficiency of the enzyme galactose-1-phosphate uridyltransferase. This is a rare and potentially lethal condition that classically presents in the first week of life once milk feeds have commenced. Affected babies may present with any or all of the following: cataracts; fulminant liver failure; prolonged jaundice; or Escherichia coli sepsis. Once the diagnosis is suspected, feeds containing galactose must be stopped immediately and replaced with a soya-based formula. The majority of babies will recover, however a number will not survive. There are long-term complications of galactosaemia, despite treatment, including learning disabilities and female infertility. It has been postulated that galactosaemia could be detected on newborn screening and this would prevent the immediate severe liver dysfunction and sepsis. To assess whether there is evidence that newborn screening for galactosaemia prevents or reduces mortality and morbidity and improves clinical outcomes in affected neonates and the quality of life in older children. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from electronic database searches, handsearches of relevant journals and conference abstract books. We also searched online trials registries and the reference lists of relevant articles and reviews.Date of the most recent search of Cochrane Cystic Fibrosis Group's Trials Register: 18 December 2017.Date of the most recent search of additional resources: 11 October 2017. Randomised controlled studies and controlled clinical studies, published or unpublished comparing the use of any newborn screening test to diagnose infants with galactosaemia and presenting a comparison between a screened population versus a non-screened population. No studies of newborn screening for galactosaemia were found. No studies were identified for inclusion in the

The cumulative risk of false-positive screening results across screening centres in the Norwegian Breast Cancer Screening Program

Energy Technology Data Exchange (ETDEWEB)

Roman, M., E-mail: Marta.Roman@kreftregisteret.no [Cancer Registry of Norway, Oslo (Norway); Department of Women and Children’s Health, Oslo University Hospital, Oslo (Norway); Skaane, P., E-mail: PERSK@ous-hf.no [Department of Radiology, Oslo University Hospital Ullevaal, University of Oslo, Oslo (Norway); Hofvind, S., E-mail: Solveig.Hofvind@kreftregisteret.no [Cancer Registry of Norway, Oslo (Norway); Oslo and Akershus University College of Applied Sciences, Faculty of Health Science, Oslo (Norway)

2014-09-15

Highlights: • We found variation in early performance measures across screening centres. • Radiologists’ performance may play a key role in the variability. • Potential to improve the effectiveness of breast cancer screening programs. • Continuous surveillance of screening centres and radiologists is essential. - Abstract: Background: Recall for assessment in mammographic screening entails an inevitable number of false-positive screening results. This study aimed to investigate the variation in the cumulative risk of a false positive screening result and the positive predictive value across the screening centres in the Norwegian Breast Cancer Screening Program. Methods: We studied 618,636 women aged 50–69 years who underwent 2,090,575 screening exams (1996–2010. Recall rate, positive predictive value, rate of screen-detected cancer, and the cumulative risk of a false positive screening result, without and with invasive procedures across the screening centres were calculated. Generalized linear models were used to estimate the probability of a false positive screening result and to compute the cumulative false-positive risk for up to ten biennial screening examinations. Results: The cumulative risk of a false-positive screening exam varied from 10.7% (95% CI: 9.4–12.0%) to 41.5% (95% CI: 34.1–48.9%) across screening centres, with a highest to lowest ratio of 3.9 (95% CI: 3.7–4.0). The highest to lowest ratio for the cumulative risk of undergoing an invasive procedure with a benign outcome was 4.3 (95% CI: 4.0–4.6). The positive predictive value of recall varied between 12.0% (95% CI: 11.0–12.9%) and 19.9% (95% CI: 18.3–21.5%), with a highest to lowest ratio of 1.7 (95% CI: 1.5–1.9). Conclusions: A substantial variation in the performance measures across the screening centres in the Norwegian Breast Cancer Screening Program was identified, despite of similar administration, procedures, and quality assurance requirements. Differences in the

The cumulative risk of false-positive screening results across screening centres in the Norwegian Breast Cancer Screening Program

International Nuclear Information System (INIS)

Roman, M.; Skaane, P.; Hofvind, S.

2014-01-01

Highlights: • We found variation in early performance measures across screening centres. • Radiologists’ performance may play a key role in the variability. • Potential to improve the effectiveness of breast cancer screening programs. • Continuous surveillance of screening centres and radiologists is essential. - Abstract: Background: Recall for assessment in mammographic screening entails an inevitable number of false-positive screening results. This study aimed to investigate the variation in the cumulative risk of a false positive screening result and the positive predictive value across the screening centres in the Norwegian Breast Cancer Screening Program. Methods: We studied 618,636 women aged 50–69 years who underwent 2,090,575 screening exams (1996–2010. Recall rate, positive predictive value, rate of screen-detected cancer, and the cumulative risk of a false positive screening result, without and with invasive procedures across the screening centres were calculated. Generalized linear models were used to estimate the probability of a false positive screening result and to compute the cumulative false-positive risk for up to ten biennial screening examinations. Results: The cumulative risk of a false-positive screening exam varied from 10.7% (95% CI: 9.4–12.0%) to 41.5% (95% CI: 34.1–48.9%) across screening centres, with a highest to lowest ratio of 3.9 (95% CI: 3.7–4.0). The highest to lowest ratio for the cumulative risk of undergoing an invasive procedure with a benign outcome was 4.3 (95% CI: 4.0–4.6). The positive predictive value of recall varied between 12.0% (95% CI: 11.0–12.9%) and 19.9% (95% CI: 18.3–21.5%), with a highest to lowest ratio of 1.7 (95% CI: 1.5–1.9). Conclusions: A substantial variation in the performance measures across the screening centres in the Norwegian Breast Cancer Screening Program was identified, despite of similar administration, procedures, and quality assurance requirements. Differences in the

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

Science.gov (United States)

Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

2017-10-13

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

Science.gov (United States)

Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

2014-01-17

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

NARCIS (Netherlands)

Damveld, R.A.; Franken, A.; Arentshorst, M.; Punt, P.J.; Klis, F.M.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.

2008-01-01

To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative a-glucan

Nutrition screening tools: an analysis of the evidence.

Science.gov (United States)

Skipper, Annalynn; Ferguson, Maree; Thompson, Kyle; Castellanos, Victoria H; Porcari, Judy

2012-05-01

In response to questions about tools for nutrition screening, an evidence analysis project was developed to identify the most valid and reliable nutrition screening tools for use in acute care and hospital-based ambulatory care settings. An oversight group defined nutrition screening and literature search criteria. A trained analyst conducted structured searches of the literature for studies of nutrition screening tools according to predetermined criteria. Eleven nutrition screening tools designed to detect undernutrition in patients in acute care and hospital-based ambulatory care were identified. Trained analysts evaluated articles for quality using criteria specified by the American Dietetic Association's Evidence Analysis Library. Members of the oversight group assigned quality grades to the tools based on the quality of the supporting evidence, including reliability and validity data. One tool, the NRS-2002, received a grade I, and 4 tools-the Simple Two-Part Tool, the Mini-Nutritional Assessment-Short Form (MNA-SF), the Malnutrition Screening Tool (MST), and Malnutrition Universal Screening Tool (MUST)-received a grade II. The MST was the only tool shown to be both valid and reliable for identifying undernutrition in the settings studied. Thus, validated nutrition screening tools that are simple and easy to use are available for application in acute care and hospital-based ambulatory care settings.

Screening physical health? Yes! But...: nurses' views on physical health screening in mental health care.

Science.gov (United States)

Happell, Brenda; Scott, David; Nankivell, Janette; Platania-Phung, Chris

2013-08-01

To explore nurses' views on the role of nurses in screening and monitoring for physical care of consumers with serious mental illness, at a regional mental health care service. People with serious mental illness experience heightened incidence of preventable and treatable physical illnesses such as cardiovascular disease and diabetes. Screening and monitoring are considered universal clinical safeguards. Nurses can potentially facilitate systematic screening, but their views on physical health care practices are rarely investigated. Qualitative exploratory study. Focus group interviews with 38 nurses of a regional mental health care service district of Australia. To facilitate discussion, participants were presented with a screening system, called the Health Improvement Profile (HIP), as an exemplar of screening of physical health risks by nurses. Inductive data analysis and theme development were guided by a thematic analysis framework. Nurses argued that treatable and preventable physical health problems were common. Four main themes were identified: screening - essential for good practice; the policy-practice gap; 'screening then what?' and, is HIP the answer? Screening and monitoring were considered crucial to proper diagnosis and treatment, however, were not performed systematically or consistently. Nurse readiness for an enhanced role in screening was shaped by: role and responsibility issues, legal liability concerns, funding and staff shortages. Participants were concerned that lack of follow up would limit effectiveness of these interventions. Screening was considered an important clinical step in effective diagnosis and treatment; however, identified barriers need to be addressed to ensure screening is part of a systemic approach to improve physical health of consumers with serious mental illness. Nurses have potential to influence improvement in physical health outcomes for consumers of mental health services. Such potential can only be realised if a

Psychometric properties and reliability of the Assessment Screen to Identify Survivors Toolkit for Gender Based Violence (ASIST-GBV): results from humanitarian settings in Ethiopia and Colombia.

Science.gov (United States)

Vu, Alexander; Wirtz, Andrea; Pham, Kiemanh; Singh, Sonal; Rubenstein, Leonard; Glass, Nancy; Perrin, Nancy

2016-01-01

Refugees and internally displaced persons who are affected by armed-conflict are at increased vulnerability to some forms of sexual violence or other types of gender-based violence. A validated, brief and easy-to-administer screening tool will help service providers identify GBV survivors and refer them to appropriate GBV services. To date, no such GBV screening tool exists. We developed the 7-item ASIST-GBV screening tool from qualitative research that included individual interviews and focus groups with GBV refugee and IDP survivors. This study presents the psychometric properties of the ASIST-GBV with female refugees living in Ethiopia and IDPs in Colombia. Several strategies were used to validate ASIST-GBV, including a 3 month implementation to validate the brief screening tool with women/girls seeking health services, aged ≥15 years in Ethiopia (N = 487) and female IDPs aged ≥ 18 years in Colombia (N = 511). High proportions of women screened positive for past-year GBV according to the ASIST-GBV: 50.6 % in Ethiopia and 63.4 % in Colombia. The factor analysis identified a single dimension, meaning that all items loaded on the single factor. Cronbach's α = 0.77. A 2-parameter logistic IRT model was used for estimating the precision and discriminating power of each item. Item difficulty varied across the continuum of GBV experiences in the following order (lowest to highest): threats of violence (0.690), physical violence (1.28), forced sex (2.49), coercive sex for survival (2.25), forced marriage (3.51), and forced pregnancy (6.33). Discrimination results showed that forced pregnancy was the item with the strongest ability to discriminate between different levels of GBV. Physical violence and forced sex also have higher levels of discrimination with threats of violence discriminating among women at the low end of the GBV continuum and coercive sex for survival among women at the mid-range of the continuum. The findings demonstrate that

Hypersexual Disorder According to the Hypersexual Disorder Screening Inventory in Help-Seeking Swedish Men and Women With Self-Identified Hypersexual Behavior.

Science.gov (United States)

Öberg, Katarina Görts; Hallberg, Jonas; Kaldo, Viktor; Dhejne, Cecilia; Arver, Stefan

2017-12-01

The Hypersexual Disorder Screening Inventory (HDSI) was developed by the American Psychiatric Association for clinical screening of hypersexual disorder (HD). To examine the distribution of the proposed diagnostic entity HD according to the HDSI in a sample of men and women seeking help for problematic hypersexuality and evaluate some psychometric properties. Data on sociodemographics, the HDSI, the Sexual Compulsivity Scale (SCS), and the Cognitive and Behavioral Outcomes of Sexual Behavior were collected online from 16 women and 64 men who self-identified as hypersexual. Respondents were recruited by advertisements offering psychological treatment for hypersexual behavior. The HDSI, covering the proposed criteria for HD. Of the entire sample, 50% fulfilled the criteria for HD. Compared with men, women scored higher on the HDSI, engaged more often in risky sexual behavior, and worried more about physical injuries and pain. Men primarily used pornography, whereas women had sexual encounters. The HD group reported a larger number of sexual specifiers, higher scores on the SCS, more negative effects of sexual behavior, and more concerns about consequences compared with the non-HD group. Sociodemographics had no influence on HD. The HDSI's core diagnostic criteria showed high internal reliability for men (α = 0.80) and women (α = 0.81). A moderate correlation between the HDSI and the SCS was found (0.51). The vast majority of the entire sample (76 of 80, 95%) fulfilled the criteria for sexual compulsivity according to the SCS. The HDSI could be used as a screening tool for HD, although further explorations of the empirical implications regarding criteria are needed, as are refinements of cutoff scores and specific sexual behaviors. Hypersexual problematic behavior causes distress and impairment and, although not included in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, HD should be endorsed as a diagnosis to develop evidence

An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

Science.gov (United States)

Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

2009-03-12

Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

Screening College Students for Hypercholesterolemia.

Science.gov (United States)

Faigel, Harris C.

1992-01-01

Describes one college's mandatory mass cholesterol screening for new students. Each year, over 30 beginning students with unknown hypercholesterolemia were detected. The program suggests that mass screening efficiently and economically identifies students who would benefit from cholesterol reduction, a modifiable risk in coronary artery disease.…

A Yeast/Drosophila Screen to Identify New Compounds Overcoming Frataxin Deficiency

Directory of Open Access Journals (Sweden)

Alexandra Seguin

2015-01-01

Full Text Available Friedreich’s ataxia (FA is a rare neurodegenerative disease which is very debilitating for the patients who progressively lose their autonomy. The lack of efficient therapeutic treatment of the disease strongly argues for urgent need to search for new active compounds that may stop the progression of the disease or prevent the appearance of the symptoms when the genetic defect is diagnosed early enough. In the present study, we used a yeast strain with a deletion of the frataxin homologue gene as a model of FA cells in a primary screen of two chemical libraries, a fraction of the French National Chemical Library (5500 compounds and the Prestwick collection (880 compounds. We ran a secondary screen on Drosophila melanogaster flies expressing reduced levels of frataxin during larval development. Half of the compounds selected in yeast appeared to be active in flies in this developmental paradigm, and one of the two compounds with highest activities in this assay partially rescued the heart dilatation phenotype resulting from heart specific depletion of frataxin. The unique complementarity of these two frataxin-deficient models, unicellular and multicellular, appears to be very efficient to select new compounds with improved selectivity, bringing significant perspectives towards improvements in FA therapy.

In vivo phage display screening for tumor vascular targets in glioblastoma identifies a llama nanobody against dynactin-1-p150Glued.

Science.gov (United States)

van Lith, Sanne A M; Roodink, Ilse; Verhoeff, Joost J C; Mäkinen, Petri I; Lappalainen, Jari P; Ylä-Herttuala, Seppo; Raats, Jos; van Wijk, Erwin; Roepman, Ronald; Letteboer, Stef J; Verrijp, Kiek; Leenders, William P J

2016-11-01

Diffuse gliomas are primary brain cancers that are characterised by infiltrative growth. Whereas high-grade glioma characteristically presents with perinecrotic neovascularisation, large tumor areas thrive on pre-existent vasculature as well. Clinical studies have revealed that pharmacological inhibition of the angiogenic process does not improve survival of glioblastoma patients. Direct targeting of tumor vessels may however still be an interesting therapeutic approach as it allows pinching off the blood supply to tumor cells. Such tumor vessel targeting requires the identification of tumor-specific vascular targeting agents (TVTAs).Here we describe a novel TVTA, C-C7, which we identified via in vivo biopanning of a llama nanobody phage display library in an orthotopic mouse model of diffuse glioma. We show that C-C7 recognizes a subpopulation of tumor blood vessels in glioma xenografts and clinical glioma samples. Additionally, C-C7 recognizes macrophages and activated endothelial cells in atherosclerotic lesions. By using C-C7 as bait in yeast-2-hybrid (Y2H) screens we identified dynactin-1-p150Glued as its binding partner. The interaction was confirmed by co-immunostainings with C-C7 and a commercial anti-dynactin-1-p150Glued antibody, and via co-immunoprecipitation/western blot studies. Normal brain vessels do not express dynactin-1-p150Glued and its expression is reduced under anti-VEGF therapy, suggesting that dynactin-1-p150Glued is a marker for activated endothelial cells.In conclusion, we show that in vivo phage display combined with Y2H screenings provides a powerful approach to identify tumor-targeting nanobodies and their binding partners. Using this combination of methods we identify dynactin-1-p150Glued as a novel targetable protein on activated endothelial cells and macrophages.

Screen or not to screen for peripheral arterial disease: guidance from a decision model.

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Vaidya, Anil; Joore, Manuela A; Ten Cate-Hoek, Arina J; Ten Cate, Hugo; Severens, Johan L

2014-01-29

Asymptomatic Peripheral Arterial Disease (PAD) is associated with greater risk of acute cardiovascular events. This study aims to determine the cost-effectiveness of one time only PAD screening using Ankle Brachial Index (ABI) test and subsequent anti platelet preventive treatment (low dose aspirin or clopidogrel) in individuals at high risk for acute cardiovascular events compared to no screening and no treatment using decision analytic modelling. A probabilistic Markov model was developed to evaluate the life time cost-effectiveness of the strategy of selective PAD screening and consequent preventive treatment compared to no screening and no preventive treatment. The analysis was conducted from the Dutch societal perspective and to address decision uncertainty, probabilistic sensitivity analysis was performed. Results were based on average values of 1000 Monte Carlo simulations and using discount rates of 1.5% and 4% for effects and costs respectively. One way sensitivity analyses were performed to identify the two most influential model parameters affecting model outputs. Then, a two way sensitivity analysis was conducted for combinations of values tested for these two most influential parameters. For the PAD screening strategy, life years and quality adjusted life years gained were 21.79 and 15.66 respectively at a lifetime cost of 26,548 Euros. Compared to no screening and treatment (20.69 life years, 15.58 Quality Adjusted Life Years, 28,052 Euros), these results indicate that PAD screening and treatment is a dominant strategy. The cost effectiveness acceptability curves show 88% probability of PAD screening being cost effective at the Willingness To Pay (WTP) threshold of 40000 Euros. In a scenario analysis using clopidogrel as an alternative anti-platelet drug, PAD screening strategy remained dominant. This decision analysis suggests that targeted ABI screening and consequent secondary prevention of cardiovascular events using low dose aspirin or

Testing Precision Screening for Breast Cancer

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An NCI research article about individualized approaches that could help identify those at risk of breast cancer who need to be screened and testing screening intervals that are appropriate for each person’s level of risk.

Influence of qualitative research on women's health screening guidelines.

Science.gov (United States)

Abadir, Anna Maria; Lang, Ariella; Klein, Talia; Abenhaim, Haim Arie

2014-01-01

Considerable time and resources are allocated to carry out qualitative research. The purpose of our study was to evaluate the availability of qualitative research on women's health screening and assess its influence on screening practice guidelines in the United States, Canada, and the United Kingdom. Medline, CINHAL, and WEB of Science databases were used to identify the availability of qualitative research conducted in the past 15 years on 3 different women's health screening topics: cervical cancer screening, breast cancer screening, and prenatal first-trimester screening. Key national practice guidelines on women's health screening were selected using the National Guideline Clearinghouse web site. Bibliometric analysis was used to determine the frequency of qualitative references cited in the guidelines. A total of 272 qualitative research papers on women's health screening was identified: 109 on cervical cancer screening, 104 on breast cancer screening, and 59 on prenatal first-trimester screening. The qualitative studies focused on health care provider perspectives as well as ethical, ethnographic, psychological, and social issues surrounding screening. Fifteen national clinical practice guidelines on women's health screening were identified. A total of 943 references was cited, only 2 of which comprised of qualitative research cited by only 1 clinical practice guideline. Although there is considerable qualitative research that has been carried out on women's health screening, its incorporation into clinical practice guidelines is minimal. Further exploration of the disconnect between the two is important for enhancing knowledge translation of qualitative research within clinical practice. Copyright © 2014 Mosby, Inc. All rights reserved.

NSC23925, identified in a high-throughput cell-based screen, reverses multidrug resistance.

Directory of Open Access Journals (Sweden)

Zhenfeng Duan

2009-10-01

Full Text Available Multidrug resistance (MDR is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically.To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1 but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 microM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors.The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients.

Third-Generation Sequencing and Analysis of Four Complete Pig Liver Esterase Gene Sequences in Clones Identified by Screening BAC Library.

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Zhou, Qiongqiong; Sun, Wenjuan; Liu, Xiyan; Wang, Xiliang; Xiao, Yuncai; Bi, Dingren; Yin, Jingdong; Shi, Deshi

2016-01-01

Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing. After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis. Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression. This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for

Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger.

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Reilly, Morgann C; Kim, Joonhoon; Lynn, Jed; Simmons, Blake A; Gladden, John M; Magnuson, Jon K; Baker, Scott E

2018-02-01

Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.

Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Reilly, Morgann C. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kim, Joonhoon [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lynn, Jed [Joint BioEnergy Institute, Emeryville, CA (United States); Wright-Patterson Air Force Base, Dayton, OH (United States); Simmons, Blake A. [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Gladden, John M. [Joint BioEnergy Institute, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States); Magnuson, Jon K. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Baker, Scott E. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2018-01-06

Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.

Nutrition screening tools: does one size fit all? A systematic review of screening tools for the hospital setting.

