Advances in identification and validation of protein targets of natural products without chemical modification.

PubMed

Chang, J; Kim, Y; Kwon, H J

2016-05-04

Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates.

Detection and Identification of Multiple Stationary Human Targets Via Bio-Radar Based on the Cross-Correlation Method

PubMed Central

Zhang, Yang; Chen, Fuming; Xue, Huijun; Li, Zhao; An, Qiang; Wang, Jianqi; Zhang, Yang

2016-01-01

Ultra-wideband (UWB) radar has been widely used for detecting human physiological signals (respiration, movement, etc.) in the fields of rescue, security, and medicine owing to its high penetrability and range resolution. In these applications, especially in rescue after disaster (earthquake, collapse, mine accident, etc.), the presence, number, and location of the trapped victims to be detected and rescued are the key issues of concern. Ample research has been done on the first issue, whereas the identification and localization of multi-targets remains a challenge. False positive and negative identification results are two common problems associated with the detection of multiple stationary human targets. This is mainly because the energy of the signal reflected from the target close to the receiving antenna is considerably stronger than those of the targets at further range, often leading to missing or false recognition if the identification method is based on the energy of the respiratory signal. Therefore, a novel method based on cross-correlation is proposed in this paper that is based on the relativity and periodicity of the signals, rather than on the energy. The validity of this method is confirmed through experiments using different scenarios; the results indicate a discernible improvement in the detection precision and identification of the multiple stationary targets. PMID:27801795

Strategies for target identification of antimicrobial natural products.

PubMed

Farha, Maya A; Brown, Eric D

2016-05-04

Covering: 2000 to 2015Despite a pervasive decline in natural product research at many pharmaceutical companies over the last two decades, natural products have undeniably been a prolific and unsurpassed source for new lead antibacterial compounds. Due to their inherent complexity, natural extracts face several hurdles in high-throughout discovery programs, including target identification. Target identification and validation is a crucial process for advancing hits through the discovery pipeline, but has remained a major bottleneck. In the case of natural products, extremely low yields and limited compound supply further impede the process. Here, we review the wealth of target identification strategies that have been proposed and implemented for the characterization of novel antibacterials. Traditionally, these have included genomic and biochemical-based approaches, which, in recent years, have been improved with modern-day technology and better honed for natural product discovery. Further, we discuss the more recent innovative approaches for uncovering the target of new antibacterial natural products, which have resulted from modern advances in chemical biology tools. Finally, we present unique screening platforms implemented to streamline the process of target identification. The different innovative methods to respond to the challenge of characterizing the mode of action for antibacterial natural products have cumulatively built useful frameworks that may advocate a renovated interest in natural product drug discovery programs.

Detection and Identification of Multiple Stationary Human Targets Via Bio-Radar Based on the Cross-Correlation Method.

PubMed

Zhang, Yang; Chen, Fuming; Xue, Huijun; Li, Zhao; An, Qiang; Wang, Jianqi; Zhang, Yang

2016-10-27

Ultra-wideband (UWB) radar has been widely used for detecting human physiological signals (respiration, movement, etc.) in the fields of rescue, security, and medicine owing to its high penetrability and range resolution. In these applications, especially in rescue after disaster (earthquake, collapse, mine accident, etc.), the presence, number, and location of the trapped victims to be detected and rescued are the key issues of concern. Ample research has been done on the first issue, whereas the identification and localization of multi-targets remains a challenge. False positive and negative identification results are two common problems associated with the detection of multiple stationary human targets. This is mainly because the energy of the signal reflected from the target close to the receiving antenna is considerably stronger than those of the targets at further range, often leading to missing or false recognition if the identification method is based on the energy of the respiratory signal. Therefore, a novel method based on cross-correlation is proposed in this paper that is based on the relativity and periodicity of the signals, rather than on the energy. The validity of this method is confirmed through experiments using different scenarios; the results indicate a discernible improvement in the detection precision and identification of the multiple stationary targets.

Identification of Target Complaints by Computer Interview: Evaluation of the Computerized Assessment System for Psychotherapy Evaluation and Research.

ERIC Educational Resources Information Center

Farrell, Albert D.; And Others

1987-01-01

Evaluated computer interview to standardize collection of target complaints. Adult outpatients (N=103) completed computer interview, unstructured intake interview, Symptoms Checklist-90, and Minnesota Multiphasic Personality Inventory. Results provided support for the computer interview in regard to reliability and validity though there was low…

Are multiple-trial experiments appropriate for eyewitness identification studies? Accuracy, choosing, and confidence across trials.

PubMed

Mansour, J K; Beaudry, J L; Lindsay, R C L

2017-12-01

Eyewitness identification experiments typically involve a single trial: A participant views an event and subsequently makes a lineup decision. As compared to this single-trial paradigm, multiple-trial designs are more efficient, but significantly reduce ecological validity and may affect the strategies that participants use to make lineup decisions. We examined the effects of a number of forensically relevant variables (i.e., memory strength, type of disguise, degree of disguise, and lineup type) on eyewitness accuracy, choosing, and confidence across 12 target-present and 12 target-absent lineup trials (N = 349; 8,376 lineup decisions). The rates of correct rejections and choosing (across both target-present and target-absent lineups) did not vary across the 24 trials, as reflected by main effects or interactions with trial number. Trial number had a significant but trivial quadratic effect on correct identifications (OR = 0.99) and interacted significantly, but again trivially, with disguise type (OR = 1.00). Trial number did not significantly influence participants' confidence in correct identifications, confidence in correct rejections, or confidence in target-absent selections. Thus, multiple-trial designs appear to have minimal effects on eyewitness accuracy, choosing, and confidence. Researchers should thus consider using multiple-trial designs for conducting eyewitness identification experiments.

The verbal facilitation effect: re-reading person descriptions as a system variable to improve identification performance.

PubMed

Sporer, Siegfried L; Kaminski, Kristina S; Davids, Maike C; McQuiston, Dawn

2016-11-01

When witnesses report a crime, police usually ask for a description of the perpetrator. Several studies suggested that verbalising faces leads to a detriment in identification performance (verbal overshadowing effect [VOE]) but the effect has been difficult to replicate. Here, we sought to reverse the VOE by inducing context reinstatement as a system variable through re-reading one's own description before an identification task. Participants (N = 208) watched a video film and were then dismissed (control group), only described the perpetrator, or described and later re-read their own descriptions before identification in either target-present or target-absent lineups after a 2-day or a 5-week delay. Identification accuracy was significantly higher after re-reading (85.0%) than in the no description control group (62.5%) irrespective of target presence. Data were internally replicated using a second target and corroborated by several small meta-analyses. Identification accuracy was related to description quality. Moreover, there was a tendency towards a verbal facilitation effect (VFE) rather than a VOE. Receiver operating characteristic (ROC) curve analyses confirm that our findings are not due to a shift in response bias but truly reflect improvement of recognition performance. Differences in the ecological validity of study paradigms are discussed.

Literature evidence in open targets - a target validation platform.

PubMed

Kafkas, Şenay; Dunham, Ian; McEntyre, Johanna

2017-06-06

We present the Europe PMC literature component of Open Targets - a target validation platform that integrates various evidence to aid drug target identification and validation. The component identifies target-disease associations in documents and ranks the documents based on their confidence from the Europe PMC literature database, by using rules utilising expert-provided heuristic information. The confidence score of a given document represents how valuable the document is in the scope of target validation for a given target-disease association by taking into account the credibility of the association based on the properties of the text. The component serves the platform regularly with the up-to-date data since December, 2015. Currently, there are a total number of 1168365 distinct target-disease associations text mined from >26 million PubMed abstracts and >1.2 million Open Access full text articles. Our comparative analyses on the current available evidence data in the platform revealed that 850179 of these associations are exclusively identified by literature mining. This component helps the platform's users by providing the most relevant literature hits for a given target and disease. The text mining evidence along with the other types of evidence can be explored visually through https://www.targetvalidation.org and all the evidence data is available for download in json format from https://www.targetvalidation.org/downloads/data .

Target identification of small molecules based on chemical biology approaches.

PubMed

Futamura, Yushi; Muroi, Makoto; Osada, Hiroyuki

2013-05-01

Recently, a phenotypic approach-screens that assess the effects of compounds on cells, tissues, or whole organisms-has been reconsidered and reintroduced as a complementary strategy of a target-based approach for drug discovery. Although the finding of novel bioactive compounds from large chemical libraries has become routine, the identification of their molecular targets is still a time-consuming and difficult process, making this step rate-limiting in drug development. In the last decade, we and other researchers have amassed a large amount of phenotypic data through progress in omics research and advances in instrumentation. Accordingly, the profiling methodologies using these datasets expertly have emerged to identify and validate specific molecular targets of drug candidates, attaining some progress in current drug discovery (e.g., eribulin). In the case of a compound that shows an unprecedented phenotype likely by inhibiting a first-in-class target, however, such phenotypic profiling is invalid. Under the circumstances, a photo-crosslinking affinity approach should be beneficial. In this review, we describe and summarize recent progress in both affinity-based (direct) and phenotypic profiling (indirect) approaches for chemical biology target identification.

