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Screening Approach (screening + approach)
Selected AbstractsA Genomic Screening Approach to the Structure-Guided Identification of Drug Candidates from Natural SourcesCHEMBIOCHEM, Issue 7 2007Andreas Hornung Dr. Abstract The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence-guided genetic screening strategy. In this "genome mining" approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties. [source] Diagnostic potential of serum protein pattern in Type 2 diabetic nephropathyDIABETIC MEDICINE, Issue 12 2007Y-H. Yang Abstract Aims Microalbuminuria is the earliest clinical sign of diabetic nephropathy (DN). However, the multifactorial nature of DN supports the application of combined markers as a diagnostic tool. Thus, another screening approach, such as protein profiling, is required for accurate diagnosis. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel method for biomarker discovery. We aimed to use SELDI and bioinformatics to define and validate a DN-specific protein pattern in serum. Methods SELDI was used to obtain protein or polypeptide patterns from serum samples of 65 patients with DN and 65 non-DN subjects. From signatures of protein/polypeptide mass, a decision tree model was established for diagnosing the presence of DN. We estimated the proportion of correct classifications from the model by applying it to a masked group of 22 patients with DN and 28 non-DN subjects. The weak cationic exchange (CM10) ProteinChip arrays were performed on a ProteinChip PBS IIC reader. Results The intensities of 22 detected peaks appeared up-regulated, whereas 24 peaks were down-regulated more than twofold (P < 0.01) in the DN group compared with the non-DN groups. The algorithm identified a diagnostic DN pattern of six protein/polypeptide masses. On masked assessment, prediction models based on these protein/polypeptides achieved a sensitivity of 90.9% and specificity of 89.3%. Conclusion These observations suggest that DN patients have a unique cluster of molecular components in serum, which are present in their SELDI profile. Identification and characterization of these molecular components will help in the understanding of the pathogenesis of DN. The serum protein signature, combined with a tree analysis pattern, may provide a novel clinical diagnostic approach for DN. [source] An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protectorFEBS JOURNAL, Issue 23 2008Ke-He Ruan It remains a challenge to achieve the stable and long-term expression (in human cell lines) of a previously engineered hybrid enzyme [triple-catalytic (Trip-cat) enzyme-2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003,14011], which links cyclo-oxygenase isoform-2 (COX-2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme's Trip-cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)-mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip-cat enzyme, in which human cyclo-oxygenase isoform-1, which has a different C-terminal sequence from COX-2, was linked to PGI2 synthase and called Trip-cat enzyme-1. Transient expression of recombinant Trip-cat enzyme-1 in HEK293 cells led to 3,5-fold higher expression capacity and better PGI2 -synthesizing activity as compared to that of the previously engineered Trip-cat enzyme-2. Furthermore, an HEK293 cell line that can stably express the active new Trip-cat enzyme-1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase-rich plasma. This study has optimized engineering of the active Trip-cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2 -producing therapeutic cell line for use against vascular diseases. [source] Molecular neonatal screening for homocystinuria in the Qatari population,HUMAN MUTATION, Issue 6 2009Johannes Zschocke Abstract We report the results of molecular neonatal screening for homocystinuria (cystathionine beta-synthase deficiency) in neonates of Qatari origin, developed in conjunction with a novel biochemical screening approach. DNA was extracted from dried blood spots (DBS); the prevalent Qatari CBS gene mutation p.R336C (c.1006C>T) and a second mutation were tested with specific TaqMan assays. Over a period of 2 years we screened 12,603 neonates and identified six affected neonates homozygous for p.R336C. There were 225 heterozygous carriers for p.R336C. One additional child with homocystinuria detected through biochemical screening was homozygous for a mutation not previously identified in Qatar. Homocystinuria in the Qatari population has an incidence of 1:1,800, the highest in the world and even higher than previously estimated. Allele frequency of the mutation p.R336C is approximately 1%, displaying a significant deviation from Hardy Weinberg equilibrium. In conclusion, first-line