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van Bokhorst-de van der Schueren, Marian A E; Guaitoli, Patrícia Realino; Jansma, Elise P; de Vet, Henrica C W

2014-02-01

Numerous nutrition screening tools for the hospital setting have been developed. The aim of this systematic review is to study construct or criterion validity and predictive validity of nutrition screening tools for the general hospital setting. A systematic review of English, French, German, Spanish, Portuguese and Dutch articles identified via MEDLINE, Cinahl and EMBASE (from inception to the 2nd of February 2012). Additional studies were identified by checking reference lists of identified manuscripts. Search terms included key words for malnutrition, screening or assessment instruments, and terms for hospital setting and adults. Data were extracted independently by 2 authors. Only studies expressing the (construct, criterion or predictive) validity of a tool were included. 83 studies (32 screening tools) were identified: 42 studies on construct or criterion validity versus a reference method and 51 studies on predictive validity on outcome (i.e. length of stay, mortality or complications). None of the tools performed consistently well to establish the patients' nutritional status. For the elderly, MNA performed fair to good, for the adults MUST performed fair to good. SGA, NRS-2002 and MUST performed well in predicting outcome in approximately half of the studies reviewed in adults, but not in older patients. Not one single screening or assessment tool is capable of adequate nutrition screening as well as predicting poor nutrition related outcome. Development of new tools seems redundant and will most probably not lead to new insights. New studies comparing different tools within one patient population are required. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

A study looking at the effectiveness of developmental screening in identifying learning disabilities in early childhood.

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Flanagan, O; Nualláin, S O

2001-05-01

This is a retrospective study of children under six years of age referred to the Brothers of Charity Early Intervention Services in County Galway, a service that caters for children under 6 years with learning disabilities. The aim in doing this study was to assess the value of routine developmental screening in identifying children with learning difficulties. This study also investigates the patterns and sources of referral to the remedial services provided by the Brothers of Charity and highlights possible avoidable delays in referral. The results showed that many children were referred for remedial services late. The reasons for late referral included late identification of some children with problems, insufficient co-ordination of community-based services and a lack of awareness of the importance of early intervention in some cases. As some communication disorders such as autism, autistic spectrum disorders and specific language delay may not express themselves until the later part of the second year of life, the 18-24 month developmental assessment is of vital importance. However identification of these disorders can present difficulties and may call for additional training for professionals involved in the developmental screening of children in that age group. The interval between initial identification and referral for remedial care in many cases was more than twelve months. We propose that, in order to minimize this time, children requiring a more in-depth assessment should be assessed by a community-based multidisciplinary team, enabling integrated assessment by the different disciplines and thus speedier referral to remedial services.

Industrial screening programs for workers

International Nuclear Information System (INIS)

Lavine, M.P.

1982-01-01

Industrial screening efforts to identify classes of workers who are more susceptible to workplace hazards, by virtue of their fertility, genetic, or lifestyle characteristics, represent a relatively new approach to reducing workplace risks. Screening has already raised some important economic, legal, social, medical, and moral questions. Employers, employees, administrative agencies, and the courts are offering different, often conflicting answers. Ultimately the acceptability of various screening schemes rests upon judgments about how a society justifies the distribution of risk. The questions that industrial screening programs raise are only partially answered by empirical evidence; the rest is a matter of values

Screening for skin cancer.

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Helfand, M; Mahon, S M; Eden, K B; Frame, P S; Orleans, C T

2001-04-01

Malignant melanoma is often lethal, and its incidence in the United States has increased rapidly over the past 2 decades. Nonmelanoma skin cancer is seldom lethal, but, if advanced, can cause severe disfigurement and morbidity. Early detection and treatment of melanoma might reduce mortality, while early detection and treatment of nonmelanoma skin cancer might prevent major disfigurement and to a lesser extent prevent mortality. Current recommendations from professional societies regarding screening for skin cancer vary. To examine published data on the effectiveness of routine screening for skin cancer by a primary care provider, as part of an assessment for the U.S. Preventive Services Task Force. We searched the MEDLINE database for papers published between 1994 and June 1999, using search terms for screening, physical examination, morbidity, and skin neoplasms. For information on accuracy of screening tests, we used the search terms sensitivity and specificity. We identified the most important studies from before 1994 from the Guide to Clinical Preventive Services, second edition, and from high-quality reviews. We used reference lists and expert recommendations to locate additional articles. Two reviewers independently reviewed a subset of 500 abstracts. Once consistency was established, the remainder were reviewed by one reviewer. We included studies if they contained data on yield of screening, screening tests, risk factors, risk assessment, effectiveness of early detection, or cost effectiveness. We abstracted the following descriptive information from full-text published studies of screening and recorded it in an electronic database: type of screening study, study design, setting, population, patient recruitment, screening test description, examiner, advertising targeted at high-risk groups or not targeted, reported risk factors of participants, and procedure for referrals. We also abstracted the yield of screening data including probabilities and numbers

Yield and Efficiency of Mental Health Screening: A Comparison of Screening Protocols at Intake to Prison.

Science.gov (United States)

Martin, Michael S; Potter, Beth K; Crocker, Anne G; Wells, George A; Colman, Ian

2016-01-01

The value of screening for mental illness has increasingly been questioned in low prevalence settings due to high false positive rates. However, since false positive rates are related to prevalence, screening may be more effective in higher prevalence settings, including correctional institutions. We compared the yield (i.e. newly detected cases) and efficiency (i.e. false positives) of five screening protocols to detect mental illness in prisons against the use of mental health history taking (the prior approach to detecting mental illness). We estimated the accuracy of the six approaches to detect an Axis I disorder among a sample of 467 newly admitted male inmates (83.1% participation rate). Mental health history taking identified only 41.0% (95% CI 32.1, 50.6) of all inmates with mental illness. Screening protocols identified between 61.9 and 85.7% of all cases, but referred between 2 and 3 additional individuals who did not have a mental illness for every additional case detected compared to the mental health history taking approach. In low prevalence settings (i.e. 10% or less) the screening protocols would have had between 4.6 and 16.2 false positives per true positive. While screening may not be practical in low prevalence settings, it may be beneficial in jails and prisons where the prevalence of mental illness is higher. Further consideration of the context in which screening is being implemented, and of the impacts of policies and clinical practices on the benefits and harms of screening is needed to determine the effectiveness of screening in these settings.

Yield and Efficiency of Mental Health Screening: A Comparison of Screening Protocols at Intake to Prison.

Directory of Open Access Journals (Sweden)

Michael S Martin

Full Text Available The value of screening for mental illness has increasingly been questioned in low prevalence settings due to high false positive rates. However, since false positive rates are related to prevalence, screening may be more effective in higher prevalence settings, including correctional institutions. We compared the yield (i.e. newly detected cases and efficiency (i.e. false positives of five screening protocols to detect mental illness in prisons against the use of mental health history taking (the prior approach to detecting mental illness.We estimated the accuracy of the six approaches to detect an Axis I disorder among a sample of 467 newly admitted male inmates (83.1% participation rate. Mental health history taking identified only 41.0% (95% CI 32.1, 50.6 of all inmates with mental illness. Screening protocols identified between 61.9 and 85.7% of all cases, but referred between 2 and 3 additional individuals who did not have a mental illness for every additional case detected compared to the mental health history taking approach. In low prevalence settings (i.e. 10% or less the screening protocols would have had between 4.6 and 16.2 false positives per true positive.While screening may not be practical in low prevalence settings, it may be beneficial in jails and prisons where the prevalence of mental illness is higher. Further consideration of the context in which screening is being implemented, and of the impacts of policies and clinical practices on the benefits and harms of screening is needed to determine the effectiveness of screening in these settings.

One more hurdle to increasing mammography screening: pubescent, adolescent, and prior mammography screening experiences.

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Thomas, Eileen; Usher, LaToya

2009-01-01

Approximately $8.1 billion dollars is spent each year in the United States alone on the treatment of breast cancer. Survival rates are dependent on access to, and utilization of, early detection services. The primary reason for disparity in breast cancer mortality is the delay in time to diagnosis, resulting in poor prognosis. Despite ongoing research to understand barriers to mammography screening, recent studies report a decrease in mammography screening among all racial groups. A qualitative approach was used to elicit information from 36 White non- Hispanic, African-American, Hispanic, and Native American women without a history of breast cancer. Women were invited to share written or audiotape-recorded narratives about experiences pertaining to their breasts and their mammography screening experiences. Major categories identified were: teasing, family norms and values, media/societal influence, body image, and mammography screening experiences. The resulting effects of these experiences left these women with feelings of shame and "conflict" regarding their breasts. The major theme identified was breast conflict. Findings suggest that breast conflict may persist throughout the lifespan and can have a negative influence on a woman's decision to participate in mammography screening. The authors hypothesize that experiences that occur during adolescence pertaining to young girls' breasts can influence a women's body image, which in turn can later in life affect health-seeking behaviors related to mammography screening. These findings have implications for public health practice in planning for breast cancer screening, education, and interventions for women from diverse racial/ethnics groups.

Q Sepharose micro-column chromatography: A simple screening method for identifying beta thalassemia traits and hemoglobin E carriers.

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Wong, Peerapon; Sritippayawan, Suchila; Suwannakhon, Narutchala; Tapprom, Akamon; Deoisares, Rawisut; Sanguansermsri, Torpong

2016-11-01

For beta thalassemia control program in pregnancy, mass screening of the carrier state by determination of the hemoglobin (Hb) A 2 and Hb E proportions and mutation analysis is a preferred method for making prenatal diagnoses. Q Sepharose micro-column chromatography, developed for the determination of Hb A 2 and Hb E for screening purposes, was compared with high performance liquid chromatography (HPLC) to ascertain its relative accuracy and reliability. Results using Q Sepharose micro-column chromatography in 350 blood specimens, including 50 samples genetically proven to be beta thalassemia heterozygotes, were compared to HPLC for validation. An additional study was conducted to test a clinical application on a large-scale survey for beta thalassemia in 1581 pregnant women and their spouses. The mean (±SD) Hb A 2 proportions in the normal and genetically proven beta thalassemia heterozygotes were 2.70±0.40% and 6.30±1.23%, respectively, as determined by Q-Sepharose micro-column chromatography, and 2.65±0.31% and 5.37±0.96%, respectively, as determined by HPLC. The mean Hb E proportions in the Hb E heterozygotes were 23.25±4.13% and 24.72±3.5% as determined by Q Sepharose micro-column chromatography and HPLC, respectively. In the large-scale survey for beta thalassemia, 23 at risk couples were detected. Seven affected fetuses were identified by prenatal diagnosis. Q Sepharose micro-column chromatography was found to be reliable, reproducible and well-suited for large-scale surveys. Additionally, by being reusable and convenient, this simple and economical chromatography method may be an alternative means to screen for beta thalassemia and Hb E carriers in the mass population. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Liposomal solubilization of new 3-hydroxy-quinolinone derivatives with promising anticancer activity: a screening method to identify maximum incorporation capacity

DEFF Research Database (Denmark)

Di Cagno, Massimiliano; Styskala, Jakub; Hlaváč, Jan

2011-01-01

Four new 3-hydroxy-quinolinone derivatives with promising anticancer activity could be solubilized using liposomes as vehicle to an extent that allows their in vitro and in vivo testing without use of toxic solvent(s). A screening method to identify the maximum incorporation capacity of hydrophobic......, resulting in a 200-500-fold increase in apparent solubility. Drug-to-lipid ratios in the range of 2-5 µg/mg were obtained. Interestingly, the four quinolinone derivatives have shown different association tendencies with liposomes, probably due to the physicochemical properties of the different group bonded...

Screening for colorectal cancer.

Science.gov (United States)

He, Jin; Efron, Jonathan E

2011-01-01

March is national colorectal cancer awareness month. It is estimated that as many as 60% of colorectal cancer deaths could be prevented if all men and women aged 50 years or older were screened routinely. In 2000, Katie Couric's televised colonoscopy led to a 20% increase in screening colonoscopies across America, a stunning rise called the "Katie Couric Effect". This event demonstrated how celebrity endorsement affects health behavior. Currently, discussion is ongoing about the optimal strategy for CRC screening, particularly the costs of screening colonoscopy. The current CRC screening guidelines are summarized in Table 2. Debates over the optimum CRC screening test continue in the face of evidence that 22 million Americans aged 50 to 75 years are not screened for CRC by any modality and 25,000 of those lives may have been saved if they had been screened for CRC. It is clear that improving screening rates and reducing disparities in underscreened communities and population subgroups could further reduce colorectal cancer morbidity and mortality. National Institutes of Health consensus identified the following priority areas to enhance the use and quality of colorectal cancer screening: Eliminate financial barriers to colorectal cancer screening and appropriate follow-up of positive results of colorectal cancer screening. Develop systems to ensure the high quality of colorectal cancer screening programs. Conduct studies to determine the comparative effectiveness of the various colorectal cancer screening methods in usual practice settings. Encouraging population adherence to screening tests and allowing patients to select the tests they prefer may do more good (as long as they choose something) than whatever procedure is chosen by the medical profession as the preferred test.

Provider Perspectives on Promoting Cervical Cancer Screening Among Refugee Women.

Science.gov (United States)

Zhang, Ying; Ornelas, India J; Do, H Hoai; Magarati, Maya; Jackson, J Carey; Taylor, Victoria M

2017-06-01

Many refugees in the United States emigrated from countries where the incidence of cervical cancer is high. Refugee women are unlikely to have been screened for cervical cancer prior to resettlement in the U.S. National organizations recommend cervical cancer screening for refugee women soon after resettlement. We sought to identify health and social service providers' perspectives on promoting cervical cancer screening in order to inform the development of effective programs to increase screening among recently resettled refugees. This study consisted of 21 in-depth key informant interviews with staff from voluntary refugee resettlement agencies, community based organizations, and healthcare clinics serving refugees in King County, Washington. Interview transcripts were analyzed to identify themes. We identified the following themes: (1) refugee women are unfamiliar with preventive care and cancer screening; (2) providers have concerns about the timing of cervical cancer education and screening; (3) linguistic and cultural barriers impact screening uptake; (4) provider factors and clinic systems facilitate promotion of screening; and (5) strategies for educating refugee women about screening. Our findings suggest that refugee women are in need of health education on cervical cancer screening during early resettlement. Frequent messaging about screening could help ensure that women receive screening within the early resettlement period. Health education videos may be effective for providing simple, low literacy messages in women's native languages. Appointments with female clinicians and interpreters, as well as clinic systems that remind clinicians to offer screening at each appointment could increase screening among refugee women.

A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer.

Directory of Open Access Journals (Sweden)

Harsh B Pathak

Full Text Available Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC. The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1 was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other

Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

Science.gov (United States)

Ali, Dalia; Hamam, Rimi; Alfayez, Musaed; Kassem, Moustapha; Aldahmash, Abdullah

2016-01-01

The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering. Significance This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase

A Drug Combination Screen Identifies Drugs Active against Amoxicillin-induced Round Bodies of Borrelia burgdorferi Persisters from an FDA Drug Library

Directory of Open Access Journals (Sweden)

Jie eFeng

2016-05-01

Full Text Available Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that are not killed by current Lyme antibiotics. To identify more effective drugs that are active against the round bodies of B. burgdorferi, we established a round body persister model induced by amoxicillin and screened the Food and Drug Administration (FDA drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide (PI viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven of these scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. While some drug candidates such as daptomycin and clofazimine overlapped with a previous screen against stationary phase B. burgdorferi persisters, additional drug candidates active against round bodies we identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even if pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease.

A Quick Drinking Screen for identifying women at risk for an alcohol-exposed pregnancy.

Science.gov (United States)

Dum, Mariam; Sobell, Linda Carter; Sobell, Mark B; Heinecke, Nicholas; Voluse, Andrew; Johnson, Kenneth

2009-09-01

Two previous studies comparing the Quick Drinking Screen (QDS) with the Timeline Followback (TLFB) found that these two instruments yielded similar reports of alcohol use for clinical and nonclinical populations of problem drinkers. The current study evaluated the correspondence between these two drinking measures with women at risk of an Alcohol-Exposed Pregnancy (AEP). Participants were 355 women who voluntarily participated in a research study during 2005 through 2007 designed to prevent AEPs. All women were screened by phone for eligibility using the QDS and approximately 2 weeks later completed a 3-month TLFB by mail. Results of this study, analyzed in 2008, paralleled previous studies showing that the QDS and the TLFB, two very different drinking measures, collected similar aggregate drinking data for women who drink heavily and are at risk of an AEP. Correspondence between the two drinking measures met acceptable levels of reliability. The present study found that the QDS has demonstrated efficacy for screening women whose level of alcohol use puts them at risk for an AEP. Although the QDS does not yield detailed drinking information, it could be used when it is not possible or necessary to gather daily drinking data.

An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

Science.gov (United States)

Casalino, Laura; Magnani, Dario; De Falco, Sandro; Filosa, Stefania; Minchiotti, Gabriella; Patriarca, Eduardo J; De Cesare, Dario

2012-03-01

The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

Diabetes screening in the workplace.

Science.gov (United States)

Gulley, Tauna; Boggs, Dusta; Mullins, Rebecca; Brock, Emily

2014-11-01

The prevalence of diabetes has increased worldwide and the pathophysiological problems associated with diabetes increase the potential for employees' physical disabilities. These complications, including neuropathy, nephropathy, and visual impairment, negatively impact the job performance of employees and compromise workplace safety. Occupational health nurses can provide diabetes screening programs to employees and identify chronic disease risk factors early. This article describes an occupational diabetes screening program at a major corporation in Belize, Central America, defines diabetes, outlines the diabetes teaching plan, and presents the demographics of the participants and results of the screening. Cultural considerations and recommendations for future occupational diabetes screenings are proposed. Copyright 2014, SLACK Incorporated.

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

Science.gov (United States)

Hassig, Christian A; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E; Brown, Susan G; Baire, Beeraiah; Michel, Andrew R; Hoye, Thomas R; Francis, Subhashree; Georg, Gunda I; Walters, Michael A; Divlianska, Daniela B; Roth, Gregory P; Wright, Amy E; Reed, John C

2014-09-01

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach. © 2014 Society for Laboratory Automation and Screening.

Using probabilistic record linkage methods to identify Australian Indigenous women on the Queensland Pap Smear Register: the National Indigenous Cervical Screening Project.

Science.gov (United States)

Whop, Lisa J; Diaz, Abbey; Baade, Peter; Garvey, Gail; Cunningham, Joan; Brotherton, Julia M L; Canfell, Karen; Valery, Patricia C; O'Connell, Dianne L; Taylor, Catherine; Moore, Suzanne P; Condon, John R

2016-02-12

To evaluate the feasibility and reliability of record linkage of existing population-based data sets to determine Indigenous status among women receiving Pap smears. This method may allow for the first ever population measure of Australian Indigenous women's cervical screening participation rates. A linked data set of women aged 20-69 in the Queensland Pap Smear Register (PSR; 1999-2011) and Queensland Cancer Registry (QCR; 1997-2010) formed the Initial Study Cohort. Two extracts (1995-2011) were taken from Queensland public hospitals data (Queensland Hospital Admitted Patient Data Collection, QHAPDC) for women, aged 20-69, who had ever been identified as Indigenous (extract 1) and had a diagnosis or procedure code relating to cervical cancer (extract 2). The Initial Study Cohort was linked to extract 1, and women with cervical cancer in the initial cohort were linked to extract 2. The proportion of women in the Initial Cohort who linked with the extracts (true -pairs) is reported, as well as the proportion of potential pairs that required clerical review. After assigning Indigenous status from QHAPDC to the PSR, the proportion of women identified as Indigenous was calculated using 4 algorithms, and compared. There were 28,872 women (2.1%) from the Initial Study Cohort who matched to an ever Indigenous record in extract 1 (n=76,831). Women with cervical cancer in the Initial Study Cohort linked to 1385 (71%) records in extract 2. The proportion of Indigenous women ranged from 2.00% to 2.08% when using different algorithms to define Indigenous status. The Final Study Cohort included 1,372,823 women (PSR n=1,374,401; QCR n=1955), and 5,062,118 records. Indigenous status in Queensland cervical screening data was successfully ascertained through record linkage, allowing for the crucial assessment of the current cervical screening programme for Indigenous women. Our study highlights the need to include Indigenous status on Pap smear request and report forms in any

Breast cancer screening implementation and reassurance

DEFF Research Database (Denmark)

Østerø, J; Siersma, Volkert Dirk; Brodersen, John

2013-01-01

difference in reported psychosocial aspects had disappeared or been reduced because of the nationwide screening implementation. METHODS: The 1000 women included in the previous survey were posted part I of the questionnaire Consequences of Screening in Breast Cancer (COS-BC1) in August 2011, nearly 5 years......BACKGROUND: Women not offered screening mammography reported higher levels of negative psychosocial aspects than women offered screening. This was demonstrated in a questionnaire survey where 1000 women were included: 500 women living in areas where the public authorities had never offered...... screening mammography and 500 women living in areas where women had been invited to screening mammography for >10 years. After this baseline survey, nationwide screening mammography was implemented. The aim of this follow-up study was to resurvey the 1000 women and to investigate if the identified...

Clinically targeted screening for congenital CMV - potential for integration into the National Hearing Screening Programme.

Science.gov (United States)

Kadambari, S; Luck, S; Davis, A; Williams, Ej; Berrington, J; Griffiths, Pd; Sharland, M

2013-10-01

Screening for a condition should only be undertaken if certain strict criteria are met. Congenital CMV (cCMV) is a leading cause of sensorineuronal hearing loss (SNHL) and meets many of these criteria, but is not currently screened for in the UK. Ganciclovir reduces CMV-induced progressive SNHL if treatment is begun in the first month of life. The Newborn Hearing Screening Programme (NHSP) has been shown to identify SNHL at the earliest possible age. The potential of integrating screening for cCMV into the NHSP is discussed to consolidate the link between screening, early diagnosis and management. The early diagnosis and treatment of cCMV may prevent a small proportion of late SNHL. In the absence of any screening programme, we provide evidence that clinically targeted screening through the NHSP is a potential option in the UK, enhancing the diagnostic pathway and enabling appropriate early treatment to reduce long-term morbidity. ©2013 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

Spent-fuel-stabilizer screening studies

International Nuclear Information System (INIS)

Wynhoff, N.; Girault, S.E.; Fish, R.L.