Mapping sites of aspirin-induced acetylations in live cells by quantitative acid-cleavable activity-based protein profiling (QA-ABPP)

PubMed Central

Wang, Jigang; Zhang, Chong-Jing; Zhang, Jianbin; He, Yingke; Lee, Yew Mun; Chen, Songbi; Lim, Teck Kwang; Ng, Shukie; Shen, Han-Ming; Lin, Qingsong

2015-01-01

Target-identification and understanding of mechanism-of-action (MOA) are challenging for development of small-molecule probes and their application in biology and drug discovery. For example, although aspirin has been widely used for more than 100 years, its molecular targets have not been fully characterized. To cope with this challenge, we developed a novel technique called quantitative acid-cleavable activity-based protein profiling (QA-ABPP) with combination of the following two parts: (i) activity-based protein profiling (ABPP) and iTRAQ™ quantitative proteomics for identification of target proteins and (ii) acid-cleavable linker-based ABPP for identification of peptides with specific binding sites. It is known that reaction of aspirin with its target proteins leads to acetylation. We thus applied the above technique using aspirin-based probes in human cancer HCT116 cells. We identified 1110 target proteins and 2775 peptides with exact acetylation sites. By correlating these two sets of data, 523 proteins were identified as targets of aspirin. We used various biological assays to validate the effects of aspirin on inhibition of protein synthesis and induction of autophagy which were elicited from the pathway analysis of Aspirin target profile. This technique is widely applicable for target identification in the field of drug discovery and biology, especially for the covalent drugs. PMID:25600173

Detection of foliage-obscured vehicle using a multiwavelength polarimetric lidar

USGS Publications Warehouse

Tan, S.; Stoker, J.; Greenlee, S.

2008-01-01

Foliage obscured man-made targets detection and identification is of great interest to many applications. In this paper, the backscattered laser signals from a multiwavelength polarimetric lidar were used to detect a vehicle hidden inside a vegetated area. The Polarimetric reflectance data from the lidar at two separate laser wavelengths at 1064 nm and 532 nm revealed distinct target characteristics from both the vehicle and the vegetation. The results from this case study demonstrated the validity of the proposed lidar detection technique. Furthermore, the results could potentially lead to a lidar detection and identification technique for a wide variety of foliage-obscured man-made targets under various application scenarios. ?? 2007 IEEE.

Working memory enhances visual perception: evidence from signal detection analysis.

PubMed

Soto, David; Wriglesworth, Alice; Bahrami-Balani, Alex; Humphreys, Glyn W

2010-03-01

We show that perceptual sensitivity to visual stimuli can be modulated by matches between the contents of working memory (WM) and stimuli in the visual field. Observers were presented with an object cue (to hold in WM or to merely attend) and subsequently had to identify a brief target presented within a colored shape. The cue could be re-presented in the display, where it surrounded either the target (on valid trials) or a distractor (on invalid trials). Perceptual identification of the target, as indexed by A', was enhanced on valid relative to invalid trials but only when the cue was kept in WM. There was minimal effect of the cue when it was merely attended and not kept in WM. Verbal cues were as effective as visual cues at modulating perceptual identification, and the effects were independent of the effects of target saliency. Matches to the contents of WM influenced perceptual sensitivity even under conditions that minimized competition for selecting the target. WM cues were also effective when targets were less likely to fall in a repeated WM stimulus than in other stimuli in the search display. There were no effects of WM on decisional criteria, in contrast to sensitivity. The findings suggest that reentrant feedback from WM can affect early stages of perceptual processing.

Identification of Direct Target Genes Using Joint Sequence and Expression Likelihood with Application to DAF-16

PubMed Central

Yu, Ron X.; Liu, Jie; True, Nick; Wang, Wei

2008-01-01

A major challenge in the post-genome era is to reconstruct regulatory networks from the biological knowledge accumulated up to date. The development of tools for identifying direct target genes of transcription factors (TFs) is critical to this endeavor. Given a set of microarray experiments, a probabilistic model called TRANSMODIS has been developed which can infer the direct targets of a TF by integrating sequence motif, gene expression and ChIP-chip data. The performance of TRANSMODIS was first validated on a set of transcription factor perturbation experiments (TFPEs) involving Pho4p, a well studied TF in Saccharomyces cerevisiae. TRANSMODIS removed elements of arbitrariness in manual target gene selection process and produced results that concur with one's intuition. TRANSMODIS was further validated on a genome-wide scale by comparing it with two other methods in Saccharomyces cerevisiae. The usefulness of TRANSMODIS was then demonstrated by applying it to the identification of direct targets of DAF-16, a critical TF regulating ageing in Caenorhabditis elegans. We found that 189 genes were tightly regulated by DAF-16. In addition, DAF-16 has differential preference for motifs when acting as an activator or repressor, which awaits experimental verification. TRANSMODIS is computationally efficient and robust, making it a useful probabilistic framework for finding immediate targets. PMID:18350157

Early Probe and Drug Discovery in Academia: A Minireview.

PubMed

Roy, Anuradha

2018-02-09

Drug discovery encompasses processes ranging from target selection and validation to the selection of a development candidate. While comprehensive drug discovery work flows are implemented predominantly in the big pharma domain, early discovery focus in academia serves to identify probe molecules that can serve as tools to study targets or pathways. Despite differences in the ultimate goals of the private and academic sectors, the same basic principles define the best practices in early discovery research. A successful early discovery program is built on strong target definition and validation using a diverse set of biochemical and cell-based assays with functional relevance to the biological system being studied. The chemicals identified as hits undergo extensive scaffold optimization and are characterized for their target specificity and off-target effects in in vitro and in animal models. While the active compounds from screening campaigns pass through highly stringent chemical and Absorption, Distribution, Metabolism, and Excretion (ADME) filters for lead identification, the probe discovery involves limited medicinal chemistry optimization. The goal of probe discovery is identification of a compound with sub-µM activity and reasonable selectivity in the context of the target being studied. The compounds identified from probe discovery can also serve as starting scaffolds for lead optimization studies.

An enhanced targeted identification strategy for the selective identification of flavonoid O-glycosides from Carthamus tinctorius by integrating offline two-dimensional liquid chromatography/linear ion-trap-Orbitrap mass spectrometry, high-resolution diagnostic product ions/neutral loss filtering and liquid chromatography-solid phase extraction-nuclear magnetic resonance.

PubMed

Yao, Chang-Liang; Yang, Wen-Zhi; Si, Wei; Shen, Yao; Zhang, Nai-Xia; Chen, Hua-Li; Pan, Hui-Qin; Yang, Min; Wu, Wan-Ying; Guo, De-An

2017-03-31

Targeted identification of potentially bioactive molecules from herbal medicines is often stymied by the insufficient chromatographic separation, ubiquitous matrix interference, and pervasive isomerism. An enhanced targeted identification strategy is presented and validated by the selective identification of flavonoid O-glycosides (FOGs) from Carthamus tinctorius. It consists of four steps: (i) enhanced separation and detection by offline two-dimensional liquid chromatography/LTQ-Orbitrap MS (offline 2D-LC/LTQ-Orbitrap MS) using collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD); (ii) improved identification of the major aglycones by acid hydrolysis and LC-SPE-NMR; (iii) simplified spectral elucidation by high-resolution diagnostic product ions/neutral loss filtering; and (iv) more convincing structural identification by matching an in-house library. An offline 2D-LC system configuring an Acchrom XAmide column and a BEH Shield RP-18 UPLC ® column enabled much better separation of the easily co-eluting components. Combined use of CID and HCD could produce complementary fragmentation information. The intensity ratios of the aglycone ion species ([Y 0 -H] - /Y 0 - and [Y 0 -2H] - /Y 0 - ) in the HCD-MS 2 spectra were found diagnostic for discriminating the aglycone subtypes and characterizing the glycosylation patterns. Five aglycone structures (kaempferol, 6-hydroxykaempferol, 6-methoxykaempferol, carthamidin, and isocarthamidin) were identified based on the 1 H-NMR data recorded by LC-SPE-NMR. Of the 107 characterized flavonoids, 80 FOGs were first reported from C. tinctorius. Unknown aglycones, pentose, and novel acyl substituents were discovered. A new compound thereof was isolated and fully identified, which could partially validate the MS-oriented identification. This integral strategy can improve the potency, efficiency, and accuracy in the detection of new compounds from medicinal herbs and other natural sources. Copyright © 2017 Elsevier B.V. All rights reserved.