molecular neonatal screening is technically feasible and may be developed as an option for presymptomatic identification of genetic disorders caused by specific mutations or a limited number of prevalent mutations. However, sensitivity for the diagnosis of disorders caused by various mutations is limited even in a homogeneous population such as Qatar. Hum Mutat 30:1,2, 2009. © 2009 Wiley-Liss, Inc. [source] ARC protects rat cardiomyocytes against oxidative stress through inhibition of caspase-2 mediated mitochondrial pathwayJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Yi-Qiang Zhang Abstract Apoptosis repressor with a CARD domain (ARC) has been demonstrated to protect heart cells against ischemia/reperfusion (I/R) injury. In this study, we investigated the mechanism by which ARC protects heart cells against oxidative stress. We monitored the extent of apoptosis and activity of multiple components of the intrinsic apoptotic pathway in rat cardiac myoblast cell line H9c2 with either reduced or increased expression of ARC during oxidative stress. Overexpression of ARC-inhibited oxidative stress-induced caspase-2/3 activation, cytochrome c release, and translocation of Bax to mitochondria. Furthermore, phosphorylation of ARC at threonine 149 was found to be critical to its function. ARC containing a T149A mutation failed to translocate to mitochondria, did not inhibit caspase-2 activation, and had a dominant negative effect against the protective effect of endogenous ARC during oxidative stress. In addition, wild-type ARC but not the T149A mutant inhibited cell death induced by overexpression of caspase-2. Using a yeast two-hybrid (YTH) screening approach and co-immunoprecipitation (Co-IP), we found that protein phosphatase 2C (PP2C) interacted with ARC and that PP2C mediated-dephosphorylation of ARC inhibited its anti-apoptotic activity. Eliminating either the N-terminal CARD domain or the C-terminal P/E domain also abolished the anti-apoptotic function of ARC, suggesting that full-length ARC is required for its apoptotic inhibition. These results indicate that ARC plays an important role in protection of H9c2 cells against oxidative stress-induced apoptosis by phosphorylation-dependent suppression of the mitochondria-mediated intrinsic pathway, partially initiated through the activation of caspase-2. J. Cell. Biochem. 99: 575,588, 2006. © 2006 Wiley-Liss, Inc. [source] Evaluation of library ranking efficacy in virtual screening,JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 1 2005Maria Kontoyianni Abstract We present the results of a comprehensive study in which we explored how the docking procedure affects the performance of a virtual screening approach. We used four docking engines and applied 10 scoring functions to the top-ranked docking solutions of seeded databases against six target proteins. The scores of the experimental poses were placed within the total set to assess whether the scoring function required an accurate pose to provide the appropriate rank for the seeded compounds. This method allows a direct comparison of library ranking efficacy. Our results indicate that the LigandFit/Ligscore1 and LigandFit/GOLD docking/scoring combinations, and to a lesser degree FlexX/FlexX, Glide/Ligscore1, DOCK/PMF (Tripos implementation), LigandFit1/Ligscore2 and LigandFit/PMF (Tripos implementation) were able to retrieve the highest number of actives at a 10% fraction of the database when all targets were looked upon collectively. We also show that the scoring functions rank the observed binding modes higher than the inaccurate poses provided that the experimental poses are available. This finding stresses the discriminatory ability of the scoring algorithms, when better poses are available, and suggests that the number of false positives can be lowered with conformers closer to bioactive ones. © 2004 Wiley Periodicals, Inc. J Comput Chem 26: 11,22, 2005 [source] Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice LeavesJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 6 2009Yin-Wan Wendy Fung Abstract Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice. We combined the RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice. We believe that the findings described here will help to elucidate the molecular mechanism(s) underlying the developmental processes of rice leaf. [source] Formation of cellular projections in neural progenitor cells depends on SK3 channel activityJOURNAL OF NEUROCHEMISTRY, Issue 5 2007Stefan Liebau Abstract Ion channels are potent modulators for developmental processes in progenitor cells. In a screening approach for different ion channels in neural progenitor cells (NPCs) we observed a 1-ethyl-2-benzimidazolinone (1-EBIO) activated