1980-11-01

A broad range of potential stabilizer materials was identified and screened for packaging spent fuel assemblies for underground storage. The screening took into consideration the thermal gradient, stress, differential thermal expansion, nuclear criticality, radiation shielding, cost, and availability. Recommended stabilizer materials for further testing include silica, quartz, mullite, zircon, bentonite, graphite, gases, lead, Zn alloys, Cu alloys, etc

Comparison of screening questionnaires to identify psoriatic arthritis in a primary-care population: a cross-sectional study.

Science.gov (United States)

Coates, L C; Savage, L; Waxman, R; Moverley, A R; Worthington, S; Helliwell, P S

2016-09-01

Many questionnaires are available for assessment of psoriatic arthritis (PsA), but there is little evidence comparing them. To test the proposed CONTEST questionnaire, which was developed to identify patients with psoriasis who have undiagnosed PsA, and compare it with the validated Psoriasis Epidemiology Screening Tool (PEST) questionnaire in a primary-care setting. A random sample of adult patients with psoriasis and no diagnosis of arthritis was identified from five general practice surgeries in Yorkshire, U.K. Consenting patients completed both questionnaires and were assessed by a dermatologist and rheumatologist. Diagnosis of PsA was made by the assessing rheumatologist. Receiver operator characteristic (ROC) curve analysis examined the sensitivity and specificity of potential cut points. In total 932 packs were sent to recruit 191 (20·5%) participants. Of these, 169 (88·5%) were confirmed to have current or previous psoriasis. Using physician diagnosis 17 (10·1%) were found to have previously undiagnosed PsA, while 90 (53·3%) had another musculoskeletal complaint and 62 (36·7%) had no musculoskeletal problems. Using ROC curve analysis, all of the questionnaires showed a significant ability to identify PsA. The area under the curve (AUC) for the CONTEST questionnaires was slightly higher than that of PEST (0·69 and 0·70 vs. 0·65), but there was no significant difference identified. Examining the sensitivities and specificities for the different cut points suggested that a PEST score ≥ 2 would perform better in this dataset, and the optimal scores for CONTEST and CONTEST plus joint manikin were 3 and 4, respectively. The accuracy of the questionnaires to identify PsA appeared similar, with a slightly higher AUC for the CONTEST questionnaires. The optimal cut points in this study appeared lower than in previous studies. © 2016 British Association of Dermatologists.

Genetic Screens in Yeast to Identify BRCA1 Modifiers

National Research Council Canada - National Science Library

Plon, Sharon E

2004-01-01

.... The yeast RAD9 protein has similar functions and sequence motifs as BRCA1 and we proposed to identify candidate modifier loci by identifying haploinsufficient mutations at a second locus that alters...

IDENTIFYING DEMENTIA IN ELDERLY POPULATION : A CAMP APPROACH

OpenAIRE

Anand P; Chaukimath; Srikanth; Koli

2015-01-01

BACKGROUND: Dementia is an emerging medico social problem affecting elderly, and poses a challenge to clinician and caregivers. It is usually identified in late stage where management becomes difficult. AIM: The aim of camp was to identify dementia in elderly population participating in screening camp. MATERIAL AND METHODS : The geriatric clinic and department of psychiatry jointly organised screening camp to detect dementia in elderly for five days in Sept...

Expanded Newborn Screening Program in Saudi Arabia: Incidence of screened disorders.

Science.gov (United States)

Alfadhel, Majid; Al Othaim, Ali; Al Saif, Saif; Al Mutairi, Fuad; Alsayed, Moeenaldeen; Rahbeeni, Zuhair; Alzaidan, Hamad; Alowain, Mohammed; Al-Hassnan, Zuhair; Saeedi, Mohamad; Aljohery, Saeed; Alasmari, Ali; Faqeih, Eissa; Alwakeel, Mansour; AlMashary, Maher; Almohameed, Sulaiman; Alzahrani, Mohammed; Migdad, Abeer; Al-Dirbashi, Osama Y; Rashed, Mohamed; Alamoudi, Mohamed; Jacob, Minnie; Alahaidib, Lujane; El-Badaoui, Fahd; Saadallah, Amal; Alsulaiman, Ayman; Eyaid, Wafaa; Al-Odaib, Ali

2017-06-01

To address the implementation of the National Newborn Screening Program (NBS) in Saudi Arabia and stratify the incidence of the screened disorders. A retrospective study conducted between 1 August 2005 and 31 December 2012, total of 775 000 newborns were screened from 139 hospitals distributed among all regions of Saudi Arabia. The NBS Program screens for 16 disorders from a selective list of inborn errors of metabolism (IEM) and endocrine disorders. Heel prick dry blood spot samples were obtained from all newborns for biochemical and immunoassay testing. Recall screening testing was performed for Initial positive results and confirmed by specific biochemical assays. A total of 743 cases were identified giving an overall incidence of 1:1043. Frequently detected disorders nationwide were congenital hypothyroidism and congenital adrenal hyperplasia with an incidence of 1:7175 and 1:7908 correspondingly. The highest incidence among the IEM was propionic acidaemia with an incidence rate of 1:14 000. The article highlights the experience of the NBS Program in Saudi Arabia and providing data on specific regional incidences of all the screened disorders included in the programme; and showed that the incidence of these disorders is one of the highest reported so far world-wide. © 2017 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).

Kinome-wide shRNA Screen Identifies the Receptor Tyrosine Kinase AXL as a Key Regulator for Mesenchymal Glioblastoma Stem-like Cells

Directory of Open Access Journals (Sweden)

Peng Cheng

2015-05-01

Full Text Available Glioblastoma is a highly lethal cancer for which novel therapeutics are urgently needed. Two distinct subtypes of glioblastoma stem-like cells (GSCs were recently identified: mesenchymal (MES and proneural (PN. To identify mechanisms to target the more aggressive MES GSCs, we combined transcriptomic expression analysis and kinome-wide short hairpin RNA screening of MES and PN GSCs. In comparison to PN GSCs, we found significant upregulation and phosphorylation of the receptor tyrosine kinase AXL in MES GSCs. Knockdown of AXL significantly decreased MES GSC self-renewal capacity in vitro and inhibited the growth of glioblastoma patient-derived xenografts. Moreover, inhibition of AXL with shRNA or pharmacologic inhibitors also increased cell death significantly more in MES GSCs. Clinically, AXL expression was elevated in the MES GBM subtype and significantly correlated with poor prognosis in multiple cancers. In conclusion, we identified AXL as a potential molecular target for novel approaches to treat glioblastoma and other solid cancers.

IDENTIFYING DEMENTIA IN ELDERLY POPULATION : A CAMP APPROACH

Directory of Open Access Journals (Sweden)

Anand P

2015-06-01

Full Text Available BACKGROUND: Dementia is an emerging medico social problem affecting elderly, and poses a challenge to clinician and caregivers. It is usually identified in late stage where management becomes difficult. AIM: The aim of camp was to identify dementia in elderly population participating in screening camp. MATERIAL AND METHODS : The geriatric clinic and department of psychiatry jointly organised screening camp to detect dementia in elderly for five days in September 2014 to commemorate world Alzheimer’s day. The invitation regarding camp was sent to all senio r citizen forums and also published in leading Kannada daily newspaper. Mini Mental Status Examination and Diagnostic and Statistical Manual of Mental Disorders, 4 th edition criteria (DSM IV was used to identify dementia. RESULTS: Elderly male participate d in camp in more number than females and dementia was identified in 36% elderly with education less than 9 th standard. Dementia was found in 18% in our study population. CONCLUSION: The camp help identify elderly suffering from dementia and also created a wareness about it. Hypertension and diabetes mellitus were common co morbidity in study population. Our study suggested organising screening camp will help identify elderly living with dementia.

Screening on urogenital Chlamydia trachomatis

Directory of Open Access Journals (Sweden)

Busse, Reinhard

2005-12-01

Full Text Available Introduction: Around 92 million urogenital infections are caused yearly by Chlamydia trachomatis worldwide [1]. The overall incidence of sexually transmitted diseases is increasing, as shown by the increases in the number of reported cases of syphilis and gonorrhea [2]. Chlamydia trachomatis infections are associated with various serious diseases in women, men and newborns, which could be, at least partially, avoided by means of early diagnosis and therapy. The Federal Joint Committee - responsible for decision-making concerning the benefit package of the German Social Health Insurance - has publicly announced the starting of deliberations on the issue of screening for Chlamydia trachomatis. Research Questions: The leading question to be answered is whether screening for Chlamydia trachomatis should be included in the German benefit basket. The aim of this report is to provide a summary of the available evidence concerning the issue of screening for Chlamydia trachomatis. Methods: The summary of published scientific evidence, including HTA reports, systematic reviews, guidelines and primary research is represented. The synthesis follows the structure given by the criteria of Wilson and Jungner [3] for the introduction of screening in a population: relevance of the condition, availability of an adequate test, effectiveness of screening, acceptance of the programme, and economical issues. A literature search was conducted for each aspect of the synthesis and the evidence has been summarised in evidence tables. Results: We identified five HTA reports from three European agencies [4], [5], [6], [7] and one from the USA [8]. In addition, we identified four guidelines from Northamerica [9], [10], [11], [12] and one from Europe [13]. A total of 56 primary research publications were included: relevance of the disease (n=26, availability of test (n=1, effectiveness of screening (n=11, acceptance of the programme (n=11, economical issues (n=7. Discussion

Screening for type 2 diabetes in a multiethnic setting using known risk factors to identify those at high risk: a cross-sectional study

Directory of Open Access Journals (Sweden)

Laura J Gray

2010-09-01

Full Text Available Laura J Gray1, Jennifer R Tringham2, Melanie J Davies3, David R Webb3, Janet Jarvis4, Timothy C Skinner5, Azhar M Farooqi1, Kamlesh Khunti11Department of Health Sciences, University of Leicester, Leicester, UK; 2Department of Diabetes, Frimley Park Hospital, Surrey, UK; 3Department of Cardiovascular Sciences, University Hospitals of Leicester, Leicester, UK; 4University Hospitals Leicester, Leicester, UK; 5Flinders University Rural Clinical School, Flinders University, Renmark, AustraliaIntroduction: Screening enables the identification of type 2 diabetes mellitus (T2DM during its asymptomatic stage and therefore allows early intervention which may lead to fewer complications and improve outcomes. A targeted screening program was carried out in a United Kingdom (UK multiethnic population to identify those with abnormal glucose tolerance.Methods: A sample of individuals aged 25–75 years (40–75 white European with at least one risk factor for T2DM were invited for screening from 17 Leicestershire (UK general practices or through a health awareness campaign. All participants received a 75 g oral glucose tolerance test, cardiovascular risk assessment, detailed medical and family histories and anthropometric measurements.Results: In the 3,225 participants who were screened. 640 (20% were found to have some form of abnormal glucose tolerance of whom 4% had T2DM, 3% impaired fasting glucose (IFG, 10% impaired glucose tolerance (IGT and 3% both IFG and IGT. The odds of detecting IGT was approximately 60% greater (confounder-adjusted odds ratios [OR] 1.67 [1.22–2.29] in the South Asian population.Conclusions: Around one in five people who had targeted screening have IGT, IFG or T2DM, with a higher prevalence in those of South Asian origin. The prevalence of undetected T2DM is lower in South Asians compared to previously published studies and maybe due to increased awareness of this group being at high risk.Keywords: type 2 diabetes, screening

Barriers to colorectal cancer screening in community health centers: A qualitative study

Directory of Open Access Journals (Sweden)

Fletcher Robert H

2008-02-01

Full Text Available Abstract Background Colorectal cancer screening rates are low among disadvantaged patients; few studies have explored barriers to screening in community health centers. The purpose of this study was to describe barriers to/facilitators of colorectal cancer screening among diverse patients served by community health centers. Methods We identified twenty-three outpatients who were eligible for colorectal cancer screening and their 10 primary care physicians. Using in-depth semi-structured interviews, we asked patients to describe factors influencing their screening decisions. For each unscreened patient, we asked his or her physician to describe barriers to screening. We conducted patient interviews in English (n = 8, Spanish (n = 2, Portuguese (n = 5, Portuguese Creole (n = 1, and Haitian Creole (n = 7. We audiotaped and transcribed the interviews, and then identified major themes in the interviews. Results Four themes emerged: 1 Unscreened patients cited lack of trust in doctors as a barrier to screening whereas few physicians identified this barrier; 2 Unscreened patients identified lack of symptoms as the reason they had not been screened; 3 A doctor's recommendation, or lack thereof, significantly influenced patients' decisions to be screened; 4 Patients, but not their physicians, cited fatalistic views about cancer as a barrier. Conversely, physicians identified competing priorities, such as psychosocial stressors or comorbid medical illness, as barriers to screening. In this culturally diverse group of patients seen at community health centers, similar barriers to screening were reported by patients of different backgrounds, but physicians perceived other factors as more important. Conclusion Further study of these barriers is warranted.

Shattering the Screen: Embodied Narrative in Digital Media

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Russell J. Cook

2017-03-01

Full Text Available This illustrated phenomenological inquiry into storytelling in screen media identifies important media transformations of experience. Viewers embody, or situate their experienced selves, according to screen requirements. A viewer’s compelled perspective on the screen causes fundamental spatio-temporal transformations of narrative experience, including horizontal stretching of screen space and time compression or leakage. Virtual media have the potential, as yet unrealized, to break out of the screen and to restore narrative to its primordial, experiential roots.

Screening for psychological distress in cancer: renewing the research agenda.

Science.gov (United States)

Salmon, Peter; Clark, Louise; McGrath, Elly; Fisher, Peter

2015-03-01

Although health policy for cancer care promotes screening of patients for emotional distress, the utility and validity of screening have been questioned. Continued research to refine detection of distress or to evaluate outcomes of screening programmes is unlikely to end this controversy. Instead, we need to identify more fundamental research questions that address the validity or utility of screening in this context. We critically and selectively review research and policy literature on psychological screening in cancer care, drawing also from research literature about the nature of psychological needs in cancer care and from relevant literature on psychological screening in mental health. We identify three broad research questions: (i) Apart from intensity of distress, what further information should screening seek about the context of distress, psychological processes that promote distress and patients' own perspective on their needs? (ii) What are the implications of the contextual dependence of disclosure of emotional feelings, given that screening questions can be asked in contexts ranging from an impersonal questionnaire to dialogue with a trusted practitioner? (iii) How should a screen be responded to, given the inherent uncertainty associated with screening results and given that distress in a cancer context can indicate instrumental as well as psychological needs? Examining these questions will mean exchanging a diagnostic framework for screening, in which health need is indicated by the presence of a psychological disorder, for a public health framework, in which health need is identified from multiple perspectives. Copyright © 2014 John Wiley & Sons, Ltd.

High-throughput screening of chemicals as functional ...

Science.gov (United States)

Identifying chemicals that provide a specific function within a product, yet have minimal impact on the human body or environment, is the goal of most formulation chemists and engineers practicing green chemistry. We present a methodology to identify potential chemical functional substitutes from large libraries of chemicals using machine learning based models. We collect and analyze publicly available information on the function of chemicals in consumer products or industrial processes to identify a suite of harmonized function categories suitable for modeling. We use structural and physicochemical descriptors for these chemicals to build 41 quantitative structure–use relationship (QSUR) models for harmonized function categories using random forest classification. We apply these models to screen a library of nearly 6400 chemicals with available structure information for potential functional substitutes. Using our Functional Use database (FUse), we could identify uses for 3121 chemicals; 4412 predicted functional uses had a probability of 80% or greater. We demonstrate the potential application of the models to high-throughput (HT) screening for “candidate alternatives” by merging the valid functional substitute classifications with hazard metrics developed from HT screening assays for bioactivity. A descriptor set could be obtained for 6356 Tox21 chemicals that have undergone a battery of HT in vitro bioactivity screening assays. By applying QSURs, we wer

Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

Science.gov (United States)

Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

2011-01-01

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

Use of anti-Müllerian hormone testing during ovarian reserve screening to identify women at risk of polycystic ovary syndrome.

Science.gov (United States)

Safier, Lauren Z; Grossman, Lisa C; Chan, Cariann W; Sauer, Mark V; Lobo, Rogerio A; Douglas, Nataki C

2016-10-01

To assess the applicability of anti-Müllerian hormone (AMH) testing in the identification of women at risk for polycystic ovary syndrome (PCOS) when AMH is used in ovarian reserve screening in the general population. A secondary analysis was undertaken of a large cross-sectional study. Women aged 27-37years, presently delaying childbearing but interested in future fertility, completed an online questionnaire to assess knowledge and attitudes about ovarian reserve testing, and underwent serum AMH testing between October 2014 and April 2015 in New York, NY, USA. For the secondary analysis, women considered to have elevated AMH levels (≥4.7ng/mL) were invited for physical examination and transvaginal ultrasonography. Among 97 women who underwent AMH testing, 32 (33.0%) had elevated AMH levels. Hyperandrogenism was reported by 8 (25.0%) women with elevated AMH and none with AMH concentrations lower than 4.7ng/mL (Ppolycystic ovaries and 13 (65.0%) were diagnosed with PCOS (Rotterdam criteria). When AMH levels are used as a screening test for fertility, elevated concentrations can identify women at risk for PCOS. Copyright © 2016 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

Antifungal chemical compounds identified using a C. elegans pathogenicity assay.

Directory of Open Access Journals (Sweden)

Julia Breger

2007-02-01

Full Text Available There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (approximately 1.2% that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as "probe compounds" and may have antifungal activity against other fungi.

Can economics be applied to prenatal screening?

OpenAIRE

Nicholas Phin

1990-01-01

This paper is a review of the economics of prenatal screening as seen from a medical point of view. The difficulties and controversies over the economic analysis are examined with specific reference to screening for Down syndrome. The aims and principles of prenatal screening are set out and discussed before reviewing the attempts that have been made to assess the costs and benefits of screening for Down syndrome. The major problem identified is the measurement and valuation of benefits. This...

Novel Abscisic Acid Antagonists Identified with Chemical Array Screening.

Science.gov (United States)

Ito, Takuya; Kondoh, Yasumitsu; Yoshida, Kazuko; Umezawa, Taishi; Shimizu, Takeshi; Shinozaki, Kazuo; Osada, Hiroyuki

2015-11-01

Abscisic acid (ABA) signaling is involved in multiple processes in plants, such as water stress control and seed dormancy. Major regulators of ABA signaling are the PYR/PYL/RCAR family receptor proteins, group A protein phosphatases 2C (PP2Cs), and subclass III of SNF1-related protein kinase 2 (SnRK2). Novel ABA agonists and antagonists to modulate the functions of these proteins would not only contribute to clarification of the signaling mechanisms but might also be used to improve crop yields. To obtain small molecules that interact with Arabidopsis ABA receptor PYR1, we screened 24 275 compounds from a chemical library at the RIKEN Natural Products Depository by using a chemical array platform. Subsequent SnRK2 and PP2C assays narrowed down the candidates to two molecules. One antagonized ABA in a competitive manner and inhibited the formation of the PYR1-ABA-PP2C ternary complex. These compounds might have potential as bioprobes to analyze ABA signaling. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Missed opportunities in primary care: the importance of identifying depression through screening, family history, and chronic disease management.

Science.gov (United States)

Maradiegue, Ann H; Khan, Fakiha

2013-02-01

This study explored the adequacy of depression screening in a community health center. The medical charts of individuals (N = 90) enrolled at a community health center were randomly selected, reviewed, and compared to current standard-of-care guidelines for four elements: family history, screening for depression, control of chronic illnesses, and missed opportunities for preventive care. Family history documentation collected by the providers was limited and 44.4% had no family history. There was no routine depression screening process, although 48.9% of the clients had red flags (warning signals) for depression. Laboratory values used for screening control of chronic disease in the medical records were: fasting glucose levels ⩽100 mg/dL (46%), total cholesterol levels ⩽200 mg/dL (38%), and blood pressure ⩽120/80 mmHg (23%). The results highlight the need to focus on depression screening as part of preventive care and the management of chronic disease in the primary care setting. Copyright 2013, SLACK Incorporated.

School scoliosis screening programme-a systematic review.

Science.gov (United States)

Sabirin, J; Bakri, R; Buang, S N; Abdullah, A T; Shapie, A

2010-12-01

A systematic review on the effectiveness and cost-effectiveness of school scoliosis screening programme was carried out. A total of 248 relevant titles were identified, 117 abstracts were screened and 28 articles were included in the results. There was fair level of evidence to suggest that school scoliosis screening programme is safe, contributed to early detection and reduction of surgery. There was also evidence to suggest that school-based scoliosis screening programme is cost-effective. Based on the above review, screening for scoliosis among school children is recommended only for high risk group such as girls at twelve years of age.

European position statement on lung cancer screening

DEFF Research Database (Denmark)

Oudkerk, Matthijs; Devaraj, Anand; Vliegenthart, Rozemarijn

2017-01-01

Lung cancer screening with low-dose CT can save lives. This European Union (EU) position statement presents the available evidence and the major issues that need to be addressed to ensure the successful implementation of low-dose CT lung cancer screening in Europe. This statement identified...... specific actions required by the European lung cancer screening community to adopt before the implementation of low-dose CT lung cancer screening. This position statement recommends the following actions: a risk stratification approach should be used for future lung cancer low-dose CT programmes...... need to set a timeline for implementing lung cancer screening....

Vision Screening

Science.gov (United States)

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.

Factors Influencing Colorectal Cancer Screening Participation

Directory of Open Access Journals (Sweden)

Antonio Z. Gimeno García

2012-01-01

Full Text Available Colorectal cancer (CRC is a major health problem worldwide. Although population-based CRC screening is strongly recommended in average-risk population, compliance rates are still far from the desirable rates. High levels of screening uptake are necessary for the success of any screening program. Therefore, the investigation of factors influencing participation is crucial prior to design and launches a population-based organized screening campaign. Several studies have identified screening behaviour factors related to potential participants, providers, or health care system. These influencing factors can also be classified in non-modifiable (i.e., demographic factors, education, health insurance, or income and modifiable factors (i.e., knowledge about CRC and screening, patient and provider attitudes or structural barriers for screening. Modifiable determinants are of great interest as they are plausible targets for interventions. Interventions at different levels (patient, providers or health care system have been tested across the studies with different results. This paper analyzes factors related to CRC screening behaviour and potential interventions designed to improve screening uptake.