The identification of new protein kinase inhibitors as targets in modern drug discovery.

PubMed

Akritopoulou-Zanze, Irini

2006-07-01

In recent years there has been great interest in developing protein kinase inhibitors as therapeutic agents for a variety of diseases. This article provides an overview on the history, development and validity of kinases as drug targets, as well as a description of kinase research, including its limitations, challenges and successes.

Targeting Conserved Genes in Penicillium Species.

PubMed

Peterson, Stephen W

2017-01-01

Polymerase chain reaction amplification of conserved genes and sequence analysis provides a very powerful tool for the identification of toxigenic as well as non-toxigenic Penicillium species. Sequences are obtained by amplification of the gene fragment, sequencing via capillary electrophoresis of dideoxynucleotide-labeled fragments or NGS. The sequences are compared to a database of validated isolates. Identification of species indicates the potential of the fungus to make particular mycotoxins.

Development and validation of real-time PCR tests for the identification of four Spodoptera species: Spodoptera eridania, Spodoptera frugiperda, Spodoptera littoralis, and Spodoptera litura (Lepidoptera: Noctuidae).

PubMed

Van de Vossenberg, B T L H; Van der Straten, M J

2014-08-01

The genus Spodoptera comprises 31 species, 4 of which are listed as quarantine pests for the European Union: Spodoptera eridania (Cramer), Spodoptera frugiperda (Smith), Spodoptera littoralis (Boisduval), and Spodoptera litura (F.). In international trade, the earlier life stages (eggs and larvae) are being intercepted at point of inspection most frequently, challenging the possibilities of morphological identification. To realize a rapid and reliable identification for all stages, we developed and validated four simplex real-time polymerase chain reaction identification tests based on the mitochondrial cytochrome b gene using dual-labeled hydrolysis probes. Method validation on dilutions of extracted DNA of the target organisms showed that low levels of template (up to 0.2-100 pg) can reliably be identified. No cross-reactivity was observed with 14 nontarget Spodoptera and 5 non-Spodoptera species in the specific Spodoptera tests. The tests showed to be repeatable, reproducible (both 100%), and robust. The new Spodoptera tests have proven to be suitable tools for routine identification of all life stages of S. eridania, S. frugiperda, S. littoralis, and S. litura.

2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification.

PubMed

Herner, András; Marjanovic, Jasmina; Lewandowski, Tracey M; Marin, Violeta; Patterson, Melanie; Miesbauer, Laura; Ready, Damien; Williams, Jon; Vasudevan, Anil; Lin, Qing

2016-11-09

Photoaffinity labels are powerful tools for dissecting ligand-protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C-H/X-H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery.

2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification

PubMed Central

2016-01-01

Photoaffinity labels are powerful tools for dissecting ligand–protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C–H/X–H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery. PMID:27740749

Plant microRNA-Target Interaction Identification Model Based on the Integration of Prediction Tools and Support Vector Machine

PubMed Central

Meng, Jun; Shi, Lin; Luan, Yushi

2014-01-01

Background Confident identification of microRNA-target interactions is significant for studying the function of microRNA (miRNA). Although some computational miRNA target prediction methods have been proposed for plants, results of various methods tend to be inconsistent and usually lead to more false positive. To address these issues, we developed an integrated model for identifying plant miRNA–target interactions. Results Three online miRNA target prediction toolkits and machine learning algorithms were integrated to identify and analyze Arabidopsis thaliana miRNA-target interactions. Principle component analysis (PCA) feature extraction and self-training technology were introduced to improve the performance. Results showed that the proposed model outperformed the previously existing methods. The results were validated by using degradome sequencing supported Arabidopsis thaliana miRNA-target interactions. The proposed model constructed on Arabidopsis thaliana was run over Oryza sativa and Vitis vinifera to demonstrate that our model is effective for other plant species. Conclusions The integrated model of online predictors and local PCA-SVM classifier gained credible and high quality miRNA-target interactions. The supervised learning algorithm of PCA-SVM classifier was employed in plant miRNA target identification for the first time. Its performance can be substantially improved if more experimentally proved training samples are provided. PMID:25051153

Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

PubMed

Vasaikar, Suhas; Bhatia, Pooja; Bhatia, Partap G; Chu Yaiw, Koon

2016-11-21

In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

Validating models of target acquisition performance in the dismounted soldier context

NASA Astrophysics Data System (ADS)

Glaholt, Mackenzie G.; Wong, Rachel K.; Hollands, Justin G.

2018-04-01

The problem of predicting real-world operator performance with digital imaging devices is of great interest within the military and commercial domains. There are several approaches to this problem, including: field trials with imaging devices, laboratory experiments using imagery captured from these devices, and models that predict human performance based on imaging device parameters. The modeling approach is desirable, as both field trials and laboratory experiments are costly and time-consuming. However, the data from these experiments is required for model validation. Here we considered this problem in the context of dismounted soldiering, for which detection and identification of human targets are essential tasks. Human performance data were obtained for two-alternative detection and identification decisions in a laboratory experiment in which photographs of human targets were presented on a computer monitor and the images were digitally magnified to simulate range-to-target. We then compared the predictions of different performance models within the NV-IPM software package: Targeting Task Performance (TTP) metric model and the Johnson model. We also introduced a modification to the TTP metric computation that incorporates an additional correction for target angular size. We examined model predictions using NV-IPM default values for a critical model constant, V50, and we also considered predictions when this value was optimized to fit the behavioral data. When using default values, certain model versions produced a reasonably close fit to the human performance data in the detection task, while for the identification task all models substantially overestimated performance. When using fitted V50 values the models produced improved predictions, though the slopes of the performance functions were still shallow compared to the behavioral data. These findings are discussed in relation to the models' designs and parameters, and the characteristics of the behavioral paradigm.

Genome-Wide Identification of CBX2 Targets: Insights in the Human Sex Development Network

PubMed Central

Eid, Wassim; Opitz, Lennart

2015-01-01

Chromobox homolog 2 (CBX2) is a chromatin modifier that plays an important role in sexual development and its disorders (disorders of sex development [DSD]), yet the exact rank and function of human CBX2 in this pathway remains unclear. Here, we performed large-scale mapping and analysis of in vivo target loci of the protein CBX2 in Sertoli-like NT-2D1 cells, using the DNA adenine methyltransferase identification technique. We identified close to 1600 direct targets for CBX2. Intriguingly, validation of selected candidate genes using qRT-PCR in cells overexpressing CBX2 or in which CBX2 has been knocked down indicated that several CBX2-responsive genes encode proteins that are involved in DSD. We further validated these effects on the candidate genes using a mutated CBX2 causing DSD in human patient. Overall, our findings suggest that CBX2 role in the sex development cascade is to stimulate the male pathway and concurrently inhibit the female pathway. These data provide fundamental insights into potential etiology of DSD. PMID:25569159

A comparison of machine learning techniques for detection of drug target articles.

PubMed

Danger, Roxana; Segura-Bedmar, Isabel; Martínez, Paloma; Rosso, Paolo

2010-12-01

Important progress in treating diseases has been possible thanks to the identification of drug targets. Drug targets are the molecular structures whose abnormal activity, associated to a disease, can be modified by drugs, improving the health of patients. Pharmaceutical industry needs to give priority to their identification and validation in order to reduce the long and costly drug development times. In the last two decades, our knowledge about drugs, their mechanisms of action and drug targets has rapidly increased. Nevertheless, most of this knowledge is hidden in millions of medical articles and textbooks. Extracting knowledge from this large amount of unstructured information is a laborious job, even for human experts. Drug target articles identification, a crucial first step toward the automatic extraction of information from texts, constitutes the aim of this paper. A comparison of several machine learning techniques has been performed in order to obtain a satisfactory classifier for detecting drug target articles using semantic information from biomedical resources such as the Unified Medical Language System. The best result has been achieved by a Fuzzy Lattice Reasoning classifier, which reaches 98% of ROC area measure. Copyright © 2010 Elsevier Inc. All rights reserved.

Large-Scale Chemical Similarity Networks for Target Profiling of Compounds Identified in Cell-Based Chemical Screens

PubMed Central

Lo, Yu-Chen; Senese, Silvia; Li, Chien-Ming; Hu, Qiyang; Huang, Yong; Damoiseaux, Robert; Torres, Jorge Z.

2015-01-01

Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60–70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/). PMID:25826798

Bridging the gap to therapeutic strategies based on connexin/pannexin biology.

PubMed

Naus, Christian C; Giaume, Christian

2016-11-29

A unique workshop was recently held focusing on enhancing collaborations leading to identify and update the development of therapeutic strategies targeting connexin/pannexin large pore channels. Basic scientists exploring the functions of these channels in various pathologies gathered together with leading pharma companies which are targeting gap junction proteins for specific therapeutic applications. This highlights how paths of discovery research can converge with therapeutic strategies in innovative ways to enhance target identification and validation.