inward current, which could be blocked by scyllatoxin (ScTX, IC50 = 2 ± 0.3 nmol/L). This initial evidence for the expression of the small conductance Ca2+ activated K+ -channel SK3 was confirmed by the detection of SK3 transcripts and protein in NPCs. Interestingly, SK3 proteins were highly expressed in non-differentiated NPCs with a focused localization in lamellipodia as well as filopodial structures. The activation of SK3 channels using 1-EBIO lead to an immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions. Both effects could be prevented by the pre-incubation of NPCs with ScTX. Our study gives first evidence that the formation and prolongation of filopodia in NPCs is, at least in part, effectively induced and regulated by SK3 channels. [source] Evaluation of hydrate-screening methodsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2008Yong Cui Abstract The purpose of this work is to evaluate the effectiveness and reliability of several common hydrate-screening techniques, and to provide guidelines for designing hydrate-screening programs for new drug candidates. Ten hydrate-forming compounds were selected as model compounds and six hydrate-screening approaches were applied to these compounds in an effort to generate their hydrate forms. The results prove that no screening approach is universally effective in finding hydrates for small organic compounds. Rather, a combination of different methods should be used to improve screening reliability. Among the approaches tested, the dynamic water vapor sorption/desorption isotherm (DVI) method and storage under high humidity (HH) yielded 60,70% success ratios, the lowest among all techniques studied. The risk of false negatives arises in particular for nonhygroscopic compounds. On the other hand, both slurry in water (Slurry) and temperature cycling of aqueous suspension (TCS) showed high success rates (90%) with some exceptions. The mixed solvent systems (MSS) procedure also achieved high success rates (90%), and was found to be more suitable for water-insoluble compounds. For water-soluble compounds, MSS may not be the best approach because recrystallization is difficult in solutions with high water activity. Finally, vapor diffusion (VD) yielded a reasonably high success ratio in finding hydrates (80%). However, this method suffers from experimental difficulty and unreliable results for either highly water-soluble or water-insoluble compounds. This study indicates that a reliable hydrate-screening strategy should take into consideration the solubility and hygroscopicity of the compounds studied. A combination of the Slurry or TCS method with the MSS procedure could provide a screening strategy with reasonable reliability. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2730,2744, 2008 [source] MULTIPLE CRITERIA SCREENING OF A LARGE WATER POLICY SUBSET SELECTION PROBLEM,JOURNAL OF THE AMERICAN WATER RESOURCES ASSOCIATION, Issue 3 2001Siamak Rajabi ABSTRACT: A new screening approach is applied to a large-scale multiple criteria water management problem to remove actions that cannot possibly be in the best subset. An inherent advantage of the approach is its ability to identify inferior actions by examining them individually, rather than within subsets. In a case study involving the selection of actions to address high water levels in the Great Lakes-St. Lawrence Basin, two statistical indicators, the mode and the mean, are used to aggregate the opinions of experts and representatives of interest groups on the impacts of actions according to various criteria. Application of the screening approach shows that some of the proposed actions can be removed as they can never be in the optimal subset, thereby reducing the size of the problem. [source] Mailed urine samples are not an effective screening approach for Chlamydia trachomatis case finding among young menJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 6 2007M Domeika Abstract Background, Frequency of testing is known to be low for sexually transmitted infections (STIs) in men aged 20,24 years. The use of mailed, home-obtained urine specimens could increase the uptake of young men and facilitate screening programmes for the detection of asymptomatic Chlamydia trachomatis. Objective, The aim of the present study is to evaluate the home screening approach as a tool for recruitment of asymptomatic men for screening of genital C. trachomatis infections. Methods, Men aged 19,24 years old (n = 1936) were invited to participate in home-based testing for genital C. trachomatis infection. Persons who agreed to be tested were provided with a testing kit. Self-collected first void urine was sent for testing to the microbiology laboratory. The test result was accessible on the study's