Novel high-resolution computed tomography-based radiomic classifier for screen-identified pulmonary nodules in the National Lung Screening Trial.

Science.gov (United States)

Peikert, Tobias; Duan, Fenghai; Rajagopalan, Srinivasan; Karwoski, Ronald A; Clay, Ryan; Robb, Richard A; Qin, Ziling; Sicks, JoRean; Bartholmai, Brian J; Maldonado, Fabien

2018-01-01

Optimization of the clinical management of screen-detected lung nodules is needed to avoid unnecessary diagnostic interventions. Herein we demonstrate the potential value of a novel radiomics-based approach for the classification of screen-detected indeterminate nodules. Independent quantitative variables assessing various radiologic nodule features such as sphericity, flatness, elongation, spiculation, lobulation and curvature were developed from the NLST dataset using 726 indeterminate nodules (all ≥ 7 mm, benign, n = 318 and malignant, n = 408). Multivariate analysis was performed using least absolute shrinkage and selection operator (LASSO) method for variable selection and regularization in order to enhance the prediction accuracy and interpretability of the multivariate model. The bootstrapping method was then applied for the internal validation and the optimism-corrected AUC was reported for the final model. Eight of the originally considered 57 quantitative radiologic features were selected by LASSO multivariate modeling. These 8 features include variables capturing Location: vertical location (Offset carina centroid z), Size: volume estimate (Minimum enclosing brick), Shape: flatness, Density: texture analysis (Score Indicative of Lesion/Lung Aggression/Abnormality (SILA) texture), and surface characteristics: surface complexity (Maximum shape index and Average shape index), and estimates of surface curvature (Average positive mean curvature and Minimum mean curvature), all with Pscreen-detected nodule characterization appears extremely promising however independent external validation is needed.

A Phenotypic Cell-Binding Screen Identifies a Novel Compound Targeting Triple-Negative Breast Cancer.

Science.gov (United States)

Chen, Luxi; Long, Chao; Youn, Jonghae; Lee, Jiyong

2018-06-11

We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug targets. Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelial-mesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

Attitudes toward newborn screening for cytomegalovirus infection.

Science.gov (United States)

Din, Erica S; Brown, Cedric J; Grosse, Scott D; Wang, Chengbin; Bialek, Stephanie R; Ross, Danielle S; Cannon, Michael J

2011-12-01

Newborns are not routinely screened for cytomegalovirus (CMV), the leading infectious cause of developmental disability. Congenital CMV satisfies a number of criteria for inclusion in newborn screening, and screening potentially offers benefits. Screening could also introduce harms such as anxiety and unnecessary costs for the families of the substantial proportion of CMV-infected children who never develop CMV-related disabilities. Our objective was to assess attitudes toward newborn screening for CMV. We analyzed responses to 5 statements about CMV and newborn screening from 3922 participants in the 2009 HealthStyles survey, a national mail survey designed to include a group similar to the US population with respect to gender, age, race/ethnicity, income, and household size. Two-step cluster analysis was performed to identify clusters of parental attitudes. The majority of respondents strongly or somewhat agreed that they would want to have their newborn tested for CMV even if it was not performed routinely (84%), they had to pay $20 (87%), or CMV-related problems never developed (84%). Nearly half (47%) of them "would worry that the CMV test would lead to unneeded doctor visits and expenses," and 32% "think CMV problems are too rare to worry about." Three clusters of parent respondents were identified on the basis of their attitudes toward CMV screening: "strongly in favor" (31%), "moderately in favor" (49%), and "weakly opposed" (20%). Among most parents, costs, worry, and anxiety associated with newborn screening for CMV would be acceptable. Although attitudes were generally favorable, a minority of the parents were weakly opposed to newborn screening for CMV.

Overdiagnosis in mammographic screening for breast cancer in Europe

DEFF Research Database (Denmark)

Puliti, Donella; Duffy, Stephen W; Miccinesi, Guido

2012-01-01

Overdiagnosis, the detection through screening of a breast cancer that would never have been identified in the lifetime of the woman, is an adverse outcome of screening. We aimed to determine an estimate range for overdiagnosis of breast cancer in European mammographic service screening programmes....

Prenatal Screening Using Maternal Markers

Directory of Open Access Journals (Sweden)

Howard Cuckle

2014-05-01

Full Text Available Maternal markers are widely used to screen for fetal neural tube defects (NTDs, chromosomal abnormalities and cardiac defects. Some are beginning to broaden prenatal screening to include pregnancy complications such as pre-eclampsia. The methods initially developed for NTDs using a single marker have since been built upon to develop high performance multi-maker tests for chromosomal abnormalities. Although cell-free DNA testing is still too expensive to be considered for routine application in public health settings, it can be cost-effective when used in combination with existing multi-maker marker tests. The established screening methods can be readily applied in the first trimester to identify pregnancies at high risk of pre-eclampsia and offer prevention though aspirin treatment. Prenatal screening for fragile X syndrome might be adopted more widely if the test was to be framed as a form of maternal marker screening.

Baseline and annual repeat rounds of screening. Implications for optimal regimens of screening

Energy Technology Data Exchange (ETDEWEB)

Henschke, Claudia I.; Salvatore, Mary; Cham, Matthew; Eber, Corey; Yip, Rowena; Yankelevitz, David F. [Icahn School of Medicine at Mount Sinai, Department of Radiology, New York, NY (United States); Powell, Charles A.; DiFabrizio, Larry [Icahn School of Medicine at Mount Sinai, Department of Medicine, New York, NY (United States); Flores, Raja; Kaufman, Andrew [Icahn School of Medicine at Mount Sinai, Department of Thoracic Surgery, New York, NY (United States); Collaboration: International Early Lung Cancer Action Program Investigators

2018-03-15

Differences in results of baseline and subsequent annual repeat rounds provide important information for optimising the regimen of screening. A prospective cohort study of 65,374 was reviewed to examine the frequency/percentages of the largest noncalcified nodule (NCN), lung cancer cell types and Kaplan-Meier (K-M) survival rates, separately for baseline and annual rounds. Of 65,374 baseline screenings, NCNs were identified in 28,279 (43.3%); lung cancer in 737 (1.1%). Of 74,482 annual repeat screenings, new NCNs were identified in 4959 (7%); lung cancer in 179 (0.24%). Only adenocarcinoma was diagnosed in subsolid NCNs. Percentages of lung cancers by cell type were significantly different (p < 0.0001) in the baseline round compared with annual rounds, reflecting length bias, as were the ratios, reflecting lead times. Long-term K-M survival rate was 100% for typical carcinoids and for adenocarcinomas manifesting as subsolid NCNs; 85% (95% CI 81-89%) for adenocarcinoma, 74% (95% CI 63-85%) for squamous cell, 48% (95% CI 34-62%) for small cell. The rank ordering by lead time was the same as the rank ordering by survival rates. The significant differences in the frequency of NCNs and frequency and aggressiveness of diagnosed cancers in baseline and annual repeat need to be recognised for an optimal regimen of screening. (orig.)

A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35–NP Interface

Energy Technology Data Exchange (ETDEWEB)

Liu, Gai; Nash, Peter J.; Johnson, Britney; Pietzsch, Colette; Ilagan, Ma. Xenia G.; Bukreyev, Alexander; Basler, Christopher F.; Bowlin, Terry L.; Moir, Donald T.; Leung, Daisy W.; Amarasinghe, Gaya K. (WU-MED); (GSU); (Texas-MED); (Microbiotix)

2017-01-24

The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP–VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.

In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity

Energy Technology Data Exchange (ETDEWEB)

Boisvert, Annie; Jones, Steven; Issop, Leeyah [The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Medicine, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Erythropel, Hanno C. [Department of Chemical Engineering, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Papadopoulos, Vassilios [The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Medicine, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Culty, Martine, E-mail: martine.culty@mcgill.ca [The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Medicine, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada)

2016-10-15

Plasticizers are indispensable additives providing flexibility and malleability to plastics. Among them, several phthalates, including di (2-ethylhexyl) phthalate (DEHP), have emerged as endocrine disruptors, leading to their restriction in consumer products and creating a need for new, safer plasticizers. The goal of this project was to use in vitro functional screening tools to select novel non-toxic plasticizers suitable for further in vivo evaluation. A panel of novel compounds with satisfactory plasticizer properties and biodegradability were tested, along with several commercial plasticizers, such as diisononyl-cyclohexane-1,2-dicarboxylate (DINCH®). MEHP, the monoester metabolite of DEHP was also included as reference compound. Because phthalates target mainly testicular function, including androgen production and spermatogenesis, we used the mouse MA-10 Leydig and C18-4 spermatogonial cell lines as surrogates to examine cell survival, proliferation, steroidogenesis and mitochondrial integrity. The most promising compounds were further assessed on organ cultures of rat fetal and neonatal testes, corresponding to sensitive developmental windows. Dose-response studies revealed the toxicity of most maleates and fumarates, while identifying several dibenzoate and succinate plasticizers as innocuous on Leydig and germ cells. Interestingly, DINCH®, a plasticizer marketed as a safe alternative to phthalates, exerted a biphasic effect on steroid production in MA-10 and fetal Leydig cells. MEHP was the only plasticizer inducing the formation of multinucleated germ cells (MNG) in organ culture. Overall, organ cultures corroborated the cell line data, identifying one dibenzoate and one succinate as the most promising candidates. The adoption of such collaborative approaches for developing new chemicals should help prevent the development of compounds potentially harmful to human health. - Highlights: • Phthalate plasticizers exert toxic effects on male reproduction

In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity

International Nuclear Information System (INIS)

Boisvert, Annie; Jones, Steven; Issop, Leeyah; Erythropel, Hanno C.; Papadopoulos, Vassilios; Culty, Martine

2016-01-01

Plasticizers are indispensable additives providing flexibility and malleability to plastics. Among them, several phthalates, including di (2-ethylhexyl) phthalate (DEHP), have emerged as endocrine disruptors, leading to their restriction in consumer products and creating a need for new, safer plasticizers. The goal of this project was to use in vitro functional screening tools to select novel non-toxic plasticizers suitable for further in vivo evaluation. A panel of novel compounds with satisfactory plasticizer properties and biodegradability were tested, along with several commercial plasticizers, such as diisononyl-cyclohexane-1,2-dicarboxylate (DINCH®). MEHP, the monoester metabolite of DEHP was also included as reference compound. Because phthalates target mainly testicular function, including androgen production and spermatogenesis, we used the mouse MA-10 Leydig and C18-4 spermatogonial cell lines as surrogates to examine cell survival, proliferation, steroidogenesis and mitochondrial integrity. The most promising compounds were further assessed on organ cultures of rat fetal and neonatal testes, corresponding to sensitive developmental windows. Dose-response studies revealed the toxicity of most maleates and fumarates, while identifying several dibenzoate and succinate plasticizers as innocuous on Leydig and germ cells. Interestingly, DINCH®, a plasticizer marketed as a safe alternative to phthalates, exerted a biphasic effect on steroid production in MA-10 and fetal Leydig cells. MEHP was the only plasticizer inducing the formation of multinucleated germ cells (MNG) in organ culture. Overall, organ cultures corroborated the cell line data, identifying one dibenzoate and one succinate as the most promising candidates. The adoption of such collaborative approaches for developing new chemicals should help prevent the development of compounds potentially harmful to human health. - Highlights: • Phthalate plasticizers exert toxic effects on male reproduction

In silico structure-based drug screening of novel antimycobacterial pharmacophores by DOCK-GOLD tandem screening

Directory of Open Access Journals (Sweden)

Junichi Taira

2017-01-01

Full Text Available Background: Enzymes responsible for cell wall development in Mycobacterium tuberculosis are considered as potential targets of anti-tuberculosis (TB agents. Mycobacterial cyclopropane mycolic acid synthase 1 (CmaA1 is essential for mycobacterial survival because of its critical role in synthesizing mycolic acids. Materials and Methods: We screened compounds that were capable of interacting with the mycobacterial CmaA1 active site using a virtual compound library with an in silico structure-based drug screening (SBDS. Following the selection of such compounds, their antimycobacterial activity was examined. Results: With the in silico SBDS, for which we also used DOCK-GOLD programs and screening methods that utilized the structural similarity between the selected active compounds, we identified two compounds with potent inhibitory effects on mycobacterial growth. The antimycobacterial effect of the compounds was comparable to that of isoniazid, which is used as a first-line anti-TB drug. Conclusion: The compounds identified through SBDS were expected to be a novel class of anti-TB pharmacophores.

Genetic screens in Caenorhabditis elegans models for neurodegenerative diseases

NARCIS (Netherlands)

Alvarenga Fernandes Sin, Olga; Michels, Helen; Nollen, Ellen A. A.

2014-01-01

Caenorhabditis elegans comprises unique features that make it an attractive model organism in diverse fields of biology. Genetic screens are powerful to identify genes and C. elegans can be customized to forward or reverse genetic screens and to establish gene function. These genetic screens can be

Determining Childhood Blood Lead Level Screening Compliance Among Physicians.

Science.gov (United States)

Haboush-Deloye, Amanda; Marquez, Erika R; Gerstenberger, Shawn L

2017-08-01

Childhood Lead Poisoning Prevention Programs throughout the U.S. have addressed childhood lead poisoning by implementing primary and secondary prevention efforts. While many programs have helped increase screening rates, in some states children under the age of six still have not been tested for lead. This study aims to identify the barriers to childhood blood lead testing and develop a strategy to increase the number of children tested. Clark County physicians who work with children six and under were surveyed about blood lead level (BLL) testing practices, particularly, adherence to Centers for Disease Control and Prevention (CDC) guidelines, and parental compliance with orders to have their children tested to determine their blood lead levels. In addition, select in-person interviews were conducted with physicians who reported high parental compliance to identify best practices and barriers. Of the 77 physicians that provided data, 48% indicated they did not follow CDC guideline compared to 52% who follow guidelines. 18 of the 30 (or 60%) physicians reported more than 80% of parents complied with doctor recommended BLL testing. Twelve physicians identified cost, lack of insurance, and absence of symptomology as persistent barriers to lead screening. This study identified barriers to childhood lead screening including inadequate parental adherence to physician-ordered screenings and physician non-compliance with screening recommendations are two primary contributors. Addressing these issues could increase screening in children and reduce the risk of lead poisoning.

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

Science.gov (United States)

Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

2016-01-01

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

Science.gov (United States)

Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

2015-01-01

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

Directory of Open Access Journals (Sweden)

Christoph Seiler

Full Text Available Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

Implementation of a Targeted Screening Program to Detect Airflow Obstruction Suggestive of Chronic Obstructive Pulmonary Disease within a Presurgical Screening Clinic

Directory of Open Access Journals (Sweden)

Chantal Robitaille

2015-01-01

Full Text Available BACKGROUND: Targeted spirometry screening for chronic obstructive pulmonary disease (COPD has been studied in primary care and community settings. Limitations regarding availability and quality of testing remain. A targeted spirometry screening program was implemented within a presurgical screening (PSS clinic to detect undiagnosed airways disease and identify patients with COPD/asthma in need of treatment optimization.

Breast Cancer Screening in an Era of Personalized Regimens

Science.gov (United States)

Onega, Tracy; Beaber, Elisabeth F.; Sprague, Brian L.; Barlow, William E.; Haas, Jennifer S.; Tosteson, Anna N.A.; Schnall, Mitchell D.; Armstrong, Katrina; Schapira, Marilyn M.; Geller, Berta; Weaver, Donald L.; Conant, Emily F.

2014-01-01

Breast cancer screening holds a prominent place in public health, health care delivery, policy, and women’s health care decisions. Several factors are driving shifts in how population-based breast cancer screening is approached, including advanced imaging technologies, health system performance measures, health care reform, concern for “overdiagnosis,” and improved understanding of risk. Maximizing benefits while minimizing the harms of screening requires moving from a “1-size-fits-all” guideline paradigm to more personalized strategies. A refined conceptual model for breast cancer screening is needed to align women’s risks and preferences with screening regimens. A conceptual model of personalized breast cancer screening is presented herein that emphasizes key domains and transitions throughout the screening process, as well as multilevel perspectives. The key domains of screening awareness, detection, diagnosis, and treatment and survivorship are conceptualized to function at the level of the patient, provider, facility, health care system, and population/policy arena. Personalized breast cancer screening can be assessed across these domains with both process and outcome measures. Identifying, evaluating, and monitoring process measures in screening is a focus of a National Cancer Institute initiative entitled PROSPR (Population-based Research Optimizing Screening through Personalized Regimens), which will provide generalizable evidence for a risk-based model of breast cancer screening, The model presented builds on prior breast cancer screening models and may serve to identify new measures to optimize benefits-to-harms tradeoffs in population-based screening, which is a timely goal in the era of health care reform. PMID:24830599

Sickle cell disease: time for a targeted neonatal screening programme.

LENUS (Irish Health Repository)

Gibbons, C

2015-02-01

Ireland has seen a steady increase in paediatric sickle cell disease (SCD). In 2005, only 25% of children with SCD were referred to the haemoglobinopathy service in their first year. A non-funded screening programme was implemented. This review aimed to assess the impact screening has had. All children referred to the haemoglobinopathy service born in Ireland after 2005 were identified. Data was collected from the medical chart and laboratory system. Information was analysed using Microsoft Excel. 77 children with SCD were identified. The median age at antibiotic commencement in the screened group was 56 days compared with 447 days in the unscreened group, p = < 0.0003. 22 (28%) of infants were born in centre\\'s that do not screen and 17 (81%) were over 6 months old at referral, compared with 14 (21%) in the screened group. 6 (27%) of those in the unscreened group presented in acute crisis compared with 2 (3%) in the screened population. The point prevalence of SCD in Ireland is 0.2% in children under 15 yr of African and Asian descent. We identified delays in referral and treatment, which reflect the lack of government funded support and policy. We suggest all maternity units commence screening for newborns at risk of SCD. It is a cost effective intervention with a number needed to screen of just 4 to prevent a potentially fatal crisis.

Screening Technologies for Target Identification in Pancreatic Cancer

Energy Technology Data Exchange (ETDEWEB)

Michl, Patrick, E-mail: michlp@med.uni-marburg.de; Ripka, Stefanie; Gress, Thomas; Buchholz, Malte [Department of Gastroenterology and Endocrinology, University Hospital, Philipps-University Marburg, Baldinger Strasse, D-35043 Marburg (Germany)

2010-12-29

Pancreatic cancer exhibits an extraordinarily high level of resistance to almost any kind of systemic therapy evaluated in clinical trials so far. Therefore, the identification of novel therapeutic targets is urgently required. High-throughput screens have emerged as an important tool to identify putative targets for diagnosis and therapy in an unbiased manner. More than a decade ago, microarray technology was introduced to identify differentially expressed genes in pancreatic cancer as compared to normal pancreas, chronic pancreatitis and other cancer types located in close proximity to the pancreas. In addition, proteomic screens have facilitated the identification of differentially secreted proteins in body fluids of pancreatic cancer patients, serving as possible biomarkers. Recently, RNA interference-based loss-of-function screens have been used to identify functionally relevant genes, whose knock-down has impact on pancreatic cancer cell viability, thereby representing potential new targets for therapeutic intervention. This review summarizes recent results of transcriptional, proteomic and functional screens in pancreatic cancer and discusses potentials and limitations of the respective technologies as well as their impact on future therapeutic developments.

Screening Technologies for Target Identification in Pancreatic Cancer

International Nuclear Information System (INIS)

Michl, Patrick; Ripka, Stefanie; Gress, Thomas; Buchholz, Malte

2010-01-01

Pancreatic cancer exhibits an extraordinarily high level of resistance to almost any kind of systemic therapy evaluated in clinical trials so far. Therefore, the identification of novel therapeutic targets is urgently required. High-throughput screens have emerged as an important tool to identify putative targets for diagnosis and therapy in an unbiased manner. More than a decade ago, microarray technology was introduced to identify differentially expressed genes in pancreatic cancer as compared to normal pancreas, chronic pancreatitis and other cancer types located in close proximity to the pancreas. In addition, proteomic screens have facilitated the identification of differentially secreted proteins in body fluids of pancreatic cancer patients, serving as possible biomarkers. Recently, RNA interference-based loss-of-function screens have been used to identify functionally relevant genes, whose knock-down has impact on pancreatic cancer cell viability, thereby representing potential new targets for therapeutic intervention. This review summarizes recent results of transcriptional, proteomic and functional screens in pancreatic cancer and discusses potentials and limitations of the respective technologies as well as their impact on future therapeutic developments

High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain.

Science.gov (United States)

Zhou, Ting; Tan, Lei; Cederquist, Gustav Y; Fan, Yujie; Hartley, Brigham J; Mukherjee, Suranjit; Tomishima, Mark; Brennand, Kristen J; Zhang, Qisheng; Schwartz, Robert E; Evans, Todd; Studer, Lorenz; Chen, Shuibing

2017-08-03

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Identifying transposon insertions and their effects from RNA-sequencing data.

Science.gov (United States)

de Ruiter, Julian R; Kas, Sjors M; Schut, Eva; Adams, David J; Koudijs, Marco J; Wessels, Lodewyk F A; Jonkers, Jos

2017-07-07

Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Barriers to Breast and Cervical Cancer Screening in Singapore: a Mixed Methods Analysis.