Identification and Expression Analyses of miRNAs from Two Contrasting Flower Color Cultivars of Canna by Deep Sequencing.

PubMed

Roy, Sribash; Tripathi, Abhinandan Mani; Yadav, Amrita; Mishra, Parneeta; Nautiyal, Chandra Shekhar

2016-01-01

miRNAs are endogenous small RNA (sRNA) that play critical roles in plant development processes. Canna is an ornamental plant belonging to family Cannaceae. Here, we report for the first time the identification and differential expression of miRNAs in two contrasting flower color cultivars of Canna, Tropical sunrise and Red president. A total of 313 known miRNAs belonging to 78 miRNA families were identified from both the cultivars. Thirty one miRNAs (17 miRNA families) were specific to Tropical sunrise and 43 miRNAs (10 miRNA families) were specific to Red president. Thirty two and 18 putative new miRNAs were identified from Tropical sunrise and Red president, respectively. One hundred and nine miRNAs were differentially expressed in the two cultivars targeting 1343 genes. Among these, 16 miRNAs families targeting 60 genes were involved in flower development related traits and five miRNA families targeting five genes were involved in phenyl propanoid and pigment metabolic processes. We further validated the expression analysis of a few miRNA and their target genes by qRT-PCR. Transcription factors were the major miRNA targets identified. Target validation of a few randomly selected miRNAs by RLM-RACE was performed but was successful with only miR162. These findings will help in understanding flower development processes, particularly the color development in Canna.

Experiment research on infrared targets signature in mid and long IR spectral bands

NASA Astrophysics Data System (ADS)

Wang, Chensheng; Hong, Pu; Lei, Bo; Yue, Song; Zhang, Zhijie; Ren, Tingting

2013-09-01

Since the infrared imaging system has played a significant role in the military self-defense system and fire control system, the radiation signature of IR target becomes an important topic in IR imaging application technology. IR target signature can be applied in target identification, especially for small and dim targets, as well as the target IR thermal design. To research and analyze the targets IR signature systematically, a practical and experimental project is processed under different backgrounds and conditions. An infrared radiation acquisition system based on a MWIR cooled thermal imager and a LWIR cooled thermal imager is developed to capture the digital infrared images. Furthermore, some instruments are introduced to provide other parameters. According to the original image data and the related parameters in a certain scene, the IR signature of interested target scene can be calculated. Different background and targets are measured with this approach, and a comparison experiment analysis shall be presented in this paper as an example. This practical experiment has proved the validation of this research work, and it is useful in detection performance evaluation and further target identification research.

Selection, application, and validation of a set of molecular descriptors for nuclear receptor ligands.

PubMed

Stewart, Eugene L; Brown, Peter J; Bentley, James A; Willson, Timothy M

2004-08-01

A methodology for the selection and validation of nuclear receptor ligand chemical descriptors is described. After descriptors for a targeted chemical space were selected, a virtual screening methodology utilizing this space was formulated for the identification of potential NR ligands from our corporate collection. Using simple descriptors and our virtual screening method, we are able to quickly identify potential NR ligands from a large collection of compounds. As validation of the virtual screening procedure, an 8, 000-membered NR targeted set and a 24, 000-membered diverse control set of compounds were selected from our in-house general screening collection and screened in parallel across a number of orphan NR FRET assays. For the two assays that provided at least one hit per set by the established minimum pEC(50) for activity, the results showed a 2-fold increase in the hit-rate of the targeted compound set over the diverse set.

A Pathway to Freedom: An Evaluation of Screening Tools for the Identification of Trafficking Victims.

PubMed

Bespalova, Nadejda; Morgan, Juliet; Coverdale, John

2016-02-01

Because training residents and faculty to identify human trafficking victims is a major public health priority, the authors review existing assessment tools. PubMed and Google were searched using combinations of search terms including human, trafficking, sex, labor, screening, identification, and tool. Nine screening tools that met the inclusion criteria were found. They varied greatly in length, format, target demographic, supporting resources, and other parameters. Only two tools were designed specifically for healthcare providers. Only one tool was formally assessed to be valid and reliable in a pilot project in trafficking victim service organizations, although it has not been validated in the healthcare setting. This toolbox should facilitate the education of resident physicians and faculty in screening for trafficking victims, assist educators in assessing screening skills, and promote future research on the identification of trafficking victims.

A kinase-focused compound collection: compilation and screening strategy.

PubMed

Sun, Dongyu; Chuaqui, Claudio; Deng, Zhan; Bowes, Scott; Chin, Donovan; Singh, Juswinder; Cullen, Patrick; Hankins, Gretchen; Lee, Wen-Cherng; Donnelly, Jason; Friedman, Jessica; Josiah, Serene

2006-06-01

Lead identification by high-throughput screening of large compound libraries has been supplemented with virtual screening and focused compound libraries. To complement existing approaches for lead identification at Biogen Idec, a kinase-focused compound collection was designed, developed and validated. Two strategies were adopted to populate the compound collection: a ligand shape-based virtual screening and a receptor-based approach (structural interaction fingerprint). Compounds selected with the two approaches were cherry-picked from an existing high-throughput screening compound library, ordered from suppliers and supplemented with specific medicinal compounds from internal programs. Promising hits and leads have been generated from the kinase-focused compound collection against multiple kinase targets. The principle of the collection design and screening strategy was validated and the use of the kinase-focused compound collection for lead identification has been added to existing strategies.

Automatic identification of variables in epidemiological datasets using logic regression.

PubMed

Lorenz, Matthias W; Abdi, Negin Ashtiani; Scheckenbach, Frank; Pflug, Anja; Bülbül, Alpaslan; Catapano, Alberico L; Agewall, Stefan; Ezhov, Marat; Bots, Michiel L; Kiechl, Stefan; Orth, Andreas

2017-04-13

For an individual participant data (IPD) meta-analysis, multiple datasets must be transformed in a consistent format, e.g. using uniform variable names. When large numbers of datasets have to be processed, this can be a time-consuming and error-prone task. Automated or semi-automated identification of variables can help to reduce the workload and improve the data quality. For semi-automation high sensitivity in the recognition of matching variables is particularly important, because it allows creating software which for a target variable presents a choice of source variables, from which a user can choose the matching one, with only low risk of having missed a correct source variable. For each variable in a set of target variables, a number of simple rules were manually created. With logic regression, an optimal Boolean combination of these rules was searched for every target variable, using a random subset of a large database of epidemiological and clinical cohort data (construction subset). In a second subset of this database (validation subset), this optimal combination rules were validated. In the construction sample, 41 target variables were allocated on average with a positive predictive value (PPV) of 34%, and a negative predictive value (NPV) of 95%. In the validation sample, PPV was 33%, whereas NPV remained at 94%. In the construction sample, PPV was 50% or less in 63% of all variables, in the validation sample in 71% of all variables. We demonstrated that the application of logic regression in a complex data management task in large epidemiological IPD meta-analyses is feasible. However, the performance of the algorithm is poor, which may require backup strategies.

Employing machine learning for reliable miRNA target identification in plants

PubMed Central

2011-01-01

Background miRNAs are ~21 nucleotide long small noncoding RNA molecules, formed endogenously in most of the eukaryotes, which mainly control their target genes post transcriptionally by interacting and silencing them. While a lot of tools has been developed for animal miRNA target system, plant miRNA target identification system has witnessed limited development. Most of them have been centered around exact complementarity match. Very few of them considered other factors like multiple target sites and role of flanking regions. Result In the present work, a Support Vector Regression (SVR) approach has been implemented for plant miRNA target identification, utilizing position specific dinucleotide density variation information around the target sites, to yield highly reliable result. It has been named as p-TAREF (plant-Target Refiner). Performance comparison for p-TAREF was done with other prediction tools for plants with utmost rigor and where p-TAREF was found better performing in several aspects. Further, p-TAREF was run over the experimentally validated miRNA targets from species like Arabidopsis, Medicago, Rice and Tomato, and detected them accurately, suggesting gross usability of p-TAREF for plant species. Using p-TAREF, target identification was done for the complete Rice transcriptome, supported by expression and degradome based data. miR156 was found as an important component of the Rice regulatory system, where control of genes associated with growth and transcription looked predominant. The entire methodology has been implemented in a multi-threaded parallel architecture in Java, to enable fast processing for web-server version as well as standalone version. This also makes it to run even on a simple desktop computer in concurrent mode. It also provides a facility to gather experimental support for predictions made, through on the spot expression data analysis, in its web-server version. Conclusion A machine learning multivariate feature tool has been implemented in parallel and locally installable form, for plant miRNA target identification. The performance was assessed and compared through comprehensive testing and benchmarking, suggesting a reliable performance and gross usability for transcriptome wide plant miRNA target identification. PMID:22206472

Employing machine learning for reliable miRNA target identification in plants.