web-page 1 week after testing. Individuals with a diagnosed infection were instructed to contact the venereal disease department. Results, The response rate was 24% (462/1936). The responders' main reason for not participating was a feeling of being safe regarding STIs (87%; 159/182). The primary reason for this feeling of safety was that the responders were in a steady relationship (59%; 107/159). Having sex outside a steady relationship was reported by 36% (90/250) of the responders. The prevalence of C. trachomatis infection among the responders was 2.02% and the reported history of chlamydial infection was 36% (34/95). Out of the responders, 92% (229/249) were, to varying degrees, concerned about getting STIs; however, the majority (72%; 174/242) estimated the risk to be low. Conclusion, Home screening using web-based answer management is a feasible tool for STI screening, which lowers the threshold for people at risk. In this particular population, however, the response rate was too low to be routinely introduced. [source] Image adaptive point-spread function estimation and deconvolution for in vivo confocal microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2006M. Von Tiedemann Abstract Visualizing deep inside the tissue of a thick biological sample often poses severe constraints on image conditions. Standard restoration techniques (denoising and deconvolution) can then be very useful, allowing one to increase the signal-to-noise ratio and the resolution of the images. In this paper, we consider the problem of obtaining a good determination of the point-spread function (PSF) of a confocal microscope, a prerequisite for applying deconvolution to three-dimensional image stacks acquired with this system. Because of scattering and optical distortion induced by the sample, the PSF has to be acquired anew for each experiment. To tackle this problem, we used a screening approach to estimate the PSF adaptively and automatically from the images. Small PSF-like structures were detected in the images, and a theoretical PSF model reshaped to match the geometric characteristics of these structures. We used numerical experiments to quantify the sensitivity of our detection method, and we demonstrated its usefulness by deconvolving images of the hearing organ acquired in vitro and in vivo. Microsc. Res. Tech. 69:10,20, 2006. © 2006 Wiley-Liss, Inc. [source] Cadmium partitioning and gene expression studies in Nicotiana tabacum and Nicotiana rusticaPHYSIOLOGIA PLANTARUM, Issue 3 2006Lucien Bovet To better understand the differences in cadmium (Cd) uptake, partitioning and gene regulation between Nicotiana tabacum and Nicotiana rustica, we compared these two species for root and leaf Cd concentrations after different Cd exposures, 109Cd root-to-shoot transport, Cd tolerance as well as differential gene expression in roots exposed or not to CdCl2 using reverse transcriptase,PCR (RT-PCR). When grown in 1 ,M CdCl2 for 7 days, N. rustica exhibited higher root and lower leaf Cd contents than N. tabacum. Data were confirmed by radiolabeling experiments, which further showed that some 109Cd accumulated in the distal part of lateral roots in N. rustica. Visual inspection of leaves suggested that N. rustica was somewhat more tolerant to high Cd exposure (50 ,M CdCl2) compared with N. tabacum. At such a high Cd concentration, Cd toxic effects on N. tabacum leaves were apparently not directly related to the homeostasis of Fe and Mn. However, the Zn levels were different in N. rustica compared with N. tabacum in absence and presence of Cd treatments. Root growth experiments revealed that N. tabacum, but not N. rustica, root length was reduced in bactoagar medium containing 20 ,M CdCl2. Complementary DNA microarrays were used as a screening approach to demonstrate by RT-PCR that some gene products were differentially regulated by Cd in N. rustica and in N. tabacum. In addition, "NtIRT1,"NtMTP1a, "NtHMA3" and "NtNAS3" were inducible by Cd in N. tabacum. Interestingly "NtIRT1" and NtMTP1a were differently expressed between the two species. Our results suggest different pathways for Cd sequestration and transport between these two species. [source] Desorption electrospray ionization mass spectrometric analysis of organophosphorus chemical warfare agents using ion mobility and tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2010Paul A. D'Agostino Desorption electrospray ionization mass spectrometry (DESI-MS) has been applied to the direct analysis of sample media for target chemicals, including chemical warfare agents (CWA), without the need for additional sample handling. During the present study, solid-phase microextraction (SPME) fibers were used to sample the headspace above five organophosphorus CWA, O -isopropyl methylphosphonofluoridate (sarin, GB), O -pinacolyl methylphosphonofluoridate (soman, GD), O -ethyl N,N -dimethyl phosphoramidocyanidate (tabun, GA), O -cyclohexyl methylphosphonofluoridate (cyclohexyl sarin, GF) and O -ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate (VX) spiked into glass headspace sampling vials. Following sampling, the SPME fibers were introduced directly into a modified ESI source, enabling rapid and safe DESI of the toxic compounds. A SYNAPT HDMSÔ instrument was used to acquire time-aligned parallel (TAP) fragmentation data, which provided both ion mobility and MSn (n,=,2 or 3) data useful for the confirmation of CWA. Unique ion mobility profiles were acquired for each compound and characteristic product ions of the ion mobility separated ions were produced in the TriwaveÔ transfer collision region. Up to six full scanning MSn spectra, containing the [M,+,H]+ ion and up to seven diagnostic product ions, were acquired for each CWA during SPME fiber analysis. A rapid screening approach, based on the developed methodology, was applied to several typical forensic media, including Dacron sampling swabs spiked with 5,µg of CWA. Background interference was minimal and the spiked CWA were readily identified within one minute on the basis of the acquired ion mobility and mass spectrometric data. Copyright © 2010 Crown in the right of Canada. Published by John Wiley & Sons, Ltd. [source] Clinical Burden of Screening Asymptomatic Patients for Coronary Artery Disease Prior to Liver TransplantationAMERICAN JOURNAL OF TRANSPLANTATION, Issue 5 2009D. Filì The aim of this study is to assess the clinical burden of silent coronary artery disease (CAD) in cirrhotic candidates for liver transplantation (LT), and to evaluate the usefulness of a CAD screening approach. Between July 1999 and January 2006, we evaluated 627 LT candidates. All of them underwent a detailed clinical history. Sixteen had a previous diagnosis of CAD or symptoms suggestive (2.5%). The remaining 611 underwent further tests according to a predefined protocol, including EKG, echocardiogram and, on the basis of CAD risk factors, heart stress tests. Selective coronary angiography (SCA) was performed in the 30 patients with positive heart stress test: in only 2 did SCA show any CAD, and in both it was subcritical disease requiring neither intervention nor contraindicating LT. The 611 screened patients continued their follow-up until study closure or death. No coronary events occurred in the study population in a mean follow-up of 32.50 months (+/, 23.67 DS). No perioperative mortality related to CAD occurred in the 233 transplanted patients. In conclusion, no prognostic advantage was achieved by following a strict CAD screening protocol, leading us to believe that the cost-effectiveness of a similar screening can be unacceptably high in our setting. [source] Using budding yeast to screen for anti-prion drugsBIOTECHNOLOGY JOURNAL, Issue 1 2006Déborah Tribouillard Abstract Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases. [source] NMR-Based Design and Evaluation of Novel Bidentate Inhibitors of the Protein Tyrosine Phosphatase YopHCHEMICAL BIOLOGY & DRUG DESIGN, Issue 1 2010Marilisa Leone We describe the use of a furanyl salicyl nitroxide derivative (,spin-labeled' compound), as a paramagnetic phosphotyrosine mimetic, to carry out a second-site screening by NMR against the PTPase YopH from Yersinia pestis. Using such a fragment-based screening approach we identified several small molecules targeting YopH that bind at sites adjacent to the spin-labeled compound. These second-site fragments were subsequently used to design and synthesize bidentate YopH inhibitors with submicromolar in vitro inhibition, selectivity against the human PTPase PTP1B, and cellular activity against Y. pseudotuberculosis. These initial compounds could result useful in elucidating the structural determinants necessary for YopH inhibition and may help in the design of even more active, selective and cell permeable compounds for the development of novel therapies against Yersiniae. [source] From Molecular Shape to Potent Bioactive Agents I: Bioisosteric Replacement of Molecular FragmentsCHEMMEDCHEM, Issue 1 2009Ewgenij Proschak Ligand-based virtual screening: By means of shape- and pharmacophore-based virtual screening, a potent PPAR,-selective activator was identified from a large compound collection with minimal experimental effort. This compound represents a scaffold-hop from known PPAR agonists and provides proof-of-concept for a novel ligand-based virtual screening approach. [source] Toxicological assessment of chemicals using Caenorhabditis elegans and optical oxygen respirometryENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2009Katherine