Science.gov (United States)

Malhotra, Chetna; Bilger, Marcel; Liu, Joy; Finkelstein, Eric

2016-01-01

In order to increase breast and cervical cancer screening uptake in Singapore, women's perceived barriers to screening need to be identified and overcome. Using data from both focus groups and surveys, we aimed to assess perceived barriers and motivations for breast and cervical cancer screening. We conducted 8 focus groups with 64 women, using thematic analysis to identify overarching themes related to women's attitudes towards screening. Based on recurring themes from focus groups, several hypotheses regarding potential barriers and motivations to screen were generated and tested through a national survey of 801 women aged 25-64. Focus group participants had misconceptions related to screening, believing that the procedures were painful. Cost was an issue, as well as efficacy and fatalism. By identifying barriers to and motivators for screening through a mixed-method design that has both nuance and external validity, this study offers valuable suggestions to policymakers to improve breast and cervical cancer screening uptake in Singapore.

A genome-wide RNAi screen identifies novel targets of neratinib resistance leading to identification of potential drug resistant genetic markers.

Science.gov (United States)

Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Wei; Ryan, Terence E

2012-04-01

Neratinib (HKI-272) is a small molecule tyrosine kinase inhibitor of the ErbB receptor family currently in Phase III clinical trials. Despite its efficacy, the mechanism of potential cellular resistance to neratinib and genes involved with it remains unknown. We have used a pool-based lentiviral genome-wide functional RNAi screen combined with a lethal dose of neratinib to discover chemoresistant interactions with neratinib. Our screen has identified a collection of genes whose inhibition by RNAi led to neratinib resistance including genes involved in oncogenesis (e.g. RAB33A, RAB6A and BCL2L14), transcription factors (e.g. FOXP4, TFEC, ZNF), cellular ion transport (e.g. CLIC3, TRAPPC2P1, P2RX2), protein ubiquitination (e.g. UBL5), cell cycle (e.g. CCNF), and genes known to interact with breast cancer-associated genes (e.g. CCNF, FOXP4, TFEC, several ZNF factors, GNA13, IGFBP1, PMEPA1, SOX5, RAB33A, RAB6A, FXR1, DDO, TFEC, OLFM2). The identification of novel mediators of cellular resistance to neratinib could lead to the identification of new or neoadjuvant drug targets. Their use as patient or treatment selection biomarkers could make the application of anti-ErbB therapeutics more clinically effective.

Can abstract screening workload be reduced using text mining? User experiences of the tool Rayyan.

Science.gov (United States)

Olofsson, Hanna; Brolund, Agneta; Hellberg, Christel; Silverstein, Rebecca; Stenström, Karin; Österberg, Marie; Dagerhamn, Jessica

2017-09-01

One time-consuming aspect of conducting systematic reviews is the task of sifting through abstracts to identify relevant studies. One promising approach for reducing this burden uses text mining technology to identify those abstracts that are potentially most relevant for a project, allowing those abstracts to be screened first. To examine the effectiveness of the text mining functionality of the abstract screening tool Rayyan. User experiences were collected. Rayyan was used to screen abstracts for 6 reviews in 2015. After screening 25%, 50%, and 75% of the abstracts, the screeners logged the relevant references identified. A survey was sent to users. After screening half of the search result with Rayyan, 86% to 99% of the references deemed relevant to the study were identified. Of those studies included in the final reports, 96% to 100% were already identified in the first half of the screening process. Users rated Rayyan 4.5 out of 5. The text mining function in Rayyan successfully helped reviewers identify relevant studies early in the screening process. Copyright © 2017 John Wiley & Sons, Ltd.

Identifying Adolescent Patients at Risk for Sexually Transmitted Infections: Development of a Brief Sexual Health Screening Survey.

Science.gov (United States)

Victor, Elizabeth C; Chung, Richard; Thompson, Robert J

2015-08-01

This study examined the association between survey responses to health behaviors, personality/psychosocial factors, and self-reported sexually transmitted infections (STIs) to create a brief survey to identify youth at risk for contracting STIs. Participants included 200 racially diverse 14- to 18-year-old patients from a pediatric primary care clinic. Two sexual behavior variables and one peer norm variable were used to differentiate subgroups of individuals at risk of contracting a STI based on reported history of STIs using probability (decision tree) analyses. These items, as well as sexual orientation and having ever had oral sex, were used to create a brief sexual health screening (BSHS) survey. Each point increase in total BSHS score was associated with exponential growth in the percentage of sexually active adolescents reporting STIs. Findings suggest that the BSHS could serve as a useful tool for clinicians to quickly and accurately detect sexual risk among adolescent patients. © The Author(s) 2014.

Quarkonium suppression: Gluonic dissociation vs. colour screening

Indian Academy of Sciences (India)

mechanism comes into play for the initial conditions taken from the self screened parton cascade model in these studies. Keywords. Quark gluon plasma; J ψ; suppression; dissociation; colour screening. PACS No. 12.38.M. 1. Introduction. The last two decades have seen hectic activity towards identifying unique signatures ...

Medical auditing of whole-breast screening ultrasonography.

Science.gov (United States)

Kim, Min Jung

2017-07-01

Since breast ultrasonography (US) has been used as an adjunctive screening modality in women with dense breasts, the need has arisen to evaluate and monitor its possible harm and benefits in comparison with other screening modalities such as mammography. Recently, the fifth edition of the Breast Imaging Reporting and Data System published by the American College of Radiology has suggested auditing methods for screening breast US. However, the method proposed therein is slightly different from how diagnostic performance was calculated in previous studies on screening breast US. In this article, the background and core aspects of medical audits of breast cancer screening will be reviewed to provide an introduction to the medical auditing of screening breast US, with the goal of helping radiologists to understand and identify potential ways to improve outcomes.

Medical auditing of whole-breast screening ultrasonography

Energy Technology Data Exchange (ETDEWEB)

Kim, Min Jung [Dept. of Radiology, Research Institute of Radiological Science, Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of)

2017-07-15

Since breast ultrasonography (US) has been used as an adjunctive screening modality in women with dense breasts, the need has arisen to evaluate and monitor its possible harm and benefits in comparison with other screening modalities such as mammography. Recently, the fifth edition of the Breast Imaging Reporting and Data System published by the American College of Radiology has suggested auditing methods for screening breast US. However, the method proposed therein is slightly different from how diagnostic performance was calculated in previous studies on screening breast US. In this article, the background and core aspects of medical audits of breast cancer screening will be reviewed to provide an introduction to the medical auditing of screening breast US, with the goal of helping radiologists to understand and identify potential ways to improve outcomes.

Pilot study on the use of data mining to identify cochlear implant candidates.

Science.gov (United States)

Grisel, Jedidiah J; Schafer, Erin; Lam, Anne; Griffin, Terry

2018-05-01

The goal of this pilot study was to determine the clinical utility of data-mining software that screens for cochlear implant (CI) candidacy. The Auditory Implant Initiative developed a software module that screens for CI candidates via integration with a software system (Noah 4) that serves as a depository for hearing test data. To identify candidates, patient audiograms from one practice were exported into the screening module. Candidates were tracked to determine if any eventually underwent implantation. After loading 4836 audiograms from the Noah 4 system, the screening module identified 558 potential CI candidates. After reviewing the data for the potential candidates, 117 were targeted and invited to an educational event. Following the event, a total of six candidates were evaluated, and two were implanted. This objective approach to identifying candidates has the potential to address the gross underutilization of CIs by removing any bias or lack of knowledge regarding the management of severe to profound sensorineural hearing loss with CIs. The screening module was an effective tool for identifying potential CI candidates at one ENT practice. On a larger scale, the screening module has the potential to impact thousands of CI candidates worldwide.

Barriers to Mammography among Inadequately Screened Women

Science.gov (United States)

Stoll, Carolyn R. T.; Roberts, Summer; Cheng, Meng-Ru; Crayton, Eloise V.; Jackson, Sherrill; Politi, Mary C.

2015-01-01

Mammography use has increased over the past 20 years, yet more than 30% of women remain inadequately screened. Structural barriers can deter individuals from screening, however, cognitive, emotional, and communication barriers may also prevent mammography use. This study sought to identify the impact of number and type of barriers on mammography…

TeraSCREEN: multi-frequency multi-mode Terahertz screening for border checks

Science.gov (United States)

Alexander, Naomi E.; Alderman, Byron; Allona, Fernando; Frijlink, Peter; Gonzalo, Ramón; Hägelen, Manfred; Ibáñez, Asier; Krozer, Viktor; Langford, Marian L.; Limiti, Ernesto; Platt, Duncan; Schikora, Marek; Wang, Hui; Weber, Marc Andree

2014-06-01

The challenge for any security screening system is to identify potentially harmful objects such as weapons and explosives concealed under clothing. Classical border and security checkpoints are no longer capable of fulfilling the demands of today's ever growing security requirements, especially with respect to the high throughput generally required which entails a high detection rate of threat material and a low false alarm rate. TeraSCREEN proposes to develop an innovative concept of multi-frequency multi-mode Terahertz and millimeter-wave detection with new automatic detection and classification functionalities. The system developed will demonstrate, at a live control point, the safe automatic detection and classification of objects concealed under clothing, whilst respecting privacy and increasing current throughput rates. This innovative screening system will combine multi-frequency, multi-mode images taken by passive and active subsystems which will scan the subjects and obtain complementary spatial and spectral information, thus allowing for automatic threat recognition. The TeraSCREEN project, which will run from 2013 to 2016, has received funding from the European Union's Seventh Framework Programme under the Security Call. This paper will describe the project objectives and approach.

Preflight screening techniques for centrifuge-simulated suborbital spaceflight.

Science.gov (United States)

Pattarini, James M; Blue, Rebecca S; Castleberry, Tarah L; Vanderploeg, James M

2014-12-01

Historically, space has been the venue of the healthy individual. With the advent of commercial spaceflight, we face the novel prospect of routinely exposing spaceflight participants (SPFs) with multiple comorbidities to the space environment. Preflight screening procedures must be developed to identify those individuals at increased risk during flight. We examined the responses of volunteers to centrifuge accelerations mimicking commercial suborbital spaceflight profiles to evaluate how potential SFPs might tolerate such forces. We evaluated our screening process for medical approval of subjects for centrifuge participation for applicability to commercial spaceflight operations. All registered subjects completed a medical questionnaire, physical examination, and electrocardiogram. Subjects with identified concerns including cardiopulmonary disease, hypertension, and diabetes were required to provide documentation of their conditions. There were 335 subjects who registered for the study, 124 who completed all prescreening, and 86 subjects who participated in centrifuge trials. Due to prior medical history, five subjects were disqualified, most commonly for psychiatric reasons or uncontrolled medical conditions. Of the subjects approved, four individuals experienced abnormal physiological responses to centrifuge profiles, including one back strain and three with anxiety reactions. The screening methods used were judged to be sufficient to identify individuals physically capable of tolerating simulated suborbital flight. Improved methods will be needed to identify susceptibility to anxiety reactions. While severe or uncontrolled disease was excluded, many subjects successfully participated in centrifuge trials despite medical histories of disease that are disqualifying under historical spaceflight screening regimes. Such screening techniques are applicable for use in future commercial spaceflight operations.

Factors That Cause Trimethoprim Resistance in Streptococcus pyogenes

Science.gov (United States)

Bergmann, René; van der Linden, Mark; Chhatwal, Gursharan S.

2014-01-01

The use of trimethoprim in treatment of Streptococcus pyogenes infections has long been discouraged because it has been widely believed that this pathogen is resistant to this antibiotic. To gain more insight into the extent and molecular basis of trimethoprim resistance in S. pyogenes, we tested isolates from India and Germany and sought the factors that conferred the resistance. Resistant isolates were identified in tests for trimethoprim or trimethoprim-sulfamethoxazole (SXT) susceptibility. Resistant isolates were screened for the known horizontally transferable trimethoprim-insensitive dihydrofolate reductase (dfr) genes dfrG, dfrF, dfrA, dfrD, and dfrK. The nucleotide sequence of the intrinsic dfr gene was determined for resistant isolates lacking the horizontally transferable genes. Based on tentative criteria, 69 out of 268 isolates (25.7%) from India were resistant to trimethoprim. Occurring in 42 of the 69 resistant isolates (60.9%), dfrF appeared more frequently than dfrG (23 isolates; 33.3%) in India. The dfrF gene was also present in a collection of SXT-resistant isolates from Germany, in which it was the only detected trimethoprim resistance factor. The dfrF gene caused resistance in 4 out of 5 trimethoprim-resistant isolates from the German collection. An amino acid substitution in the intrinsic dihydrofolate reductase known from trimethoprim-resistant Streptococcus pneumoniae conferred resistance to S. pyogenes isolates of emm type 102.2, which lacked other aforementioned dfr genes. Trimethoprim may be more useful in treatment of S. pyogenes infections than previously thought. However, the factors described herein may lead to the rapid development and spread of resistance of S. pyogenes to this antibiotic agent. PMID:24492367

A genetic screen for anchorage-independent proliferation in mammalian cells identifies a membrane-bound neuregulin.

Directory of Open Access Journals (Sweden)

Davide Danovi

2010-07-01

Full Text Available Anchorage-independent proliferation is a hallmark of oncogenic transformation and is thought to be conducive to proliferation of cancer cells away from their site of origin. We have previously reported that primary Schwann cells expressing the SV40 Large T antigen (LT are not fully transformed in that they maintain a strict requirement for attachment, requiring a further genetic change, such as oncogenic Ras, to gain anchorage-independence. Using the LT-expressing cells, we performed a genetic screen for anchorage-independent proliferation and identified Sensory and Motor Neuron Derived Factor (SMDF, a transmembrane class III isoform of Neuregulin 1. In contrast to oncogenic Ras, SMDF induced enhanced proliferation in normal primary Schwann cells but did not trigger cellular senescence. In cooperation with LT, SMDF drove anchorage-independent proliferation, loss of contact inhibition and tumourigenicity. This transforming ability was shared with membrane-bound class III but not secreted class I isoforms of Neuregulin, indicating a distinct mechanism of action. Importantly, we show that despite being membrane-bound signalling molecules, class III neuregulins transform via a cell intrinsic mechanism, as a result of constitutive, elevated levels of ErbB signalling at high cell density and in anchorage-free conditions. This novel transforming mechanism may provide new targets for cancer therapy.

Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors

Directory of Open Access Journals (Sweden)

Victor M. Bii

2016-10-01

Full Text Available Identifying novel genes that drive tumor metastasis and drug resistance has significant potential to improve patient outcomes. High-throughput sequencing approaches have identified cancer genes, but distinguishing driver genes from passengers remains challenging. Insertional mutagenesis screens using replication-incompetent retroviral vectors have emerged as a powerful tool to identify cancer genes. Unlike replicating retroviruses and transposons, replication-incompetent retroviral vectors lack additional mutagenesis events that can complicate the identification of driver mutations from passenger mutations. They can also be used for almost any human cancer due to the broad tropism of the vectors. Replication-incompetent retroviral vectors have the ability to dysregulate nearby cancer genes via several mechanisms including enhancer-mediated activation of gene promoters. The integrated provirus acts as a unique molecular tag for nearby candidate driver genes which can be rapidly identified using well established methods that utilize next generation sequencing and bioinformatics programs. Recently, retroviral vector screens have been used to efficiently identify candidate driver genes in prostate, breast, liver and pancreatic cancers. Validated driver genes can be potential therapeutic targets and biomarkers. In this review, we describe the emergence of retroviral insertional mutagenesis screens using replication-incompetent retroviral vectors as a novel tool to identify cancer driver genes in different cancer types.

Phenotypic Screening Identifies Synergistically Acting Natural Product Enhancing the Performance of Biomaterial Based Wound Healing

Directory of Open Access Journals (Sweden)

Srinivasan Sivasubramanian

2017-07-01

Full Text Available The potential of multifunctional wound heal biomaterial relies on the optimal content of therapeutic constituents as well as the desirable physical, chemical, and biological properties to accelerate the healing process. Formulating biomaterials such as amnion or collagen based scaffolds with natural products offer an affordable strategy to develop dressing material with high efficiency in healing wounds. Using image based phenotyping and quantification, we screened natural product derived bioactive compounds for modulators of types I and III collagen production from human foreskin derived fibroblast cells. The identified hit was then formulated with amnion to develop a biomaterial, and its biophysical properties, in vitro and in vivo effects were characterized. In addition, we performed functional profiling analyses by PCR array to understand the effect of individual components of these materials on various genes such as inflammatory mediators including chemokines and cytokines, growth factors, fibroblast stimulating markers for collagen secretion, matrix metalloproteinases, etc., associated with wound healing. FACS based cell cycle analyses were carried out to evaluate the potential of biomaterials for induction of proliferation of fibroblasts. Western blot analyses was done to examine the effect of biomaterial on collagen synthesis by cells and compared to cells grown in the presence of growth factors. This work demonstrated an uncomplicated way of identifying components that synergistically promote healing. Besides, we demonstrated that modulating local wound environment using biomaterials with bioactive compounds could enhance healing. This study finds that the developed biomaterials offer immense scope for healing wounds by means of their skin regenerative features such as anti-inflammatory, fibroblast stimulation for collagen secretion as well as inhibition of enzymes and markers impeding the healing, hydrodynamic properties complemented

Dynamic Contrast-Enhanced MRI of Cervical Cancers: Temporal Percentile Screening of Contrast Enhancement Identifies Parameters for Prediction of Chemoradioresistance

International Nuclear Information System (INIS)

Andersen, Erlend K.F.; Hole, Knut Håkon; Lund, Kjersti V.; Sundfør, Kolbein; Kristensen, Gunnar B.; Lyng, Heidi; Malinen, Eirik

2012-01-01

Purpose: To systematically screen the tumor contrast enhancement of locally advanced cervical cancers to assess the prognostic value of two descriptive parameters derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Methods and Materials: This study included a prospectively collected cohort of 81 patients who underwent DCE-MRI with gadopentetate dimeglumine before chemoradiotherapy. The following descriptive DCE-MRI parameters were extracted voxel by voxel and presented as histograms for each time point in the dynamic series: normalized relative signal increase (nRSI) and normalized area under the curve (nAUC). The first to 100th percentiles of the histograms were included in a log-rank survival test, resulting in p value and relative risk maps of all percentile–time intervals for each DCE-MRI parameter. The maps were used to evaluate the robustness of the individual percentile–time pairs and to construct prognostic parameters. Clinical endpoints were locoregional control and progression-free survival. The study was approved by the institutional ethics committee. Results: The p value maps of nRSI and nAUC showed a large continuous region of percentile–time pairs that were significantly associated with locoregional control (p < 0.05). These parameters had prognostic impact independent of tumor stage, volume, and lymph node status on multivariate analysis. Only a small percentile–time interval of nRSI was associated with progression-free survival. Conclusions: The percentile–time screening identified DCE-MRI parameters that predict long-term locoregional control after chemoradiotherapy of cervical cancer.

A genome-wide RNAi screen identifies novel targets of neratinib sensitivity leading to neratinib and paclitaxel combination drug treatments.

Science.gov (United States)

Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Yan; McGraw, John; Woods, Matthew; Murray, Stuart; Eckert, Amy; Liu, Wei; Ryan, Terence E

2011-06-01

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.

Early Detection and Screening for Breast Cancer.

Science.gov (United States)

Coleman, Cathy

2017-05-01

To review the history, current status, and future trends related to breast cancer screening. Peer-reviewed articles, web sites, and textbooks. Breast cancer remains a complex, heterogeneous disease. Serial screening with mammography is the most effective method to detect early stage disease and decrease mortality. Although politics and economics may inhibit organized mammography screening programs in many countries, the judicious use of proficient clinical and self-breast examination can also identify small tumors leading to reduced morbidity. Oncology nurses have exciting opportunities to lead, facilitate, and advocate for delivery of high-quality screening services targeting individuals and communities. A practical approach is needed to translate the complexities and controversies surrounding breast cancer screening into improved care outcomes. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteome screening of pleural effusions identifies galectin 1 as a diagnostic biomarker and highlights several prognostic biomarkers for malignant mesothelioma.

Science.gov (United States)

Mundt, Filip; Johansson, Henrik J; Forshed, Jenny; Arslan, Sertaç; Metintas, Muzaffer; Dobra, Katalin; Lehtiö, Janne; Hjerpe, Anders

2014-03-01

Malignant mesothelioma is an aggressive asbestos-induced cancer, and affected patients have a median survival of approximately one year after diagnosis. It is often difficult to reach a conclusive diagnosis, and ancillary measurements of soluble biomarkers could increase diagnostic accuracy. Unfortunately, few soluble mesothelioma biomarkers are suitable for clinical application. Here we screened the effusion proteomes of mesothelioma and lung adenocarcinoma patients to identify novel soluble mesothelioma biomarkers. We performed quantitative mass-spectrometry-based proteomics using isobaric tags for quantification and used narrow-range immobilized pH gradient/high-resolution isoelectric focusing (pH 4-4.25) prior to analysis by means of nano liquid chromatography coupled to MS/MS. More than 1,300 proteins were identified in pleural effusions from patients with malignant mesothelioma (n = 6), lung adenocarcinoma (n = 6), or benign mesotheliosis (n = 7). Data are available via ProteomeXchange with identifier PXD000531. The identified proteins included a set of known mesothelioma markers and proteins that regulate hallmarks of cancer such as invasion, angiogenesis, and immune evasion, plus several new candidate proteins. Seven candidates (aldo-keto reductase 1B10, apolipoprotein C-I, galectin 1, myosin-VIIb, superoxide dismutase 2, tenascin C, and thrombospondin 1) were validated by enzyme-linked immunosorbent assays in a larger group of patients with mesothelioma (n = 37) or metastatic carcinomas (n = 25) and in effusions from patients with benign, reactive conditions (n = 16). Galectin 1 was identified as overexpressed in effusions from lung adenocarcinoma relative to mesothelioma and was validated as an excellent predictor for metastatic carcinomas against malignant mesothelioma. Galectin 1, aldo-keto reductase 1B10, and apolipoprotein C-I were all identified as potential prognostic biomarkers for malignant mesothelioma. This analysis of the effusion proteome

A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

Science.gov (United States)

Mori, Tadashi; Hidaka, Masafumi; Ikuji, Hiroko; Yoshizawa, Ibuki; Toyohara, Haruhiko; Okuda, Toru; Uchida, Chiyoko; Asano, Tomoichiro; Yotsu-Yamashita, Mari; Uchida, Takafumi

2014-01-01

The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

Derivation and validation of REASON: a risk score identifying candidates to screen for peripheral arterial disease using ankle brachial index.