PubMed

Jha, Ashwani; Shankar, Ravi

2011-12-29

miRNAs are ~21 nucleotide long small noncoding RNA molecules, formed endogenously in most of the eukaryotes, which mainly control their target genes post transcriptionally by interacting and silencing them. While a lot of tools has been developed for animal miRNA target system, plant miRNA target identification system has witnessed limited development. Most of them have been centered around exact complementarity match. Very few of them considered other factors like multiple target sites and role of flanking regions. In the present work, a Support Vector Regression (SVR) approach has been implemented for plant miRNA target identification, utilizing position specific dinucleotide density variation information around the target sites, to yield highly reliable result. It has been named as p-TAREF (plant-Target Refiner). Performance comparison for p-TAREF was done with other prediction tools for plants with utmost rigor and where p-TAREF was found better performing in several aspects. Further, p-TAREF was run over the experimentally validated miRNA targets from species like Arabidopsis, Medicago, Rice and Tomato, and detected them accurately, suggesting gross usability of p-TAREF for plant species. Using p-TAREF, target identification was done for the complete Rice transcriptome, supported by expression and degradome based data. miR156 was found as an important component of the Rice regulatory system, where control of genes associated with growth and transcription looked predominant. The entire methodology has been implemented in a multi-threaded parallel architecture in Java, to enable fast processing for web-server version as well as standalone version. This also makes it to run even on a simple desktop computer in concurrent mode. It also provides a facility to gather experimental support for predictions made, through on the spot expression data analysis, in its web-server version. A machine learning multivariate feature tool has been implemented in parallel and locally installable form, for plant miRNA target identification. The performance was assessed and compared through comprehensive testing and benchmarking, suggesting a reliable performance and gross usability for transcriptome wide plant miRNA target identification.

Activity-based proteome profiling of potential cellular targets of Orlistat--an FDA-approved drug with anti-tumor activities.

PubMed

Yang, Peng-Yu; Liu, Kai; Ngai, Mun Hong; Lear, Martin J; Wenk, Markus R; Yao, Shao Q

2010-01-20

Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental THL structure to maintain the native biological properties of Orlistat, while providing the necessary functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike, cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis. Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers of their biological targets.

Target Acquisition: Human Observer Performance Studies and TARGAC Model Validation

DTIC Science & Technology

1994-06-01

complete Scenario I run were displayed on a monitor. A transparent sheet was attached to the face of the monitor, the approach route was sketched on...track, passed a mark on the monitor face , the target was at a stop-sign location. The IRIG-B time of that instance was noted, and, since the distance of...route right 100- 80- 60 - 40 C.) 20 [- ion, A20 recognitio 0 0 . identification,C.) . 1I . I - - -• , 4) 1000 2000 3000 4000 SP Target G Q Target I0

EOID Model Validation and Performance Prediction

DTIC Science & Technology

2002-09-30

Our long-term goal is to accurately predict the capability of the current generation of laser-based underwater imaging sensors to perform Electro ... Optic Identification (EOID) against relevant targets in a variety of realistic environmental conditions. The two most prominent technologies in this area

Identification and validation nucleolin as a target of curcumol in nasopharyngeal carcinoma cells.

PubMed

Wang, Juan; Wu, Jiacai; Li, Xumei; Liu, Haowei; Qin, Jianli; Bai, Zhun; Chi, Bixia; Chen, Xu

2018-06-30

Identification of the specific protein target(s) of a drug is a critical step in unraveling its mechanisms of action (MOA) in many natural products. Curcumol, isolated from well known Chinese medicinal plant Curcuma zedoary, has been shown to possess multiple biological activities. It can inhibit nasopharyngeal carcinoma (NPC) proliferation and induce apoptosis, but its target protein(s) in NPC cells remains unclear. In this study, we employed a mass spectrometry-based chemical proteomics approach reveal the possible protein targets of curcumol in NPC cells. Cellular thermal shift assay (CETSA), molecular docking and cell-based assay was used to validate the binding interactions. Chemical proteomics capturing uncovered that NCL is a target of curcumol in NPC cells, Molecular docking showed that curcumol bound to NCL with an -7.8 kcal/mol binding free energy. Cell function analysis found that curcumol's treatment leads to a degradation of NCL in NPC cells, and it showed slight effects on NP69 cells. In conclusion, our results providing evidences that NCL is a target protein of curcumol. We revealed that the anti-cancer effects of curcumol in NPC cells are mediated, at least in part, by NCL inhibition. Many natural products showed high bioactivity, while their mechanisms of action (MOA) are very poor or completely missed. Understanding the MOA of natural drugs can thoroughly exploit their therapeutic potential and minimize their adverse side effects. Identification of the specific protein target(s) of a drug is a critical step in unraveling its MOA. Compound-centric chemical proteomics is a classic chemical proteomics approach which integrates chemical synthesis with cell biology and mass spectrometry (MS) to identify protein targets of natural products determine the drug mechanism of action, describe its toxicity, and figure out the possible cause of off-target. It is an affinity-based chemical proteomics method to identify small molecule-protein interactions through affinity chromatography approach coupled with mass spectrometry, has been conventionally used to identify target proteins and has yielded good results. Curcumol, has shown effective inhibition on Nasopharyngeal Carcinoma (NPC) Cells, interacted with NCL and then initiated the anti-tumor biological effect. This research demonstrated the effectiveness of chemical proteomics approaches in natural drugs molecular target identification, revealing and understanding of the novel mechanism of actions of curcumol is crucial for cancer prevention and treatment in nasopharynx cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

miRTar2GO: a novel rule-based model learning method for cell line specific microRNA target prediction that integrates Ago2 CLIP-Seq and validated microRNA-target interaction data.

PubMed

Ahadi, Alireza; Sablok, Gaurav; Hutvagner, Gyorgy

2017-04-07

MicroRNAs (miRNAs) are ∼19-22 nucleotides (nt) long regulatory RNAs that regulate gene expression by recognizing and binding to complementary sequences on mRNAs. The key step in revealing the function of a miRNA, is the identification of miRNA target genes. Recent biochemical advances including PAR-CLIP and HITS-CLIP allow for improved miRNA target predictions and are widely used to validate miRNA targets. Here, we present miRTar2GO, which is a model, trained on the common rules of miRNA-target interactions, Argonaute (Ago) CLIP-Seq data and experimentally validated miRNA target interactions. miRTar2GO is designed to predict miRNA target sites using more relaxed miRNA-target binding characteristics. More importantly, miRTar2GO allows for the prediction of cell-type specific miRNA targets. We have evaluated miRTar2GO against other widely used miRNA target prediction algorithms and demonstrated that miRTar2GO produced significantly higher F1 and G scores. Target predictions, binding specifications, results of the pathway analysis and gene ontology enrichment of miRNA targets are freely available at http://www.mirtar2go.org. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of a computer-assisted forensic radiographic identification method using the lateral cervical and lumbar spine.

PubMed

Derrick, Sharon M; Raxter, Michelle H; Hipp, John A; Goel, Priya; Chan, Elaine F; Love, Jennifer C; Wiersema, Jason M; Akella, N Shastry

2015-01-01

Medical examiners and coroners (ME/C) in the United States hold statutory responsibility to identify deceased individuals who fall under their jurisdiction. The computer-assisted decedent identification (CADI) project was designed to modify software used in diagnosis and treatment of spinal injuries into a mathematically validated tool for ME/C identification of fleshed decedents. CADI software analyzes the shapes of targeted vertebral bodies imaged in an array of standard radiographs and quantifies the likelihood that any two of the radiographs contain matching vertebral bodies. Six validation tests measured the repeatability, reliability, and sensitivity of the method, and the effects of age, sex, and number of radiographs in array composition. CADI returned a 92-100% success rate in identifying the true matching pair of vertebrae within arrays of five to 30 radiographs. Further development of CADI is expected to produce a novel identification method for use in ME/C offices that is reliable, timely, and cost-effective. © 2014 American Academy of Forensic Sciences.

Drug Discovery for Neglected Diseases: Molecular Target-Based and Phenotypic Approaches

PubMed Central

2013-01-01

Drug discovery for neglected tropical diseases is carried out using both target-based and phenotypic approaches. In this paper, target-based approaches are discussed, with a particular focus on human African trypanosomiasis. Target-based drug discovery can be successful, but careful selection of targets is required. There are still very few fully validated drug targets in neglected diseases, and there is a high attrition rate in target-based drug discovery for these diseases. Phenotypic screening is a powerful method in both neglected and non-neglected diseases and has been very successfully used. Identification of molecular targets from phenotypic approaches can be a way to identify potential new drug targets. PMID:24015767

EOID System Model Validation, Metrics, and Synthetic Clutter Generation

DTIC Science & Technology

2003-09-30

Our long-term goal is to accurately predict the capability of the current generation of laser-based underwater imaging sensors to perform Electro ... Optic Identification (EOID) against relevant targets in a variety of realistic environmental conditions. The models will predict the impact of

Refining the accuracy of validated target identification through coding variant fine-mapping in type 2 diabetes.