Schouest Abstract Oxygen consumption is indicative of an organism's metabolic state, whereby alterations in respiration rate can result from the presence of different stimuli. Here, we develop a novel approach based on quenched fluorescence oxygen sensing and respirometry method for toxicity screening assays using the nematode Caenorhabditis elegans. Previously, C. elegans was established as a useful model in soil and aquatic toxicology studies. For existing toxicology screening approaches with C. elegans, however, the endpoint is lethality. In addition, the assay time frame for the existing approaches is considerably longer than that for the approach described here. We present a sensitive, robust, high-throughput platform using standard laboratory equipment for toxicological studies by measuring respiration rate in C. elegans animals using a phosphorescent probe. [source] Five novel frameshift mutations in exon 3 and 4 of the MECP2 gene identified in Rett patients: Consequences for the molecular diagnosis strategyHUMAN MUTATION, Issue 3 2001Thierry Bienvenu Abstract Rett syndrome (RTT) is a severe progressive neurological disorder that affects almost exclusively females. The gene responsible for this disorder, MECP2, was recently identified by candidate gene strategy. Mutations were detected in 70-85% of RTT cases. We report here five novel frameshift mutations (named 345delC, 895del202, 989ins18del8, 996insAG and 1124del53) in exon 3 and 4 of the MECP2 gene. To avoid the missing of few small deletions in RTT patients using classical mutation screening approaches, we suggest that screening of the mutations in the MECP2 gene in RTT girls should include at least a large PCR to amplify exon 4 entirely. Hum Mutat 18:251,252, 2001. © 2001 Wiley-Liss, Inc. [source] Consumer attitudes towards self-referral with early signs of cancer: implications for symptom awareness campaignsINTERNATIONAL JOURNAL OF NONPROFIT & VOLUNTARY SECTOR MARKETING, Issue 4 2007Douglas Eadie Traditionally, secondary prevention programmes have employed mass screening approaches to assess for asymptomatic signs of cancer. It has been suggested that early detection strategies, involving public education and self-referral may prove more cost-effective, with low-risk populations for cancers with symptomatic presentation. The success of public education approaches is dependent on careful consideration of the psycho-social factors of self-examination and referral. This paper presents the findings from an exploratory study, using qualitative methods with an at-risk population of older people living in deprived communities in west-central Scotland. The study examines consumer perceptions of the early detection of cancer and the cultural barriers to self-referral, as well as response to aspects of communication strategy. The implications for design of symptom awareness campaigns, including use of message appeals, specification of target symptoms, identification of target audience and selection of communication channels, are discussed. Copyright © 2007 John Wiley & Sons, Ltd. [source] Comparison of different methods of bacterial detection in blood componentsISBT SCIENCE SERIES: THE INTERNATIONAL JOURNAL OF INTRACELLULAR TRANSPORT, Issue 1 2009M. Schmidt Background, Over the last two decades, the residual risk of acquiring a transfusion-transmitted viral infection has been reduced to less than 1 : 1 000 000 via improvements in different techniques (e.g. donor selection, leuco-depletion, introduction of 3rd or 4th generation enzyme-linked immunosorbent assays and mini-pool nucleic acid testing (MP-NAT). In contrast, the risk for transfusion-associated bacterial infections has remained fairly stable, and is estimated to be in a range between 1 : 2000 and 1 : 3000. Platelets are at an especially higher risk for bacterial contamination, because they are stored at room temperature, which provides good culture conditions for a broad range of bacterial strains. To improve bacterial safety of blood products, different detection systems have been developed that can be divided into culture systems like BacT/ALERT or Pall eBDS, rapid detection systems like NAT systems, immunoassays and systems based on the FACS technique. Culture systems are used for routine bacterial screening of platelets in many countries, whereas rapid detection systems so far are mainly used in experimental spiking studies. Nevertheless, pathogen-reduction systems are currently available for platelet concentrates and plasma, and are under investigation for erythrocytes. Methods, In this review, the functional principles of the different assays are described and discussed with regard to their analytical sensitivity, analytical specificity, diagnostic