Science.gov (United States)

Ramos, Rafel; Baena-Díez, Jose Miguel; Quesada, Miquel; Solanas, Pascual; Subirana, Isaac; Sala, Joan; Alzamora, Maite; Forès, Rosa; Masiá, Rafel; Elosua, Roberto; Grau, María; Cordón, Ferran; Pera, Guillem; Rigo, Fernando; Martí, Ruth; Ponjoan, Anna; Cerezo, Carlos; Brugada, Ramon; Marrugat, Jaume

2011-02-01

The recommendation of screening with ankle brachial index (ABI) in asymptomatic individuals is controversial. The aims of the present study were to develop and validate a pre-screening test to select candidates for ABI measurement in the Spanish population 50-79 years old, and to compare its predictive capacity to current Inter-Society Consensus (ISC) screening criteria. Two population-based cross-sectional studies were used to develop (n = 4046) and validate (n = 3285) a regression model to predict ABI guidelines, and similar sensitivity. This resulted in fewer patients screened per diagnosis of ABI < 0.9 (10.6 vs. 8.75) and a lower proportion of the population aged 50-79 years candidate to ABI screening (63.3% vs. 55.0%). This model provides accurate ABI < 0.9 risk estimates for ages 50-79, with a better predictive capacity than that of ISC criteria. Its use could reduce possible harms and unnecessary work-ups of ABI screening as a risk stratification strategy in primary prevention of peripheral vascular disease. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

CRISPR genetic screens to discover host-virus interactions.

Science.gov (United States)

McDougall, William M; Perreira, Jill M; Reynolds, Erin C; Brass, Abraham L

2018-04-01

Viruses impose an immense burden on human health. With the goal of treating and preventing viral infections, researchers have carried out genetic screens to improve our understanding of viral dependencies and identify potential anti-viral strategies. The emergence of CRISPR genetic screening tools has facilitated this effort by enabling host-virus screens to be undertaken in a more versatile and fidelitous manner than previously possible. Here we review the growing number of CRISPR screens which continue to increase our understanding of host-virus interactions. Copyright © 2018 Elsevier B.V. All rights reserved.

Diabetic retinopathy screening: global and local perspective.

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Gangwani, R A; Lian, J X; McGhee, S M; Wong, D; Li, K Kw

2016-10-01

Diabetes mellitus has become a global epidemic. It causes significant macrovascular complications such as coronary artery disease, peripheral artery disease, and stroke; as well as microvascular complications such as retinopathy, nephropathy, and neuropathy. Diabetic retinopathy is known to be the leading cause of blindness in the working-age population and may be asymptomatic until vision loss occurs. Screening for diabetic retinopathy has been shown to reduce blindness by timely detection and effective laser treatment. Diabetic retinopathy screening is being done worldwide either as a national screening programme or hospital-based project or as a community-based screening programme. In this article, we review different methods of screening including grading used to detect the severity of sight-threatening retinopathy and the newer screening methods. This review also includes the method of systematic screening being carried out in Hong Kong, a system that has helped to identify diabetic retinopathy among all attendees in public primary care clinics using a Hong Kong-wide public patients' database.

Walkthrough screening evaluation field guide

International Nuclear Information System (INIS)

Eder, S.J.; Eli, M.W.; Salmon, M.W.

1993-11-01

The US Department of Energy (DOE) has a large inventory of existing facilities. Many of these facilities were not designed and constructed to current natural phenomena hazard (NPH) criteria. The NPH events include earthquakes, extreme winds and tornadoes, and floods. DOE Order 5480.28 establishes policy and requirements for NPH mitigation for DOE facilities. DOE is conducting a multiyear project to develop evaluation guidelines for assessing the condition and determining the need for upgrades at DOE facilities. One element of the NPH evaluation guidelines' development involves the existing systems and components at DOE facilities. This effort is described in detail in a cited reference. In the interim period prior to availability of the final guidelines, DOE facilities are encouraged to implement an NPH walk through screening evaluation process by which systems and components that need attention can be rapidly identified. Guidelines for conducting the walk through screening evaluations are contained herein. The result of the NPH walk through screening evaluation should be a prioritized list of systems and components that need further action. Simple and inexpensive fixes for items identified in the walk through as marginal or inadequate should be implemented without further study. By implementing an NPH walk through screening evaluation, DOE facilities may realize significant reduction in risk from NPH in the short term

Relativity Screens for Misvalued Medical Services: Impact on Noninvasive Diagnostic Radiology.

Science.gov (United States)

Rosenkrantz, Andrew B; Silva, Ezequiel; Hawkins, C Matthew

2017-11-01

In 2006, the AMA/Specialty Society Relative Value Scale Update Committee (RUC) introduced ongoing relativity screens to identify potentially misvalued medical services for payment adjustments. We assess the impact of these screens upon the valuation of noninvasive diagnostic radiology services. Data regarding relativity screens and relative value unit (RVU) changes were obtained from the 2016 AMA Relativity Assessment Status Report. All global codes in the 2016 Medicare Physician Fee Schedule with associated work RVUs were classified as noninvasive diagnostic radiology services versus remaining services. The frequency of having ever undergone a screen was compared between the two groups. Screened radiology codes were further evaluated regarding the RVU impact of subsequent revaluation. Of noninvasive diagnostic radiology codes, 46.0% (201 of 437) were screened versus 22.2% (1,460 of 6,575) of remaining codes (P < .001). Most common screens for which radiology codes were identified as potentially misvalued were (1) high expenditures (27.5%) and (2) high utilization (25.6%). The modality and body region most likely to be identified in a screen were CT (82.1%) and breast (90.9%), respectively. Among screened radiology codes, work RVUs, practice expense RVUs, and nonfacility total RVUs decreased in 20.3%, 65.9%, and 75.3%, respectively. All screened CT, MRI, brain, and spine codes exhibited decreased total RVUs. Policymakers' ongoing search for potentially misvalued medical services has disproportionately impacted noninvasive diagnostic radiology services, risking the introduction of unintended or artificial shifts in physician practice. Copyright © 2017 American College of Radiology. Published by Elsevier Inc. All rights reserved.

Elder abuse telephone screen reliability and validity.

Science.gov (United States)

Buri, Hilary M; Daly, Jeanette M; Jogerst, Gerald J

2009-01-01

(a) To identify reliable and valid questions that identify elder abuse, (b) to assess the reliability and validity of extant self-reported elder abuse screens in a high-risk elderly population, and (c) to describe difficulties of completing and interpreting screens in a high-need elderly population. All elders referred to research-trained social workers in a community service agency were asked to participate. Of the 70 elders asked, 49 participated, 44 completed the first questionnaire, and 32 completed the duplicate second questionnaire. A research assistant administered the telephone questionnaires. Twenty-nine (42%) persons were judged abused, 12 (17%) had abuse reported, and 4 (6%) had abuse substantiated. The elder abuse screen instruments were not found to be predictive of assessed abuse or as predictors of reported abuse; the measures tended toward being inversely predictive. Two questions regarding harm and taking of belongings were significantly different for the assessed abused group. In this small group of high-need community-dwelling elders, the screens were not effective in discriminating between abused and nonabused groups. Better instruments are needed to assess for elder abuse.

Development and analysis of a low-cost screening tool to identify and classify hearing loss in children: a proposal for developing countries

Directory of Open Access Journals (Sweden)

Alessandra Giannella Samelli

2011-01-01

Full Text Available OBJECTIVE: A lack of attention has been given to hearing health in primary care in developing countries. A strategy involving low-cost screening tools may fill the current gap in hearing health care provided to children. Therefore, it is necessary to establish and adopt lower-cost procedures that are accessible to underserved areas that lack other physical or human resources that would enable the identification of groups at risk for hearing loss. The aim of this study was to develop and analyze the efficacy of a low-cost screening tool to identify and classify hearing loss in children. METHODS: A total of 214 2-to-10 year-old children participated in this study. The study was conducted by providing a questionnaire to the parents and comparing the answers with the results of a complete audiological assessment. Receiver operating characteristic (ROC curves were constructed, and discriminant analysis techniques were used to classify each child based on the total score. RESULTS: We found conductive hearing loss in 39.3% of children, sensorineural hearing loss in 7.4% and normal hearing in 53.3%. The discriminant analysis technique provided the following classification rule for the total score on the questionnaire: 0 to 4 points - normal hearing; 5 to 7 points - conductive hearing loss; over 7 points - sensorineural hearing loss. CONCLUSION: Our results suggest that the questionnaire could be used as a screening tool to classify children with normal hearing or hearing loss and according to the type of hearing loss based on the total questionnaire score

Barriers and facilitators of mental health screening in pregnancy.

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Kingston, Dawn; Austin, Marie-Paule; Heaman, Maureen; McDonald, Sheila; Lasiuk, Gerri; Sword, Wendy; Giallo, Rebecca; Hegadoren, Kathy; Vermeyden, Lydia; van Zanten, Sander Veldhuyzen; Kingston, Joshua; Jarema, Karly; Biringer, Anne

2015-11-01

Access to mental health services during pregnancy is most commonly mobilized through formal mental health screening. However, few studies to date have identified barriers and facilitators that affect pregnant women's responses to mental health screening. The objective was to identify barriers and facilitators that influence pregnant women's responses to the screening process and factors associated with their identification. This multi-site, cross-sectional survey recruited pregnant women >16 years of age who spoke/read English in Alberta, Canada. Main outcomes were barriers and facilitators of mental health screening. Descriptive statistics were generated to identify the most common barriers and facilitators and multivariable logistic regression models were conducted to determine factors associated with barriers and facilitators. Study participation rate was 92% (460/500). Women's most common barriers were: significant others normalizing their emotional difficulties; desiring to handle mental health problems on their own; preferring to discuss feelings with significant others; and not knowing what emotions were 'normal'. Women who identified these barriers were more likely not to have been treated previously for mental illness, were primiparous, and could not be completely honest with their provider. Main facilitators were provider characteristics (sensitive, interested), reassurance that mental healthcare is a part of routine prenatal care, hearing that other women have emotional problems during pregnancy and knowing that help was available. The sample comprised largely Caucasian, well-educated, and partnered women, which limits generalizability of the findings. Personal and stigma-related barriers influence pregnant women's responses to mental health screening. Efforts to minimize barriers and enhance facilitators should be explored as potential strategies for optimizing prenatal mental health screening. Copyright © 2015 Elsevier B.V. All rights reserved.

Hazard screening application guide

International Nuclear Information System (INIS)

1992-06-01

The basic purpose of hazard screening is to group precesses, facilities, and proposed modifications according to the magnitude of their hazards so as to determine the need for and extent of follow on safety analysis. A hazard is defined as a material, energy source, or operation that has the potential to cause injury or illness in human beings. The purpose of this document is to give guidance and provide standard methods for performing hazard screening. Hazard screening is applied to new and existing facilities and processes as well as to proposed modifications to existing facilities and processes. The hazard screening process evaluates an identified hazards in terms of the effects on people, both on-site and off-site. The process uses bounding analyses with no credit given for mitigation of an accident with the exception of certain containers meeting DOT specifications. The process is restricted to human safety issues only. Environmental effects are addressed by the environmental program. Interfaces with environmental organizations will be established in order to share information

Identifying specific non-attending groups in breast cancer screening - population-based registry study of participation and socio-demography

Directory of Open Access Journals (Sweden)

Jensen Line

2012-11-01

Full Text Available Abstract Background A population-based breast cancer screening programme was implemented in the Central Denmark Region in 2008–09. The objective of this registry-based study was to examine the association between socio-demographic characteristics and screening participation and to examine whether the group of non-participants can be regarded as a homogeneous group of women. Method Participation status was obtained from a regional database for all women invited to the first screening round in the Central Denmark Region in 2008–2009 (n=149,234. Participation data was linked to registries containing socio-demographic information. Distance to screening site was calculated using ArcGIS. Participation was divided into ‘participants’ and ‘non-participants’, and non-participants were further stratified into ‘active non-participants’ and ‘passive non-participants’ based on whether the woman called and cancelled her participation or was a ‘no-show’. Results The screening participation rate was 78.9%. In multivariate analyses, non-participation was associated with older age, immigrant status, low OECD-adjusted household income, high and low level education compared with middle level education, unemployment, being unmarried, distance to screening site >20 km, being a tenant and no access to a vehicle. Active and passive non-participants comprised two distinct groups with different socio-demographic characteristics, with passive non-participants being more socially deprived compared with active non-participants. Conclusion Non-participation was associated with low social status e.g. low income, unemployment, no access to vehicle and status as tenant. Non-participants were also more likely than participants to be older, single, and of non-Danish origin. Compared to active non-participants, passive non-participants were characterized by e.g. lower income and lower educational level. Different interventions might be warranted to increase

A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library.

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Feng, Jie; Shi, Wanliang; Zhang, Shuo; Sullivan, David; Auwaerter, Paul G; Zhang, Ying

2016-01-01

Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi

PHYTOCHEMICAL SCREENING AND THROMBOLYTIC ACTIVITY ...

African Journals Online (AJOL)

2014-12-31

Dec 31, 2014 ... followed by chemical screening, random selection followed by one or more biological ... These were identified by characteristic color changes .... [1] Samuelsson, G. Drugs of Natural Origin: A Textbook of Pharmacognosy.

A genome-wide siRNA screen identifies proteasome addiction as a vulnerability of basal-like triple-negative breast cancer cells

Science.gov (United States)

Petrocca, Fabio; Altschuler, Gabriel; Tan, Shen Mynn; Mendillo, Marc L.; Yan, Haoheng; Jerry, D. Joseph; Kung, Andrew L.; Hide, Winston; Ince, Tan A.; Lieberman, Judy

2013-01-01

Summary Basal-like triple negative breast cancers (TNBC) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes - basal-like BPLER and myoepithelial HMLER. Expression of the screen’s 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence. PMID:23948298

Biochemical and molecular characteristics of patients with organic acidaemias and urea cycle disorders identified through newborn screening.

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Barends, M; Pitt, J; Morrissy, S; Tzanakos, N; Boneh, A

2014-01-01

In recent years it has become clear that newborn screening (NBS) programmes using tandem mass spectrometry identify "patients" with "classical" inborn errors of metabolism who are asymptomatic. This observation raises issues regarding medicalization of "non-diseases," potentially unnecessary treatment and unnecessary anxiety to parents. This study aims to identify possible markers that may assist in predicting the need for treatment of infants with "classical" organic acidaemias (OA) and urea cycle disorders (UCD) diagnosed through NBS. Medical records of all patients with classical OA and UCD detected through the Victorian NBS programme from February 2002 to January 2014, or diagnosed clinically between 1990 and January 2002 were retrospectively reviewed. Neonatal presentation did not always predict the need for on-going strict treatment. Blood concentrations of amino acids and acyl-carnitines and the changes thereof in follow-up samples correlated with severity in citrullinaemia-I, possibly isovaleric acidaemia but not in argininosuccinic aciduria or propionic acidaemia. Some specific mutations correlate with "attenuated" citrullinaemia-I. Gender may affect clinical outcome in propionic acidaemia. Changes in blood concentration of certain metabolites (amino acids, acyl-carnitines) in the first weeks of life may be predictive of the need for treatment in some disorders but not in others. Mutation analysis may be predictive in some disorders but whether or not this should be considered as second-tier testing in NBS should be discussed separately. Copyright © 2014 Elsevier Inc. All rights reserved.

Fabry Disease: prevalence of affected males and heterozygotes with pathogenic GLA mutations identified by screening renal, cardiac and stroke clinics, 1995-2017.

Science.gov (United States)

Doheny, Dana; Srinivasan, Ram; Pagant, Silvere; Chen, Brenden; Yasuda, Makiko; Desnick, Robert J

2018-04-01

Fabry Disease (FD), an X linked lysosomal storage disease due to pathogenic α-galactosidase A ( GLA ) mutations, results in two major subtypes, the early-onset Type 1 'Classic' and the Type 2 'Later-Onset' phenotypes. To identify previously unrecognised patients, investigators screened cardiac, renal and stroke clinics by enzyme assays. However, some screening studies did not perform confirmatory GLA mutation analyses, and many included recently recognised 'benign/likely-benign' variants, thereby inflating prevalence estimates. Online databases were searched for all FD screening studies in high-risk clinics (1995-2017). Studies reporting GLA mutations were re-analysed for pathogenic mutations, sex and phenotype. Phenotype-specific and sex-specific prevalence rates were determined. Of 67 studies, 63 that screened 51363patients (33943M and 17420F) and provided GLA mutations were reanalysed for disease-causing mutations. Of reported GLA mutations, benign variants occurred in 47.9% of males and 74.1% of females. The following were the revised prevalence estimates: among 36820 (23954M and 12866F) haemodialysis screenees, 0.21% males and 0.15% females; among 3074 (2031M and 1043F) renal transplant screenees, 0.25% males and no females; among 5491 (4054M and 1437F) cardiac screenees, 0.94% males and 0.90% females; and among 5978 (3904M and 2074F) stroke screenees, 0.13% males and 0.14% females. Among male and female screenees with pathogenic mutations, the type 1 Classic phenotype was predominant (~60%), except more male cardiac patients (75%) had type 2 Later-Onset phenotype. Compared with previous findings, reanalysis of 63 studies increased the screenee numbers (~3.4-fold), eliminated 20 benign/likely benign variants, and provided more accurate sex-specific and phenotype-specific prevalence estimates, ranging from ~0.13% of stroke to ~0.9% of cardiac male or female screenees. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article

Can a Risk Factor Based Approach Safely Reduce Screening for Retinopathy of Prematurity?

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K. M. Friddle

2017-01-01

Full Text Available Objective. Current American retinopathy of prematurity (ROP screening guidelines is imprecise for infants ≥ 30 weeks with birth weights between 1500 and 2000 g. Our objective was to evaluate a risk factor based approach for screening premature infants at low risk for severe ROP. Study Design. We performed a 13-year review from Intermountain Health Care (IHC data. All neonates born at ≤32 weeks were reviewed to determine ROP screening and/or development of severe ROP. Severe ROP was defined by stage ≥ 3 or need for laser therapy. Regression analysis was used to identify significant risk factors for severe ROP. Results. We identified 4607 neonates ≤ 32 weeks gestation. Following exclusion for death, with no retinal exam or incomplete data, 2791 (61% were included in the study. Overall, severe ROP occurred in 260 (9.3%, but only 11/1601 ≥ 29 weeks (0.7%. All infants with severe ROP ≥ 29 weeks had at least 2 identified ROP risk factors. Implementation of this risk based screening strategy to the IHC population over the timeline of this study would have eliminated screening in 21% (343/1601 of the screened population. Conclusions. Limiting ROP screening for infants ≥ 29 and ≤ 32 weeks to only those with clinical risk factors could significantly reduce screening exams while identifying all infants with severe ROP.

Patient-completed or symptom-based screening tools for endometriosis: a scoping review.

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Surrey, Eric; Carter, Cathryn M; Soliman, Ahmed M; Khan, Shahnaz; DiBenedetti, Dana B; Snabes, Michael C

2017-08-01

The objective of this review was to evaluate existing patient-completed screening questionnaires and/or symptom-based predictive models with respect to their potential for use as screening tools for endometriosis in adult women. Validated instruments were of particular interest. We conducted structured searches of PubMed and targeted searches of the gray literature to identify studies reporting on screening instruments used in endometriosis. Studies were screened according to inclusion and exclusion criteria that followed the PICOS (population, intervention, comparison, outcomes, study design) framework. A total of 16 studies were identified, of which 10 described measures for endometriosis in general, 2 described measures for endometriosis at specific sites, and 4 described measures for deep-infiltrating endometriosis. Only 1 study evaluated a questionnaire that was solely patient-completed. Most measures required physician, imaging, or laboratory assessments in addition to patient-completed questionnaires, and several measures relied on complex scoring. Validation for use as a screening tool in adult women with potential endometriosis was lacking in all studies, as most studies focused on diagnosis versus screening. This literature review did not identify any fully validated, symptom-based, patient-reported questionnaires for endometriosis screening in adult women.

Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

Science.gov (United States)

Fabrizio, Paola; Hoon, Shawn; Shamalnasab, Mehrnaz; Galbani, Abdulaye; Wei, Min; Giaever, Guri; Nislow, Corey; Longo, Valter D

2010-07-15

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Ethical issues with colorectal cancer screening-a systematic review.

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Hofmann, Bjørn

2017-06-01

Colorectal cancer (CRC) screening is widely recommended and implemented. However, sometimes CRC screening is not implemented despite good evidence, and some types of CRC screening are implemented despite lack of evidence. The objective of this article is to expose and elucidate relevant ethical issues in the literature on CRC screening that are important for open and transparent deliberation on CRC screening. An axiological question-based method is used for exposing and elucidating ethical issues relevant in HTA. A literature search in MEDLINE, Embase, PsycINFO, PubMed Bioethics subset, ISI Web of Knowledge, Bioethics Literature Database (BELIT), Ethics in Medicine (ETHMED), SIBIL Base dati di bioetica, LEWI Bibliographic Database on Ethics in the Sciences and Humanities, and EUROETHICS identified 870 references of which 114 were found relevant according to title and abstract. The content of the included papers were subject to ethical analysis to highlight the ethical issues, concerns, and arguments. A wide range of important ethical issues were identified. The main benefits are reduced relative CRC mortality rate, and potentially incidence rate, but there is no evidence of reduced absolute mortality rate. Potential harms are bleeding, perforation, false test results, overdetection, overdiagnosis, overtreatment (including unnecessary removal of polyps), and (rarely) death. Other important issues are related to autonomy and informed choice equity, justice, medicalization, and expanding disease. A series of important ethical issues have been identified and need to be addressed in open and transparent deliberation on CRC screening. © 2016 John Wiley & Sons, Ltd.

Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors within the ToxCast Phase I and II Chemical Libraries

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High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limi...

Genetic and pharmacological screens converge in identifying FLIP, BCL2 and IAP proteins as key regulators of sensitivity to the TRAIL-inducing anti-cancer agent ONC201/TIC10

OpenAIRE

Allen, Joshua E.; Prabhu, Varun V.; Talekar, Mala; van den Heuvel, AP; Lim, Bora; Dicker, David T.; Fritz, Jennifer L.; Beck, Adam; El-Deiry, Wafik S.

2015-01-01

ONC201/TIC10 is a small molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway inducer KSR1. Unexpectedly, KSR1 sile...

Measuring lip force by oral screens. Part 1: Importance of screen size and individual variability.

Science.gov (United States)

Wertsén, Madeleine; Stenberg, Manne

2017-06-01

To reduce drooling and facilitate food transport in rehabilitation of patients with oral motor dysfunction, lip force can be trained using an oral screen. Longitudinal studies evaluating the effect of training require objective methods. The aim of this study was to evaluate a method for measuring lip strength, to investigate normal values and fluctuation of lip force in healthy adults on 1 occasion and over time, to study how the size of the screen affects the force, to evaluate the most appropriate measure of reliability, and to identify force performed in relation to gender. Three different sizes of oral screens were used to measure the lip force for 24 healthy adults on 3 different occasions, during a period of 6 months, using an apparatus based on strain gauge. The maximum lip force as evaluated with this method depends on the area of the screen size. By calculating the projected area of the screen, the lip force could be normalized to an oral screen pressure quantity expressed in kPa, which can be used for comparing measurements from screens with different sizes. Both the mean value and standard deviation were shown to vary between individuals. The study showed no differences regarding gender and only small variation with age. Normal variation over time (months) may be up to 3 times greater than the standard error of measurement at a certain occasion. The lip force increases in relation to the projected area of the screen. No general standard deviation can be assigned to the method and all measurements should be analyzed individually based on oral screen pressure to compensate for different screen sizes.

The first three years of screening for medium chain acyl-CoA dehydrogenase deficiency (MCADD by newborn screening ontario

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Fisher Lawrence

2010-11-01

Full Text Available Abstract Background Medium chain acyl-CoA dehydrogenase deficiency (MCADD is a disorder of mitochondrial fatty acid oxidation and is one of the most common inborn errors of metabolism. Identification of MCADD via newborn screening permits the introduction of interventions that can significantly reduce associated morbidity and mortality. This study reports on the first three years of newborn screening for MCADD in Ontario, Canada. Methods Newborn Screening Ontario began screening for MCADD in April 2006, by quantification of acylcarnitines (primarily octanoylcarnitine, C8 in dried blood spots using tandem mass spectrometry. Babies with positive screening results were referred to physicians at one of five regional Newborn Screening Treatment Centres, who were responsible for diagnostic evaluation and follow-up care. Results From April 2006 through March 2009, approximately 439 000 infants were screened for MCADD in Ontario. Seventy-four infants screened positive, with a median C8 level of 0.68 uM (range 0.33-30.41 uM. Thirty-one of the screen positive infants have been confirmed to have MCADD, while 36 have been confirmed to be unaffected. Screening C8 levels were higher among infants with MCADD (median 8.93 uM compared to those with false positive results (median 0.47 uM. Molecular testing was available for 29 confirmed cases of MCADD, 15 of whom were homozygous for the common c.985A > G mutation. Infants homozygous for the common mutation tended to have higher C8 levels (median 12.13 uM relative to compound heterozygotes for c.985A > G and a second detectable mutation (median 2.01 uM. Eight confirmed mutation carriers were identified among infants in the false positive group. The positive predictive value of a screen positive for MCADD was 46%. The estimated birth prevalence of MCADD in Ontario is approximately 1 in 14 000. Conclusions The birth prevalence of MCADD and positive predictive value of the screening test were similar to those

Screen for Disordered Eating: Improving the accuracy of eating disorder screening in primary care.

Science.gov (United States)

Maguen, Shira; Hebenstreit, Claire; Li, Yongmei; Dinh, Julie V; Donalson, Rosemary; Dalton, Sarah; Rubin, Emma; Masheb, Robin

To develop a primary care eating disorder screen with greater accuracy and greater potential for generalizability, compared to existing screens. Cross-sectional survey to assess discriminative accuracy of a new screen, Screen for Disordered Eating (SDE), compared to Eating Disorders Screen for Primary Care (EDS-PC) and SCOFF screener, using prevalence rates of Binge Eating Disorder (BED), Bulimia Nervosa (BN), Anorexia Nervosa (AN), and Any Eating Disorder (AED), as measured by the Eating Disorder Examination Questionnaire (EDE-Q). The SDE correctly classified 87.2% (CI: 74.3%-95.2%) of BED cases, all cases of BN and AN, and 90.5% (CI: 80.4%-96.4%) of AED cases. Sensitivity estimates were higher than the SCOFF, which correctly identified 69.6% (CI: 54.2%-82.3%) of BED, 77.8% (CI: 40.0%-97.2%) of BN, 37.5% (CI: 8.52%-75.5%) of AN, and 66.1% (CI: 53%-77.7%) of AED. While the EDS-PC had slightly higher sensitivity than the SDE, the SDE had better specificity. The SDE outperformed the SCOFF in classifying true cases, the EDS-PC in classifying true non-cases, and the EDS-PC in distinguishing cases from non-cases. The SDE is the first screen, inclusive of BED, valid for detecting eating disorders in primary care. Findings have broad implications to address eating disorder screening in primary care settings. Published by Elsevier Inc.

Screening for pulmonary arterial hypertension in systemic sclerosis

Directory of Open Access Journals (Sweden)

J-L. Vachiéry

2009-09-01

Full Text Available The onset and progression of pulmonary arterial hypertension (PAH in patients with systemic sclerosis (SSc can be particularly aggressive; however, effective treatments are available. Therefore, early identification of patients with suspected PAH, confirmation of diagnosis, and intervention is essential. PAH may be challenging to diagnose in its earliest stages, particularly in populations that have multiple causes of breathlessness, and, therefore, screening is required. The optimal screening tools and methodology are, as yet, unknown, and this is confounded by a lack of consensus over which patients to screen. Current practice favours annual screening of all SSc patients using Doppler echocardiography to detect elevated right heart pressures. This will typically identify most patients with the various forms of pulmonary hypertension found in SSc. The optimum thresholds for Doppler echocardiography are still subject to investigation, especially for patients with mild pulmonary hypertension, and this technique may, therefore, yield a significant number of false-positives and a currently unknown number of false-negatives. Confirmatory right heart catheterisation remains necessary in all suspected cases. Further research is needed to identify the optimal tools and the screening approach with greatest specificity and selectivity.

Current controversies in prenatal diagnosis 2: Cell-free DNA prenatal screening should be used to identify all chromosome abnormalities.

Science.gov (United States)

Chitty, Lyn S; Hudgins, Louanne; Norton, Mary E

2018-02-01

Noninvasive prenatal testing (NIPT) using cell-free DNA (cfDNA) from maternal serum has been clinically available since 2011. This technology has revolutionized our ability to screen for the common aneuploidies trisomy 21 (Down syndrome), trisomy 18, and trisomy 13. More recently, clinical laboratories have offered screening for other chromosome abnormalities including sex chromosome abnormalities and copy number variants (CNV) without little published data on the sensitivity, specificity, and positive predictive value. In this debate, the pros and cons of performing prenatal screening via cfDNA for all chromosome abnormalities is discussed. At the time of the debate in 2017, the general consensus was that the literature does not yet support using this technology to screen for all chromosome abnormalities and that education is key for both providers and the patients so that the decision-making process is as informed as possible. © 2018 John Wiley & Sons, Ltd.

Screening methods for post-stroke visual impairment: a systematic review.

Science.gov (United States)

Hanna, Kerry Louise; Hepworth, Lauren Rachel; Rowe, Fiona

2017-12-01

To provide a systematic overview of the various tools available to screen for post-stroke visual impairment. A review of the literature was conducted including randomised controlled trials, controlled trials, cohort studies, observational studies, systematic reviews and retrospective medical note reviews. All languages were included and translation was obtained. Participants included adults ≥18 years old diagnosed with a visual impairment as a direct cause of a stroke. We searched a broad range of scholarly online resources and hand-searched articles registers of published, unpublished and on-going trials. Search terms included a variety of MESH terms and alternatives in relation to stroke and visual conditions. Study selection was performed by two authors independently. The quality of the evidence and risk of bias were assessed using the STROBE, GRACE and PRISMA statements. A total of 25 articles (n = 2924) were included in this review. Articles appraised reported on tools screening solely for visual impairments or for general post-stroke disabilities inclusive of vision. The majority of identified tools screen for visual perception including visual neglect (VN), with few screening for visual acuity (VA), visual field (VF) loss or ocular motility (OM) defects. Six articles reported on nine screening tools which combined visual screening assessment alongside screening for general stroke disabilities. Of these, three included screening for VA; three screened for VF loss; three screened for OM defects and all screened for VN. Two tools screened for all visual impairments. A further 19 articles were found which reported on individual vision screening tests in stroke populations; two for VF loss; 11 for VN and six for other visual perceptual defects. Most tools cannot accurately account for those with aphasia or communicative deficits, which are common problems following a stroke. There is currently no standardised visual screening tool which can accurately

Fetal alcohol-spectrum disorders: identifying at-risk mothers

Directory of Open Access Journals (Sweden)

Montag AC

2016-07-01

Full Text Available Annika C Montag Department of Pediatrics, Division of Dysmorphology and Teratology, University of California San Diego, San Diego, CA, USA Abstract: Fetal alcohol-spectrum disorders (FASDs are a collection of physical and neuro­behavioral disabilities caused by prenatal exposure to alcohol. To prevent or mitigate the costly effects of FASD, we must identify mothers at risk for having a child with FASD, so that we may reach them with interventions. Identifying mothers at risk is beneficial at all time points, whether prior to pregnancy, during pregnancy, or following the birth of the child. In this review, three approaches to identifying mothers at risk are explored: using characteristics of the mother and her pregnancy, using laboratory biomarkers, and using self-report assessment of alcohol-consumption risk. At present, all approaches have serious limitations. Research is needed to improve the sensitivity and specificity of biomarkers and screening instruments, and to link them to outcomes as opposed to exposure. Universal self-report screening of all women of childbearing potential should ideally be incorporated into routine obstetric and gynecologic care, followed by brief interventions, including education and personalized feedback for all who consume alcohol, and referral to treatment as indicated. Effective biomarkers or combinations of biomarkers may be used during pregnancy and at birth to determine maternal and fetal alcohol exposure. The combination of self-report and biomarker screening may help identify a greater proportion of women at risk for having a child with FASD, allowing them to access information and treatment, and empowering them to make decisions that benefit their children. Keywords: fetal alcohol-spectrum disorder (FASD, alcohol, pregnancy, screening, biomarkers, SBIRT

Screening concepts for the isolation of biosurfactant producing microorganisms.

Science.gov (United States)

Walter, Vanessa; Syldatk, Christoph; Hausmann, Rudolf

2010-01-01

This chapter gives an overview of current methods for the isolation of biosurfactant producing microbes. The common screening methods for biosurfactants are presented. Sampling and isolation of bacteria are the basis for screening of biosurfactant producing microbes. Hydrocarbon-contaminated sites are the most promising for the isolation of biosurfactant producing microbes, but many strains have also been isolated from undisturbed sites. In subsequent steps the isolates have to be characterized in order to identify the strains which are interesting for a further investigation. Several techniques have been developed for identifying biosurfactant producing strains. Most of them are directly based on the surface or interfacial activity of the culture supernatant. Apart from that, some screening methods explore the hydrophobicity of the cell surface. This trait also gives an indication on biosurfactant production. In recent years automation and miniaturization have led to the development of high throughput methods for screening. High throughput screening (HTS) for analyzing large amounts of potential candidates or whole culture collections is reflected in the end. However, no new principals have been introduced by HTS methods.

Older Adults’ Views and Communication Preferences About Cancer Screening Cessation

Science.gov (United States)

Schoenborn, Nancy L.; Lee, Kimberley; Pollack, Craig E.; Armacost, Karen; Dy, Sydney M.; Bridges, John F. P.; Xue, Qian-Li; Wolff, Antonio C.; Boyd, Cynthia

2017-01-01

IMPORTANCE Older adults with limited life expectancy are frequently screened for cancer even though it exposes them to risks of screening with minimal benefit. Patient preferences may be an important contributor to continued screening. OBJECTIVE To examine older adults’ views on the decision to stop cancer screening when life expectancy is limited and to identify older adults’ preferences for how clinicians should communicate recommendations to cease cancer screening. DESIGN, SETTING, AND PARTICIPANTS In this semistructured interview study, we interviewed 40 community-dwelling older adults (≥ 65 years) recruited at 4 clinical programs affiliated with an urban academic medical center. MAIN OUTCOMES AND MEASURE We transcribed the audio recorded discussions and analyzed the transcripts using standard techniques of qualitative content analysis to identify major themes and subthemes. RESULTS The participants’ average age was 75.7 years. Twenty-three participants (57.5%) were female; 25 (62.5%) were white. Estimated life expectancy was less than 10 years for 19 participants (47.5%). We identified 3 key themes. First, participants were amenable to stopping cancer screening, especially in the context of a trusting relationship with their clinician. Second, although many participants supported using age and health status to individualize the screening decision, they did not often understand the role of life expectancy. All except 2 participants objected to a Choosing Wisely statement about not recommending cancer screening in those with limited life expectancy, often believing that clinicians cannot accurately predict life expectancy. Third, participants preferred that clinicians explain a recommendation to stop screening by incorporating individual health status but were divided on whether life expectancy should be mentioned. Specific wording of life expectancy was important; many felt the language of “you may not live long enough to benefit from this test” was

Screening from the epidemiological point of view

International Nuclear Information System (INIS)

Becker, N.

2002-01-01

The scope of screening is to identify disease in a clinically inapparent stage in order to treat more effectively. However, the required application of a test to large subsets of the population implies risks which cannot be ignored. Thus, the introduction of a test for use in screening without previously established evidence of a benefit which exceeds potential harm by far cannot be justified. With regard to cancer, evidence of effectiveness has been proven for screening on cervical (Papsmear), breast (mammography) and colorectal (faecal occult blood test) cancer. The routine application of a screening procedure which has been proven effective provides the benefit in terms of mortality reduction seen in the trials only if the entire screening chain, from the test over potentially required assessment to treatment, is offered with highest quality and is under permanent quality control. (orig.) [de

Transcranial Doppler Screening Among Children and Adolescents With Sickle Cell Anemia.

Science.gov (United States)

Reeves, Sarah L; Madden, Brian; Freed, Gary L; Dombkowski, Kevin J

2016-06-01

With transcranial Doppler (TCD) screening, we can identify children and adolescents with sickle cell anemia who are at the highest risk of stroke. An accurate claims-based method for identifying children and adolescents with sickle cell anemia was recently developed and validated that establishes the necessary groundwork to enable large population-based assessments of health services utilization among children and adolescents with sickle cell anemia using administrative claims data. To assess the feasibility of using administrative claims data to identify and describe the receipt of TCD screening among children and adolescents with sickle cell anemia and to characterize opportunities for intervention. Retrospective cross-sectional study using Medicaid claims data from 2005 to 2010. Medicaid claims data were obtained from the following states: Florida, Illinois, Louisiana, Michigan, South Carolina, and Texas. Children and adolescents 2 to 16 years of age with sickle cell anemia were identified by the presence of 3 or more Medicaid claims with a diagnosis of sickle cell anemia within a calendar year (2005-2010). A total of 4775 children and adolescents contributed 10 787 person-years throughout the study period. Data were analyzed in 2015. A subset of children and adolescents enrolled for 2 or more consecutive years was identified to examine potential predictors of TCD screening, which included age, sex, previous receipt of TCD screening, state of residence, and health services utilization (well-child visits, outpatient visits, emergency department visits, and inpatient visits). Receipt of TCD screening was assessed by year and state. Using logistic regression with generalized estimating equations, we included associated predictors in a multivariable model to estimate odds of TCD screening. For a total of 4775 children and adolescents 2 to 16 years of age, TCD screening rates increased over the 6-year study period from 22% to 44% (P sickle cell anemia (50%) was

Evaluation of a workplace hemochromatosis screening program.

Science.gov (United States)

Stave, G M; Mignogna, J J; Powell, G S; Hunt, C M

1999-05-01

Hemochromatosis is a common inherited disorder of iron metabolism with significant health consequences for the employed population. Although screening for hemochromatosis has been recommended, workplace screening programs remain uncommon. In the first year of a newly initiated corporate screening program, 1968 employees were tested. The screening algorithm included measurement of serum iron and transferrin and subsequent ferritin levels in those employees with elevated iron/transferrin ratios. Thirteen percent of men and 21% of women had elevated iron/transferrin ratios. Of these, 14 men and 2 women had elevated ferritin levels. Of these 16, three had liver biopsies and all three have hemochromatosis. The cost of the screening program was $27,850. The cost per diagnosis was $9283 and the cost per year of life saved was $928. These costs compare very favorably with other common workplace screening programs. Several barriers to obtaining definitive diagnoses on all patients with a positive screening result were identified; strategies to overcome these barriers would further enhance the cost effectiveness of the program. We conclude that workplace hemochromatosis screening is highly cost effective and should be incorporated into health promotion/disease prevention programs.

Development of smartphone application that aids stroke screening and identifying nearby acute stroke care hospitals.

Science.gov (United States)

Nam, Hyo Suk; Heo, JoonNyung; Kim, Jinkwon; Kim, Young Dae; Song, Tae Jin; Park, Eunjeong; Heo, Ji Hoe

2014-01-01

The benefits of thrombolytic treatment are time-dependent. We developed a smartphone application that aids stroke patient self-screening and hospital selection, and may also decrease hospital arrival time. The application was developed for iPhone and Android smartphones. Map data for the application were adopted from the open map. For hospital registration, a web page (http://stroke119.org) was developed using PHP and MySQL. The Stroke 119 application includes a stroke screening tool and real-time information on nearby hospitals that provide thrombolytic treatment. It also provides information on stroke symptoms, thrombolytic treatment, and prescribed actions when stroke is suspected. The stroke screening tool was adopted from the Cincinnati Prehospital Stroke Scale and is displayed in a cartoon format. If the user taps a cartoon image that represents abnormal findings, a pop-up window shows that the user may be having a stroke, informs the user what to do, and directs the user to call emergency services. Information on nearby hospitals is provided in map and list views, incorporating proximity to the user's location using a Global Positioning System (a built-in function of smartphones). Users can search for a hospital according to specialty and treatment levels. We also developed a web page for hospitals to register in the system. Neurology training hospitals and hospitals that provide acute stroke care in Korea were invited to register. Seventy-seven hospitals had completed registration. This application may be useful for reducing hospital arrival times for thrombolytic candidates.

A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

Directory of Open Access Journals (Sweden)

Magdalena L Circu

Full Text Available Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF or acidic extracellular pH (pHe, increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes

Impact of Prostate-specific Antigen (PSA) Screening Trials and Revised PSA Screening Guidelines on Rates of Prostate Biopsy and Postbiopsy Complications.

Science.gov (United States)

Gershman, Boris; Van Houten, Holly K; Herrin, Jeph; Moreira, Daniel M; Kim, Simon P; Shah, Nilay D; Karnes, R Jeffrey

2017-01-01

Prostate biopsy and postbiopsy complications represent important risks of prostate-specific antigen (PSA) screening. Although landmark randomized trials and updated guidelines have challenged routine PSA screening, it is unclear whether these publications have affected rates of biopsy or postbiopsy complications. To evaluate whether publication of the 2008 and 2012 US Preventive Services Task Force (USPSTF) recommendations, the 2009 European Randomized Study of Screening for Prostate Cancer and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, or the 2013 American Urological Association (AUA) guidelines was associated with changes in rates of biopsy or postbiopsy complications, and to identify predictors of postbiopsy complications. This quasiexperimental study used administrative claims of 5279315 commercially insured US men aged ≥40 yr from 2005 to 2014, of whom 104584 underwent biopsy. Publications on PSA screening. Interrupted time-series analysis was used to evaluate the association of publications with rates of biopsy and 30-d complications. Logistic regression was performed to identify predictors of complications. From 2005 to 2014, biopsy rates fell 33% from 64.1 to 42.8 per 100000 person-months, with immediate reductions following the 2008 USPSTF recommendations (-10.1; 95% confidence interval [CI], -17.1 to -3.0; pprostate-specific antigen screening; however, the relative morbidity of biopsy continues to increase. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Genetic Screens in Yeast to Identify BRCA1 Modifiers

National Research Council Canada - National Science Library

Plon, Sharon E

2005-01-01

.... The yeast RAD9 protein has similar functions and sequence motifs as BRCA1 and we proposed to identify haploinsufficient mutations at a second locus that alters the chromosome loss rate of our rad9-/- diploid strains...