PubMed

Mahajan, Anubha; Wessel, Jennifer; Willems, Sara M; Zhao, Wei; Robertson, Neil R; Chu, Audrey Y; Gan, Wei; Kitajima, Hidetoshi; Taliun, Daniel; Rayner, N William; Guo, Xiuqing; Lu, Yingchang; Li, Man; Jensen, Richard A; Hu, Yao; Huo, Shaofeng; Lohman, Kurt K; Zhang, Weihua; Cook, James P; Prins, Bram Peter; Flannick, Jason; Grarup, Niels; Trubetskoy, Vassily Vladimirovich; Kravic, Jasmina; Kim, Young Jin; Rybin, Denis V; Yaghootkar, Hanieh; Müller-Nurasyid, Martina; Meidtner, Karina; Li-Gao, Ruifang; Varga, Tibor V; Marten, Jonathan; Li, Jin; Smith, Albert Vernon; An, Ping; Ligthart, Symen; Gustafsson, Stefan; Malerba, Giovanni; Demirkan, Ayse; Tajes, Juan Fernandez; Steinthorsdottir, Valgerdur; Wuttke, Matthias; Lecoeur, Cécile; Preuss, Michael; Bielak, Lawrence F; Graff, Marielisa; Highland, Heather M; Justice, Anne E; Liu, Dajiang J; Marouli, Eirini; Peloso, Gina Marie; Warren, Helen R; Afaq, Saima; Afzal, Shoaib; Ahlqvist, Emma; Almgren, Peter; Amin, Najaf; Bang, Lia B; Bertoni, Alain G; Bombieri, Cristina; Bork-Jensen, Jette; Brandslund, Ivan; Brody, Jennifer A; Burtt, Noël P; Canouil, Mickaël; Chen, Yii-Der Ida; Cho, Yoon Shin; Christensen, Cramer; Eastwood, Sophie V; Eckardt, Kai-Uwe; Fischer, Krista; Gambaro, Giovanni; Giedraitis, Vilmantas; Grove, Megan L; de Haan, Hugoline G; Hackinger, Sophie; Hai, Yang; Han, Sohee; Tybjærg-Hansen, Anne; Hivert, Marie-France; Isomaa, Bo; Jäger, Susanne; Jørgensen, Marit E; Jørgensen, Torben; Käräjämäki, Annemari; Kim, Bong-Jo; Kim, Sung Soo; Koistinen, Heikki A; Kovacs, Peter; Kriebel, Jennifer; Kronenberg, Florian; Läll, Kristi; Lange, Leslie A; Lee, Jung-Jin; Lehne, Benjamin; Li, Huaixing; Lin, Keng-Hung; Linneberg, Allan; Liu, Ching-Ti; Liu, Jun; Loh, Marie; Mägi, Reedik; Mamakou, Vasiliki; McKean-Cowdin, Roberta; Nadkarni, Girish; Neville, Matt; Nielsen, Sune F; Ntalla, Ioanna; Peyser, Patricia A; Rathmann, Wolfgang; Rice, Kenneth; Rich, Stephen S; Rode, Line; Rolandsson, Olov; Schönherr, Sebastian; Selvin, Elizabeth; Small, Kerrin S; Stančáková, Alena; Surendran, Praveen; Taylor, Kent D; Teslovich, Tanya M; Thorand, Barbara; Thorleifsson, Gudmar; Tin, Adrienne; Tönjes, Anke; Varbo, Anette; Witte, Daniel R; Wood, Andrew R; Yajnik, Pranav; Yao, Jie; Yengo, Loïc; Young, Robin; Amouyel, Philippe; Boeing, Heiner; Boerwinkle, Eric; Bottinger, Erwin P; Chowdhury, Rajiv; Collins, Francis S; Dedoussis, George; Dehghan, Abbas; Deloukas, Panos; Ferrario, Marco M; Ferrières, Jean; Florez, Jose C; Frossard, Philippe; Gudnason, Vilmundur; Harris, Tamara B; Heckbert, Susan R; Howson, Joanna M M; Ingelsson, Martin; Kathiresan, Sekar; Kee, Frank; Kuusisto, Johanna; Langenberg, Claudia; Launer, Lenore J; Lindgren, Cecilia M; Männistö, Satu; Meitinger, Thomas; Melander, Olle; Mohlke, Karen L; Moitry, Marie; Morris, Andrew D; Murray, Alison D; de Mutsert, Renée; Orho-Melander, Marju; Owen, Katharine R; Perola, Markus; Peters, Annette; Province, Michael A; Rasheed, Asif; Ridker, Paul M; Rivadineira, Fernando; Rosendaal, Frits R; Rosengren, Anders H; Salomaa, Veikko; Sheu, Wayne H-H; Sladek, Rob; Smith, Blair H; Strauch, Konstantin; Uitterlinden, André G; Varma, Rohit; Willer, Cristen J; Blüher, Matthias; Butterworth, Adam S; Chambers, John Campbell; Chasman, Daniel I; Danesh, John; van Duijn, Cornelia; Dupuis, Josée; Franco, Oscar H; Franks, Paul W; Froguel, Philippe; Grallert, Harald; Groop, Leif; Han, Bok-Ghee; Hansen, Torben; Hattersley, Andrew T; Hayward, Caroline; Ingelsson, Erik; Kardia, Sharon L R; Karpe, Fredrik; Kooner, Jaspal Singh; Köttgen, Anna; Kuulasmaa, Kari; Laakso, Markku; Lin, Xu; Lind, Lars; Liu, Yongmei; Loos, Ruth J F; Marchini, Jonathan; Metspalu, Andres; Mook-Kanamori, Dennis; Nordestgaard, Børge G; Palmer, Colin N A; Pankow, James S; Pedersen, Oluf; Psaty, Bruce M; Rauramaa, Rainer; Sattar, Naveed; Schulze, Matthias B; Soranzo, Nicole; Spector, Timothy D; Stefansson, Kari; Stumvoll, Michael; Thorsteinsdottir, Unnur; Tuomi, Tiinamaija; Tuomilehto, Jaakko; Wareham, Nicholas J; Wilson, James G; Zeggini, Eleftheria; Scott, Robert A; Barroso, Inês; Frayling, Timothy M; Goodarzi, Mark O; Meigs, James B; Boehnke, Michael; Saleheen, Danish; Morris, Andrew P; Rotter, Jerome I; McCarthy, Mark I

2018-04-01

We aggregated coding variant data for 81,412 type 2 diabetes cases and 370,832 controls of diverse ancestry, identifying 40 coding variant association signals (P < 2.2 × 10 -7 ); of these, 16 map outside known risk-associated loci. We make two important observations. First, only five of these signals are driven by low-frequency variants: even for these, effect sizes are modest (odds ratio ≤1.29). Second, when we used large-scale genome-wide association data to fine-map the associated variants in their regional context, accounting for the global enrichment of complex trait associations in coding sequence, compelling evidence for coding variant causality was obtained for only 16 signals. At 13 others, the associated coding variants clearly represent 'false leads' with potential to generate erroneous mechanistic inference. Coding variant associations offer a direct route to biological insight for complex diseases and identification of validated therapeutic targets; however, appropriate mechanistic inference requires careful specification of their causal contribution to disease predisposition.

Compressed domain ECG biometric with two-lead features

NASA Astrophysics Data System (ADS)

Lee, Wan-Jou; Chang, Wen-Whei

2016-07-01

This study presents a new method to combine ECG biometrics with data compression within a common JPEG2000 framework. We target the two-lead ECG configuration that is routinely used in long-term heart monitoring. Incorporation of compressed-domain biometric techniques enables faster person identification as it by-passes the full decompression. Experiments on public ECG databases demonstrate the validity of the proposed method for biometric identification with high accuracies on both healthy and diseased subjects.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

PubMed

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-05-06

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification

PubMed Central

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-01-01

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines. PMID:27150638

Identification of control targets in Boolean molecular network models via computational algebra.