sensitivity, diagnostic specificity and clinical efficiency. The detection methods were clustered into three groups: (i) detection systems currently used for routine screening of blood products, (ii) experimental detection systems ready to use for routine screening of blood products, and (iii) new experimental detection systems that need to be investigated in additional spiking studies and clinical trials. Results, A recent International Society of Blood Transfusion international forum reported on bacterial detection methods in 12 countries. Eight countries have implemented BacT/ALERT into blood donor screening, whereas in three countries only quality controls were done by culture methods. In one country, shelf-life was reduced to 3 days, so no bacterial screening was implemented. Screening data with culture methods can be used to investigate the prevalence of bacterial contamination in platelets. Differing results between the countries could be explained by different test definitions and different test strategies. Nevertheless, false-negative results causing severe transfusion-related septic reactions have been reported all over the world due to a residual risk of sample errors. Rapid screening systems NAT and FACS assays have improved over the last few years and are now ready to be implemented in routine screening. Non-specific amplification in NAT can be prevented by pre-treatment with Sau3AI, filtration of NAT reagents, or reduction of the number of polymerase chain reaction cycles. FACS systems offer easy fully automated handling and a handling time of only 5 min, which could be an option for re-testing day-5 platelets. New screening approaches like immunoassays, detection of bacterial adenosine triphosphate, or detection of esterase activity need to be investigated in additional studies. Conclusion, Bacterial screening of blood products, especially platelets, can be done with a broad range of technologies. The ideal system should be able to detect one colony-forming unit per blood bag without a delay in the release process. Currently, we are far away from such an ideal screening system. Nevertheless, pathogen-inactivation systems are available, but a system for all blood components will not be expected in the next few years. Therefore, existing culture systems should be complemented by rapid systems like NAT or FACS especially for day-5 platelets. [source] Illuminating the host , How RNAi screens shed light on host-pathogen interactionsBIOTECHNOLOGY JOURNAL, Issue 6 2009Miguel Prudêncio Abstract Over millions of years pathogens have coevolved with their respective hosts utilizing host cell functions for survival and replication. Despite remarkable progress in developing antibiotics and vaccination strategies in the last century, infectious diseases still remain a severe threat to human health. Meanwhile, genomic research offers a new era of data-generating platforms that will dramatically enhance our knowledge of pathogens and the diseases they cause. Improvements in gene knockdown studies by RNA interference (RNAi) combined with recent developments in instrumentation and image analysis enable the use of high-throughput screening approaches to elucidate host gene functions exploited by pathogens. Although only a few RNAi-based screens focusing on host genes have been reported so far, these studies have already uncovered hundreds of genes not previously known to be involved in pathogen infection. This review describes recent progress in RNAi screening approaches, highlighting both the limitations and the tremendous potential of RNAi-based screens for the identification of essential host cell factors during infection. [source] Chemically Induced Cardiomyogenesis of Mouse Embryonic Stem CellsCHEMBIOCHEM, Issue 2 2010Albrecht Berkessel Prof. Dr. Abstract A transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of ,-myosine heavy chain (,-MHC) promoter (p,-MHC-EGFP) was used to investigate the effects of (thio)urea and cinchona alkaloid derivatives on cardiomyogenesis. The screening of the compounds yielded cardiomyogenesis inducing substances with good (IV-5, V-4) to very good activities (II-16, IV-8), as determined by a 50 to 80,% increase in the EGFP fluorescence compared to untreated cells. Time-dependent screening approaches in which compounds were added at different developmental stages of the ES cells appeared to be of limited suitability for the identification of potential cellular targets. [source] Mapping the Druggable Allosteric Space of G-Protein Coupled Receptors: a Fragment-Based Molecular Dynamics ApproachCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2010Anthony Ivetac To address the problem of specificity in G-protein coupled receptor (GPCR) drug discovery, there has been