Imaging of dihydrofolate reductase fusion gene expression in xenografts of human liver metastases of colorectal cancer in living rats

Energy Technology Data Exchange (ETDEWEB)

Mayer-Kuckuk, Philipp; Bertino, Joseph R.; Banerjee, Debabrata [Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); The Cancer Institute of New Jersey, Robert Wood Johnson Medical School/UMDNJ, 195 Little Albany Street, NJ 08903, New Brunswick (United States); Doubrovin, Mikhail; Blasberg, Ronald; Tjuvajev, Juri Gelovani [Department of Neurooncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Gusani, Niraj J.; Fong, Yuman [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Gade, Terence; Koutcher, Jason A. [Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Balatoni, Julius; Finn, Ronald [Radiochemistry/Cyclotron Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Akhurst, Tim; Larson, Steven [Nuclear Medicine Service, Memorial Sloan-Kettering Cancer Center, New York, NY (United States)

2003-09-01

Radionuclide imaging has been demonstrated to be feasible to monitor transgene expression in vivo. We hypothesized that a potential application of this technique is to non-invasively detect in deep tissue, such as cancer cells metastatic to the liver, a specific molecular response following systemic drug treatment. Utilizing human colon adenocarcinoma cells derived from a patient's liver lesion we first developed a nude rat xenograft model for colorectal cancer metastatic to the liver. Expression of a dihydrofolate reductase-herpes simplex virus 1 thymidine kinase fusion (DHFR-HSV1 TK) transgene in the hepatic tumors was monitored in individual animals using the tracer [{sup 124}I]2'-fluoro-2'-deoxy-5-iodouracil-{beta}-d-arabinofuranoside (FIAU) and a small animal micro positron emission tomograph (microPET), while groups of rats were imaged using the tracer [{sup 131}I]FIAU and a clinical gamma camera. Growth of the human metastatic colorectal cancer cells in the rat liver was detected using magnetic resonance imaging and confirmed by surgical inspection. Single as well as multiple lesions of different sizes and sites were observed in the liver of the animals. Next, using a subset of rats bearing hepatic tumors, which were retrovirally bulk transduced to express the DHFR-HSV1 TK transgene, we imaged the fusion protein expression in the hepatic tumor of living rats using the tracer [{sup 124}I]FIAU and a microPET. The observed deep tissue signals were highly specific for the tumors expressing the DHFR-HSV1 TK fusion protein compared with parental untransduced tumors and other tissues as determined by gamma counting of tissue samples. A subsequent study used the tracer [{sup 131}I]FIAU and a gamma camera to monitor two groups of transduced hepatic tumor-bearing rats. Prior to imaging, one group was treated with trimetrexate to exploit DHFR-mediated upregulation of the fusion gene product. Imaging in the living animal as well as subsequent gamma

The performance of blood pressure-to-height ratio as a screening measure for identifying children and adolescents with hypertension: a meta-analysis.

Science.gov (United States)

Ma, Chunming; Liu, Yue; Lu, Qiang; Lu, Na; Liu, Xiaoli; Tian, Yiming; Wang, Rui; Yin, Fuzai

2016-02-01

The blood pressure-to-height ratio (BPHR) has been shown to be an accurate index for screening hypertension in children and adolescents. The aim of the present study was to perform a meta-analysis to assess the performance of BPHR for the assessment of hypertension. Electronic and manual searches were performed to identify studies of the BPHR. After methodological quality assessment and data extraction, pooled estimates of the sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, area under the receiver operating characteristic curve and summary receiver operating characteristics were assessed systematically. The extent of heterogeneity for it was assessed. Six studies were identified for analysis. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio values of BPHR, for assessment of hypertension, were 96% [95% confidence interval (CI)=0.95-0.97], 90% (95% CI=0.90-0.91), 10.68 (95% CI=8.03-14.21), 0.04 (95% CI=0.03-0.07) and 247.82 (95% CI=114.50-536.34), respectively. The area under the receiver operating characteristic curve was 0.9472. The BPHR had higher diagnostic accuracies for identifying hypertension in children and adolescents.

High-throughput drug screen identifies chelerythrine as a selective inducer of death in a TSC2-null setting.

Science.gov (United States)

Medvetz, Doug; Sun, Yang; Li, Chenggang; Khabibullin, Damir; Balan, Murugabaskar; Parkhitko, Andrey; Priolo, Carmen; Asara, John M; Pal, Soumitro; Yu, Jane; Henske, Elizabeth P

2015-01-01

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress-dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors. This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress-dependent cell death, and provide critical proof of concept that TSC2-deficient cells can be therapeutically targeted without the use of a rapalog to induce a cell death response. ©2014 American Association for Cancer Research.

A synthetic interaction screen identifies factors selectively required for proliferation and TERT transcription in p53-deficient human cancer cells.

Directory of Open Access Journals (Sweden)

Li Xie

Full Text Available Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53- human cancer cells. We find that compared to p53-competent (p53+ human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.

Prospects for population screening and diagnosis of lung cancer

DEFF Research Database (Denmark)

Field, John K; Oudkerk, Matthijs; Pedersen, Jesper Holst

2013-01-01

Deaths from lung cancer exceed those from any other type of malignancy, with 1·5 million deaths in 2010. Prevention and smoking cessation are still the main methods to reduce the death toll. The US National Lung Screening Trial, which compared CT screening with chest radiograph, yielded a mortality......, and pooled analysis of European CT screening trials, we discuss the main topics that will need consideration. These unresolved issues are risk prediction models to identify patients for CT screening; radiological protocols that use volumetric analysis for indeterminate nodules; options for surgical resection...

Computer-Aided Solvent Screening for Biocatalysis

DEFF Research Database (Denmark)

Abildskov, Jens; Leeuwen, M.B. van; Boeriu, C.G.

2013-01-01

constrained properties related to chemical reaction equilibrium, substrate and product solubility, water solubility, boiling points, toxicity and others. Two examples are provided, covering the screening of solvents for lipase-catalyzed transesterification of octanol and inulin with vinyl laurate....... Esterification of acrylic acid with octanol is also addressed. Solvents are screened and candidates identified, confirming existing experimental results. Although the examples involve lipases, the method is quite general, so there seems to be no preclusion against application to other biocatalysts....

Evaluating the evidence: direct-to-consumer screening tests advertised online.

Science.gov (United States)

Lovett, Kimberly M; Mackey, Timothy K; Liang, Bryan A

2012-09-01

Unsupervised online direct-to-consumer (DTC) access to medical services has rapidly expanded to medical screening tests, which have not been critically evaluated for their evidence basis. The objective of this study is to identify the scope of online-advertised DTC screening tests, outline the evidence for use of available DTC testing and suggest regulatory reform to address the relevant issues. An observational study of website advertisements, testing services and counselling/follow-up services for DTC testing was conducted. Data were collected from websites between 4 April and 1 June 2011. Each website was assessed for tests offered, advertised indications and availability of counselling/follow-up services. Advertised testing indications were compared with US Preventive Services Task Force recommendations and/or specialty guidelines and categorized as Supported, Against, Insufficient Evidence or No Guidance. Of 20 companies identified as offering DTC screening tests, 95% (19/20) do not clearly offer pretest counselling, post-test counselling and/or test follow-up. One hundred and twenty-seven different tests were identified. Only 19/127 (15%) could be Supported for screening in a target group selected for testing; 38/127 (30%) were given recommendations to avoid use in specific target group(s) selected for testing ('Against recommendations'); 29/127 (23%) had Insufficient Evidence of value, and for 64/127 (50%) No Guidance could be given. Only 4/127 (3%) tests were Supported for general screening use. Virtually all identified medical tests advertised and offered DTC are not recommended for use in screening by evidence-based guidelines. Limited oversight may lead to inaccurate self-diagnosis, treatment and wasted health resources.

Screening specifications for Gulf Coast salt domes

International Nuclear Information System (INIS)

Brunton, G.D.; Laughon, R.B.; McClain, W.C.

1978-01-01

A reconnaissance survey of the salt domes of Mississippi, Louisiana, and east Texas is being planned to identify study areas for potential sites for radioactive waste disposal. Preliminary screening specifications were derived for each of the geological evaluation criteria by application of the significant factors that will have an impact on the reconnaissance survey. The procedure for the derivation of each screening specification is discussed. The screening specifications are the official OWI values to be used for the first-cut acceptance for salt dome study areas along the Gulf Coast. The derivation of the screening specifications is illustrated by (1) a statement of the geological evaluation criterion, (2) a discussion of the pertinent factors affecting the criterion, and (3) the evaluation of the value of the specification

Nudging in screening: Literature review and ethical guidance.

Science.gov (United States)

Hofmann, Bjørn; Stanak, Michal

2018-03-27

Nudging is the purposeful alteration of choices presented to people that aims to make them choose in predicted ways. While nudging has been used to assure high uptake and good outcome of screening programs, it has been criticized for being paternalistic, undermining free choice, and shared decision making. Accordingly, the objective of this study is to explore a) nudging strategies identified in screening, b) arguments for and against nudging; and on basis of this, to c) suggest a tentative conclusion on how to handle nudging in screening. Literature searches in Ovid MEDLINE and PsycINFO for combinations of screening and nudging. Screening based on content analysis of titles, abstracts, and articles. 239 references were identified and 109 were included. Several forms of nudging were identified: framed information, default bias, or authority bias. Uptake and public health outcome were the most important goals. Arguments for nudging were bounded rationality, unavoidability, and beneficence, while lack of transparency, crowding out of intrinsic values, and paternalism were arguments against it. The analysis indicates that nudging can be acceptable for screenings with (high quality) evidence for high benefit-harm ratio (beneficence), where nudging does not infringe other ethical principles, such as justice and non-maleficence. In particular, nudging should not only focus on attendance rates, but also on making people "better choosers." Four specific recommendations follow from the review and the analysis: 1) Nudging should be addressed in an explicit and transparent manner. 2) The means of nudging have to be in proportion to the benefit-harm ratio. 3) Disagreement on the evidence for either benefits or harms warrants special care. 4) Assessing and assuring the intended outcome of nudging appears to be crucial, as it can be context dependent. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel colchicine-site binders with a cyclohexanedione scaffold identified through a ligand-based virtual screening approach.

Science.gov (United States)

Canela, María-Dolores; Pérez-Pérez, María-Jesús; Noppen, Sam; Sáez-Calvo, Gonzalo; Díaz, J Fernando; Camarasa, María-José; Liekens, Sandra; Priego, Eva-María

2014-05-22

Vascular disrupting agents (VDAs) constitute an innovative anticancer therapy that targets the tumor endothelium, leading to tumor necrosis. Our approach for the identification of new VDAs has relied on a ligand 3-D shape similarity virtual screening (VS) approach using the ROCS program as the VS tool and as query colchicine and TN-16, which both bind the α,β-tubulin dimer. One of the hits identified, using TN-16 as query, has been explored by the synthesis of its structural analogues, leading to 2-(1-((2-methoxyphenyl)amino)ethylidene)-5-phenylcyclohexane-1,3-dione (compound 16c) with an IC50 = 0.09 ± 0.01 μM in HMEC-1 and BAEC, being 100-fold more potent than the initial hit. Compound 16c caused cell cycle arrest in the G2/M phase and interacted with the colchicine-binding site in tubulin, as confirmed by a competition assay with N,N'-ethylenebis(iodoacetamide) and by fluorescence spectroscopy. Moreover, 16c destroyed an established endothelial tubular network at 1 μM and inhibited the migration and invasion of human breast carcinoma cells at 0.4 μM. In conclusion, our approach has led to a new chemotype of promising antiproliferative compounds with antimitotic and potential VDA properties.

Italian Euromelanoma Day Screening Campaign (2005-2007) and the planning of melanoma screening strategies.

Science.gov (United States)

Seidenari, Stefania; Benati, Elisa; Ponti, Giovanni; Borsari, Stefania; Ferrari, Chiara; Albertini, Giuseppe; Altomare, Gianfranco; Arcangeli, Fabio; Aste, Nicola; Bernengo, Maria Grazia; Bongiorno, Maria Rita; Borroni, Giovanni; Calvieri, Stefano; Chimenti, Sergio; Cusano, Francesco; Fracchiolla, Claudio; Gaddoni, Giuseppe; Girolomoni, Giampiero; Guarneri, Biagio; Lanzoni, Anna; Lombardi, Mara; Lotti, Torello; Mariotti, Antonio; Marsili, Franco; Micali, Giuseppe; Parodi, Aurora; Peris, Ketty; Peserico, Andrea; Quaglino, Pietro; Santini, Marcello; Schiavon, Sergio; Tonino, Camillo; Trevisan, Giusto; Tribuzi, Paola; Valentini, Paolo; Vena, Gino A; Virgili, Annarosa

2012-01-01

Although no study has definitively shown that unfocused screening of skin cancer is effective, many campaigns have been organized with the aim of increasing awareness on melanoma risk factors. The objective of this study was to analyse the results of the Skin Cancer Screening Day in Italy during the period 2005-2007, to determine the priorities for melanoma control plans in a Mediterranean country. A total of 5002 patients were screened by dermatologists in 31 cities. Individuals who considered themselves to have many naevi and those with a family history of melanoma showed a higher number of common and atypical naevi. Ten melanomas, 20 basal cell carcinomas and two squamous cell carcinomas were histopathologically confirmed. Our observations provide the following suggestions for melanoma prevention strategies: (a) an unfocused campaign is suitable to inform the public about the importance of self-examination of the skin, but is not useful to identify a larger number of melanomas; and (b) melanoma screening campaigns should focus on a selected population, which meets rigorous risk criteria to maintain higher cost-effectiveness. The financial support to effective melanoma screening programmes could be increased, especially in southern populations where lower levels of self-surveillance and socioeconomic conditions represent risk factors for late identification of melanoma.

Consensus review of discordant findings maximizes cancer detection rate in double-reader screening mammography: Irish National Breast Screening Program experience.

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Shaw, Colette M; Flanagan, Fidema L; Fenlon, Helen M; McNicholas, Michelle M

2009-02-01

To assesses consensus review of discordant screening mammography findings in terms of its sensitivity, safety, and effect on overall performance in the first 6 years of operation of the Irish National Breast Screening Program (NBSP). Women who participated in the Irish NBSP gave written informed consent for use of their data for auditing purposes. Local ethics committee approval was obtained. The study population consisted of women who participated in the Irish NBSP and underwent initial screening mammography at one of the two screening centers serving the eastern part of Ireland between 2000 and 2005. Independent double reading of mammograms was performed. When the readers disagreed regarding referral, the case was reviewed by a consensus panel. Of the 128 569 screenings performed, 1335 (1%) were discussed by consensus. Of the 1335 cases discussed by consensus, 606 (45.39%) were recalled for further assessment. This resulted in an overall recall rate of 4.41%. In those recalled to assessment, 71 cases of malignant disease were diagnosed (ductal carcinoma in situ, n = 24; invasive cancer, n = 47). The remaining 729 patients were returned to biennial screening. Of these 729 patients, seven had false-negative findings that were identified in the subsequent screening round. Use of the highest reader recall method, in which a patient is recalled if her findings are deemed abnormal by either reader, could potentially increase the cancer detection rate by 0.6 per 1000 women screened but would increase the recall rate by 12.69% and the number of false-positive findings by 15.37%. The consensus panel identified 71 (7.33%) of 968 cancers diagnosed. Consensus review substantially reduced the number of cases recalled and was associated with a low false-negative rate.

Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities

DEFF Research Database (Denmark)

Radic-Sarikas, Branka; Tsafou, Kalliopi P; Emdal, Kristina B.

2017-01-01

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any...... including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong...

Development of a screening tool for detecting undernutrition and dietary inadequacy among rural elderly in Malaysia: simple indices to identify individuals at high risk.

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Shahar, S; Dixon, R A; Earland, J

1999-11-01

Undernutrition and the consumption of poor diets are prevalent among elderly people in developing countries. Recognising the importance of the early identification of individuals at high nutritional risk, this study aimed to develop a simple tool for screening. A cross-sectional study was conducted on 11 randomly selected villages among the 62 in Mersing District, Malaysia. Undernutrition was assessed using body mass index, plasma albumin and haemoglobin on 285 subjects. Dietary inadequacy (a count of nutrients falling below two-thirds of the Recommended Dietary Allowances) was examined for 337 subjects. Logistic regression analysis was performed to identify significant predictors of undernutrition and dietary inadequacy from social and health factors, and to derive appropriate indices based on these predictions. The multivariate predictors of undernutrition were 'no joint disease', 'smoker', 'no hypertension', 'depended on others for economic resource', 'respiratory disease', 'perceived weight loss' and 'chewing difficulty', with a joint sensitivity of 56% and specificity of 84%. The equivalent predictors of dietary inadequacy were 'unable to take public transport', 'loss of appetite', 'chewing difficulty', 'no regular fruit intake' and 'regularly taking less than three meals per day', with a joint sensitivity of 77% and specificity of 47%. These predictions, with minor modification to simplify operational use, led to the production of a simple screening tool. The tool can be used by public health professionals or community workers or leaders as a simple and rapid instrument to screen individual at high risk of undernutrition and/or dietary inadequacy.

CT screening for lung cancer. Update 2008

International Nuclear Information System (INIS)

Henschke, C.I.; Yip, R.; Yankelevitz, D.F.

2009-01-01

Screening for a cancer should be considered when the cancer is significant in terms of incidence and mortality, treatment of early stage disease is better than treatment of late stage disease, and there is a screening regimen that provides for earlier diagnosis rather than later, symptom-prompted diagnosis. Lung cancer qualifies as it kills more people than any other cancer worldwide. In the United States it kills more people than colon, breast, and prostate cancer combined and more women than breast cancer. The fundamental concepts of screening are presented. Screening for a cancer is a repetitive process, starting with the baseline round followed by repeat rounds of screening at set intervals. The regimen of screening defines the initial diagnostic test and the sequence of tests to be performed leading to a rule-in diagnosis of the cancer. The regimen should provide lead time of the diagnosis of the cancer. The regimen for the first, baseline round may be different from the regimen for the repeat rounds as the former is inherently different from the subsequent repeat rounds. Baseline screening identifies a greater proportion of cancers with a longer latent (asymptomatic) phase than repeat screening, called length bias. Length bias exists for any screening program, regardless of the design of the study or the cancer. Repeat rounds of screening identify the same proportion of cancer diagnoses found in absence of screening for people having the same risk of the cancer and these repeat rounds of screening can be pooled. It is also a consequence of length bias that cancers found in repeat rounds are earlier in their latent phase than those of the baseline round, a less frequently mentioned consequence. Overdiagnosis bias, another bias of screening, can occur in two ways: a cancer' detected by the screening, pathologically proven, that is not life-threatening even when not resected and a genuine life-threatening cancer that is diagnosed and treated but the person dies

Racial and ethnic disparities in U.S. cancer screening rates

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The percentage of U.S. citizens screened for cancer remains below national targets, with significant disparities among racial and ethnic populations, according to the first federal study to identify cancer screening disparities among Asian and Hispanic gr

Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

Directory of Open Access Journals (Sweden)

Paola Fabrizio

2010-07-01

Full Text Available The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Structural comparison of complexes of methotrexate analogues with Lactobacillus casei dihydrofolate reductase by two-dimensional 1H NMR at 500 MHz

International Nuclear Information System (INIS)

Hammond, S.J.; Birdsall, B.; Feeney, J.; Searle, M.S.; Roberts, G.C.K.; Cheung, H.T.A.

1987-01-01

The authors have used two-dimensional (2D) NMR methods to examine complexes of Lactobacillus casei dihydrofolate reductase and methotrexate (MTX) analogues having structural modifications of the benzoyl ring and also the glutamic acid moiety. Assignments of the 1 H signals in the spectra of the various complexes were made by comparison of their 2D spectra with those complexes containing methotrexate where we have previously assigned resonances from 32 of the 162 amino acid residues. In the complexes formed with the dihalomethotrexate analogues, the glutamic acid and pteridine ring moieties were shown to bind to the enzyme in a manner similar to that found in the methotrexate-enzyme complex. Perturbations in 1 H chemical shifts of protons in Phe-49, Leu-54, and Leu-27 and the methotrexate H7 and NMe protons were observed in the different complexes and were accounted for by changes in orientation of the benzoyl ring in the various complexes. Binding of oxidized or reduced coenzyme to the binary complexes did not result in different shifts for Leu-27, Leu-54, or Leu-19 protons, and thus, the orientation of the benzoyl ring of the methotrexate analogues is not perturbed greatly by the presence of either oxidized or reduced coenzyme. In the complex with the γ-monoamide analog, the 1 H signals of assigned residues in the protein had almost identical shifts with the corresponding protons in the methotrexate-enzyme complex for all residues except His-28 and, to a lesser extent, Leu-27. This indicates that while the His-28 interaction with the MTX γ-CO 2 - is no longer present in this complex with the γ-amide, there has not been a major change in the overall structure of the two complexes. This behavior contrasts to that of the α-amide complex where 1 H signals from protons in several amino acid residues are different compared with their values in the complex formed with methotrexate

Population-based cancer screening programmes in low-income and middle-income countries: regional consultation of the International Cancer Screening Network in India.

Science.gov (United States)

Sivaram, Sudha; Majumdar, Gautam; Perin, Douglas; Nessa, Ashrafun; Broeders, Mireille; Lynge, Elsebeth; Saraiya, Mona; Segnan, Nereo; Sankaranarayanan, Rengaswamy; Rajaraman, Preetha; Trimble, Edward; Taplin, Stephen; Rath, G K; Mehrotra, Ravi

2018-02-01

The reductions in cancer morbidity and mortality afforded by population-based cancer screening programmes have led many low-income and middle-income countries to consider the implementation of national screening programmes in the public sector. Screening at the population level, when planned and organised, can greatly benefit the population, whilst disorganised screening can increase costs and reduce benefits. The International Cancer Screening Network (ICSN) was created to share lessons, experience, and evidence regarding cancer screening in countries with organised screening programmes. Organised screening programmes provide screening to an identifiable target population and use multidisciplinary delivery teams, coordinated clinical oversight committees, and regular review by a multidisciplinary evaluation board to maximise benefit to the target population. In this Series paper, we report outcomes of the first regional consultation of the ICSN held in Agartala, India (Sept 5-7, 2016), which included discussions from cancer screening programmes from Denmark, the Netherlands, USA, and Bangladesh. We outline six essential elements of population-based cancer screening programmes, and share recommendations from the meeting that policy makers might want to consider before implementation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Estimating time and travel costs incurred in clinic based screening: flexible sigmoidoscopy screening for colorectal cancer.