PubMed

Murrugarra, David; Veliz-Cuba, Alan; Aguilar, Boris; Laubenbacher, Reinhard

2016-09-23

Many problems in biomedicine and other areas of the life sciences can be characterized as control problems, with the goal of finding strategies to change a disease or otherwise undesirable state of a biological system into another, more desirable, state through an intervention, such as a drug or other therapeutic treatment. The identification of such strategies is typically based on a mathematical model of the process to be altered through targeted control inputs. This paper focuses on processes at the molecular level that determine the state of an individual cell, involving signaling or gene regulation. The mathematical model type considered is that of Boolean networks. The potential control targets can be represented by a set of nodes and edges that can be manipulated to produce a desired effect on the system. This paper presents a method for the identification of potential intervention targets in Boolean molecular network models using algebraic techniques. The approach exploits an algebraic representation of Boolean networks to encode the control candidates in the network wiring diagram as the solutions of a system of polynomials equations, and then uses computational algebra techniques to find such controllers. The control methods in this paper are validated through the identification of combinatorial interventions in the signaling pathways of previously reported control targets in two well studied systems, a p53-mdm2 network and a blood T cell lymphocyte granular leukemia survival signaling network. Supplementary data is available online and our code in Macaulay2 and Matlab are available via http://www.ms.uky.edu/~dmu228/ControlAlg . This paper presents a novel method for the identification of intervention targets in Boolean network models. The results in this paper show that the proposed methods are useful and efficient for moderately large networks.

Biased and unbiased strategies to identify biologically active small molecules.

PubMed

Abet, Valentina; Mariani, Angelica; Truscott, Fiona R; Britton, Sébastien; Rodriguez, Raphaël

2014-08-15

Small molecules are central players in chemical biology studies. They promote the perturbation of cellular processes underlying diseases and enable the identification of biological targets that can be validated for therapeutic intervention. Small molecules have been shown to accurately tune a single function of pluripotent proteins in a reversible manner with exceptional temporal resolution. The identification of molecular probes and drugs remains a worthy challenge that can be addressed by the use of biased and unbiased strategies. Hypothesis-driven methodologies employs a known biological target to synthesize complementary hits while discovery-driven strategies offer the additional means of identifying previously unanticipated biological targets. This review article provides a general overview of recent synthetic frameworks that gave rise to an impressive arsenal of biologically active small molecules with unprecedented cellular mechanisms. Copyright © 2014. Published by Elsevier Ltd.

Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies

PubMed Central

Euskirchen, Ghia M.; Rozowsky, Joel S.; Wei, Chia-Lin; Lee, Wah Heng; Zhang, Zhengdong D.; Hartman, Stephen; Emanuelsson, Olof; Stolc, Viktor; Weissman, Sherman; Gerstein, Mark B.; Ruan, Yijun; Snyder, Michael

2007-01-01

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides ≥50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%–86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies. PMID:17568005

Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV)

PubMed Central

Bonavia, Aurelio; Franti, Michael; Pusateri Keaney, Erin; Kuhen, Kelli; Seepersaud, Mohindra; Radetich, Branko; Shao, Jian; Honda, Ayako; Dewhurst, Janetta; Balabanis, Kara; Monroe, James; Wolff, Karen; Osborne, Colin; Lanieri, Leanne; Hoffmaster, Keith; Amin, Jakal; Markovits, Judit; Broome, Michelle; Skuba, Elizabeth; Cornella-Taracido, Ivan; Joberty, Gerard; Bouwmeester, Tewis; Hamann, Lawrence; Tallarico, John A.; Tommasi, Ruben; Compton, Teresa; Bushell, Simon M.

2011-01-01

The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action. PMID:21502533

Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV).

PubMed

Bonavia, Aurelio; Franti, Michael; Pusateri Keaney, Erin; Kuhen, Kelli; Seepersaud, Mohindra; Radetich, Branko; Shao, Jian; Honda, Ayako; Dewhurst, Janetta; Balabanis, Kara; Monroe, James; Wolff, Karen; Osborne, Colin; Lanieri, Leanne; Hoffmaster, Keith; Amin, Jakal; Markovits, Judit; Broome, Michelle; Skuba, Elizabeth; Cornella-Taracido, Ivan; Joberty, Gerard; Bouwmeester, Tewis; Hamann, Lawrence; Tallarico, John A; Tommasi, Ruben; Compton, Teresa; Bushell, Simon M

2011-04-26

The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.

Comprehensive analytical strategy for biomonitoring of pesticides in urine by liquid chromatography–orbitrap high resolution masss pectrometry.

PubMed

Roca, M; Leon, N; Pastor, A; Yusà, V

2014-12-29

In this study we propose an analytical strategy that combines a target approach for the quantitative analysis of contemporary pesticide metabolites with a comprehensive post-target screening for the identification of biomarkers of exposure to environmental contaminants in urine using liquid chromatography coupled to high-resolution mass spectrometry (LC–HRMS). The quantitative method for the target analysis of 29 urinary metabolites of organophosphate (OP) insecticides, synthetic pyrethroids, herbicides and fungicides was validated after a previous statistical optimization of the main factors governing the ion source ionization and a fragmentation study using the high energy collision dissociation (HCD) cell. The full scan accurate mass data were acquired with a resolving power of 50,000 FWHM (scan speed, 2 Hz), in both ESI+ and ESI− modes, and with and without HCD-fragmentation. The method – LOQ was lower than 3.2 μg L−1 for the majority of the analytes. For post-target screening a customized theoretical database was built, for the identification of 60 metabolites including pesticides, PAHs, phenols, and other metabolites of environmental pollutants. For identification purposes, accurate exact mass with less than 5 ppm, and diagnostic ions including isotopes and/or fragments were used. The analytical strategy was applied to 20 urine sample collected from children living in Valencia Region. Eleven target metabolites were detected with concentrations ranging from 1.18 to 131 μg L−1. Likewise, several compounds were tentatively identified in the post-target analysis belonging to the families of phthalates, phenols and parabenes. The proposed strategy is suitable for the determination of target pesticide biomarkers in urine in the framework of biomonitoring studies, and appropriate for the identification of other non-target metabolites.

Colour-cueing in visual search.

PubMed

Laarni, J

2001-02-01

Several studies have shown that people can selectively attend to stimulus colour, e.g., in visual search, and that preknowledge of a target colour can improve response speed/accuracy. The purpose was to use a form-identification task to determine whether valid colour precues can produce benefits and invalid cues costs. The subject had to identify the orientation of a "T"-shaped element in a ring of randomly-oriented "L"s when either two or four of the elements were differently coloured. Contrary to Moore and Egeth's (1998) recent findings, colour-based attention did affect performance under data-limited conditions: Colour cues produced benefits when processing load was high; when the load was reduced, they incurred only costs. Surprisingly, a valid colour cue succeeded in improving performance in the high-load condition even when its validity was reduced to the chance level. Overall, the results suggest that knowledge of a target colour does not facilitate the processing of the target, but makes it possible to prioritize it.

Molecular identification of python species: development and validation of a novel assay for forensic investigations.

PubMed

Ciavaglia, Sherryn A; Tobe, Shanan S; Donnellan, Stephen C; Henry, Julianne M; Linacre, Adrian M T

2015-05-01

Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

PubMed

Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

2014-01-01

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

NASA Astrophysics Data System (ADS)

Wright, Megan H.; Clough, Barbara; Rackham, Mark D.; Rangachari, Kaveri; Brannigan, James A.; Grainger, Munira; Moss, David K.; Bottrill, Andrew R.; Heal, William P.; Broncel, Malgorzata; Serwa, Remigiusz A.; Brady, Declan; Mann, David J.; Leatherbarrow, Robin J.; Tewari, Rita; Wilkinson, Anthony J.; Holder, Anthony A.; Tate, Edward W.

2014-02-01

Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase.

Hierarchical virtual screening for the discovery of new molecular scaffolds in antibacterial hit identification

PubMed Central

Ballester, Pedro J.; Mangold, Martina; Howard, Nigel I.; Robinson, Richard L. Marchese; Abell, Chris; Blumberger, Jochen; Mitchell, John B. O.

2012-01-01

One of the initial steps of modern drug discovery is the identification of small organic molecules able to inhibit a target macromolecule of therapeutic interest. A small proportion of these hits are further developed into lead compounds, which in turn may ultimately lead to a marketed drug. A commonly used screening protocol used for this task is high-throughput screening (HTS). However, the performance of HTS against antibacterial targets has generally been unsatisfactory, with high costs and low rates of hit identification. Here, we present a novel computational methodology that is able to identify a high proportion of structurally diverse inhibitors by searching unusually large molecular databases in a time-, cost- and resource-efficient manner. This virtual screening methodology was tested prospectively on two versions of an antibacterial target (type II dehydroquinase from Mycobacterium tuberculosis and Streptomyces coelicolor), for which HTS has not provided satisfactory results and consequently practically all known inhibitors are derivatives of the same core scaffold. Overall, our protocols identified 100 new inhibitors, with calculated Ki ranging from 4 to 250 μM (confirmed hit rates are 60% and 62% against each version of the target). Most importantly, over 50 new active molecular scaffolds were discovered that underscore the benefits that a wide application of prospectively validated in silico screening tools is likely to bring to antibacterial hit identification. PMID:22933186

Hierarchical virtual screening for the discovery of new molecular scaffolds in antibacterial hit identification.