tremendous recent interest in allosteric drugs that bind at sites topographically distinct from the orthosteric site. Unfortunately, structure-based drug design of allosteric GPCR ligands has been frustrated by the paucity of structural data for allosteric binding sites, making a strong case for predictive computational methods. In this work, we map the surfaces of the ,1 (,1AR) and ,2 (,2AR) adrenergic receptor structures to detect a series of five potentially druggable allosteric sites. We employ the FTMAP algorithm to identify ,hot spots' with affinity for a variety of organic probe molecules corresponding to drug fragments. Our work is distinguished by an ensemble-based approach, whereby we map diverse receptor conformations taken from molecular dynamics (MD) simulations totaling approximately 0.5 ,s. Our results reveal distinct pockets formed at both solvent-exposed and lipid-exposed cavities, which we interpret in light of experimental data and which may constitute novel targets for GPCR drug discovery. This mapping data can now serve to drive a combination of fragment-based and virtual screening approaches for the discovery of small molecules that bind at these sites and which may offer highly selective therapies. [source] A High-Content, Cell-Based Screen Identifies Micropolyin, A New Inhibitor of Microtubule DynamicsCHEMICAL BIOLOGY & DRUG DESIGN, Issue 6 2009Manu De Rycker High-content cell-based screens provide a powerful tool to identify new chemicals that interfere with complex biological processes. Here, we describe the identification of a new inhibitor of microtubule dynamics (micropolyin) using a high-content screen. Integrated high-resolution imaging allowed for fast selection of hits and progression to target identification. Treatment of cells with micropolyin efficiently causes a pro-metaphase arrest, with abnormal spindle morphology and with the spindle assembly checkpoint activated. The arrest appears to result from interference of micropolyin with microtubule dynamics. We show in vitro that tubulin is indeed the target of micropolyin and that micropolyin inhibits microtubule polymerization. Our results demonstrate the power of high-content image- and cell-based screening approaches to identify potential new drug candidates. As our approach is unbiased, it should allow for discovery of new targets that may otherwise be overlooked. [source] The Emerging Therapeutic Potential of Histone Methyltransferase and Demethylase InhibitorsCHEMMEDCHEM, Issue 10 2009Astrid Spannhoff Dr. Abstract Epigenetics is defined as heritable changes to the transcriptome that are independent of changes in the genome. The biochemical modifications that govern epigenetics are DNA methylation and posttranslational histone modifications. Among the histone modifications, acetylation and deacetylation are well characterized, whereas the fields of histone methylation and especially demethylation are still in their infancy. This is particularly true with regard to drug discovery. There is strong evidence that these modifications play an important role in the maintenance of transcription as well as in the development of certain diseases. This article gives an overview of the mechanisms of action of histone methyltransferases and demethylases, their role in the formation of certain diseases, and available inhibitors. Special emphasis is placed on the strategies that led to the first inhibitors which are currently available and the screening approaches that were used in that process. [source] Virtual Screening and Biological Characterization of Novel Histone Arginine Methyltransferase PRMT1 InhibitorsCHEMMEDCHEM, Issue 1 2009Ralf Heinke Abstract Lysine and arginine methyltransferases participate in the posttranslational modification of histones and regulate key cellular functions. Protein arginine methyltransferase,1 (PRMT1) has been identified as an essential component of mixed lineage leukemia (MLL) oncogenic complexes, revealing its potential as a novel therapeutic target in human cancer. The first potent arginine methyltransferase inhibitors were recently discovered by random- and target-based screening approaches. Herein we report virtual and biological screening for novel inhibitors of PRMT1. Structure-based virtual screening (VS) of the Chembridge database composed of 328,000 molecules was performed with a combination of ligand- and target-based in,silico approaches. Nine inhibitors were identified from the top-scored docking solutions; these were experimentally tested using human PRMT1 and an antibody-based assay with a time-resolved fluorescence readout. Among several aromatic amines, an aliphatic amine and an amide were also found to be active in the micromolar range. [source] | |||||||||||||||||||