PubMed

Ballester, Pedro J; Mangold, Martina; Howard, Nigel I; Robinson, Richard L Marchese; Abell, Chris; Blumberger, Jochen; Mitchell, John B O

2012-12-07

One of the initial steps of modern drug discovery is the identification of small organic molecules able to inhibit a target macromolecule of therapeutic interest. A small proportion of these hits are further developed into lead compounds, which in turn may ultimately lead to a marketed drug. A commonly used screening protocol used for this task is high-throughput screening (HTS). However, the performance of HTS against antibacterial targets has generally been unsatisfactory, with high costs and low rates of hit identification. Here, we present a novel computational methodology that is able to identify a high proportion of structurally diverse inhibitors by searching unusually large molecular databases in a time-, cost- and resource-efficient manner. This virtual screening methodology was tested prospectively on two versions of an antibacterial target (type II dehydroquinase from Mycobacterium tuberculosis and Streptomyces coelicolor), for which HTS has not provided satisfactory results and consequently practically all known inhibitors are derivatives of the same core scaffold. Overall, our protocols identified 100 new inhibitors, with calculated K(i) ranging from 4 to 250 μM (confirmed hit rates are 60% and 62% against each version of the target). Most importantly, over 50 new active molecular scaffolds were discovered that underscore the benefits that a wide application of prospectively validated in silico screening tools is likely to bring to antibacterial hit identification.

Clinically Detectable Dental Identifiers Observed in Intra-oral Photographs and Extra-oral Radiographs, Validated for Human Identification Purposes.

PubMed

Angelakopoulos, Nikolaos; Franco, Ademir; Willems, Guy; Fieuws, Steffen; Thevissen, Patrick

2017-07-01

Screening the prevalence and pattern of dental identifiers contributes toward the process of human identification. This research investigated the uniqueness of clinical dental identifiers in photographs and radiographs. Panoramic and lateral cephalometric radiographs and five intra-oral photographs of 1727 subjects were used. In a target set, two observers examined different subjects. In a subset, both observers examined the same subjects (source set). The distance between source and target subjects was quantified for each identifier. The percentage of subjects in the target set being at least as close as the correct subject was assessed. The number of molars (34.6%), missing teeth (42%), and displaced teeth (59.9%) were the most unique identifiers in photographs and panoramic and lateral cephalometric radiographs, respectively. The pattern of rotated teeth (14.9%) was the most unique in photographs, while displaced teeth was in panoramic (37.6%) and lateral cephalometric (54.8%) radiographs. Morphological identifiers were the most unique, highlighting their importance for human identifications. © 2016 American Academy of Forensic Sciences.

Drug target identification using network analysis: Taking active components in Sini decoction as an example

NASA Astrophysics Data System (ADS)

Chen, Si; Jiang, Hailong; Cao, Yan; Wang, Yun; Hu, Ziheng; Zhu, Zhenyu; Chai, Yifeng

2016-04-01

Identifying the molecular targets for the beneficial effects of active small-molecule compounds simultaneously is an important and currently unmet challenge. In this study, we firstly proposed network analysis by integrating data from network pharmacology and metabolomics to identify targets of active components in sini decoction (SND) simultaneously against heart failure. To begin with, 48 potential active components in SND against heart failure were predicted by serum pharmacochemistry, text mining and similarity match. Then, we employed network pharmacology including text mining and molecular docking to identify the potential targets of these components. The key enriched processes, pathways and related diseases of these target proteins were analyzed by STRING database. At last, network analysis was conducted to identify most possible targets of components in SND. Among the 25 targets predicted by network analysis, tumor necrosis factor α (TNF-α) was firstly experimentally validated in molecular and cellular level. Results indicated that hypaconitine, mesaconitine, higenamine and quercetin in SND can directly bind to TNF-α, reduce the TNF-α-mediated cytotoxicity on L929 cells and exert anti-myocardial cell apoptosis effects. We envisage that network analysis will also be useful in target identification of a bioactive compound.

Drug target identification using network analysis: Taking active components in Sini decoction as an example

PubMed Central

Chen, Si; Jiang, Hailong; Cao, Yan; Wang, Yun; Hu, Ziheng; Zhu, Zhenyu; Chai, Yifeng

2016-01-01

Identifying the molecular targets for the beneficial effects of active small-molecule compounds simultaneously is an important and currently unmet challenge. In this study, we firstly proposed network analysis by integrating data from network pharmacology and metabolomics to identify targets of active components in sini decoction (SND) simultaneously against heart failure. To begin with, 48 potential active components in SND against heart failure were predicted by serum pharmacochemistry, text mining and similarity match. Then, we employed network pharmacology including text mining and molecular docking to identify the potential targets of these components. The key enriched processes, pathways and related diseases of these target proteins were analyzed by STRING database. At last, network analysis was conducted to identify most possible targets of components in SND. Among the 25 targets predicted by network analysis, tumor necrosis factor α (TNF-α) was firstly experimentally validated in molecular and cellular level. Results indicated that hypaconitine, mesaconitine, higenamine and quercetin in SND can directly bind to TNF-α, reduce the TNF-α-mediated cytotoxicity on L929 cells and exert anti-myocardial cell apoptosis effects. We envisage that network analysis will also be useful in target identification of a bioactive compound. PMID:27095146

Exploring the Process of Energy Generation in Pathophysiology by Targeted Metabolomics: Performance of a Simple and Quantitative Method.

PubMed

Riera-Borrull, Marta; Rodríguez-Gallego, Esther; Hernández-Aguilera, Anna; Luciano, Fedra; Ras, Rosa; Cuyàs, Elisabet; Camps, Jordi; Segura-Carretero, Antonio; Menendez, Javier A; Joven, Jorge; Fernández-Arroyo, Salvador

2016-01-01

Abnormalities in mitochondrial metabolism and regulation of energy balance contribute to human diseases. The consequences of high fat and other nutrient intake, and the resulting acquired mitochondrial dysfunction, are essential to fully understand common disorders, including obesity, cancer, and atherosclerosis. To simultaneously and noninvasively measure and quantify indirect markers of mitochondrial function, we have developed a method based on gas chromatography coupled to quadrupole-time of flight mass spectrometry and an electron ionization interface, and validated the system using plasma from patients with peripheral artery disease, human cancer cells, and mouse tissues. This approach was used to increase sensibility in the measurement of a wide dynamic range and chemical diversity of multiple intermediate metabolites used in energy metabolism. We demonstrate that our targeted metabolomics method allows for quick and accurate identification and quantification of molecules, including the measurement of small yet significant biological changes in experimental samples. The apparently low process variability required for its performance in plasma, cell lysates, and tissues allowed a rapid identification of correlations between interconnected pathways. Our results suggest that delineating the process of energy generation by targeted metabolomics can be a valid surrogate for predicting mitochondrial dysfunction in biological samples. Importantly, when used in plasma, targeted metabolomics should be viewed as a robust and noninvasive source of biomarkers in specific pathophysiological scenarios.

Exploring the Process of Energy Generation in Pathophysiology by Targeted Metabolomics: Performance of a Simple and Quantitative Method

NASA Astrophysics Data System (ADS)

Riera-Borrull, Marta; Rodríguez-Gallego, Esther; Hernández-Aguilera, Anna; Luciano, Fedra; Ras, Rosa; Cuyàs, Elisabet; Camps, Jordi; Segura-Carretero, Antonio; Menendez, Javier A.; Joven, Jorge; Fernández-Arroyo, Salvador

2016-01-01

Abnormalities in mitochondrial metabolism and regulation of energy balance contribute to human diseases. The consequences of high fat and other nutrient intake, and the resulting acquired mitochondrial dysfunction, are essential to fully understand common disorders, including obesity, cancer, and atherosclerosis. To simultaneously and noninvasively measure and quantify indirect markers of mitochondrial function, we have developed a method based on gas chromatography coupled to quadrupole-time of flight mass spectrometry and an electron ionization interface, and validated the system using plasma from patients with peripheral artery disease, human cancer cells, and mouse tissues. This approach was used to increase sensibility in the measurement of a wide dynamic range and chemical diversity of multiple intermediate metabolites used in energy metabolism. We demonstrate that our targeted metabolomics method allows for quick and accurate identification and quantification of molecules, including the measurement of small yet significant biological changes in experimental samples. The apparently low process variability required for its performance in plasma, cell lysates, and tissues allowed a rapid identification of correlations between interconnected pathways. Our results suggest that delineating the process of energy generation by targeted metabolomics can be a valid surrogate for predicting mitochondrial dysfunction in biological samples. Importantly, when used in plasma, targeted metabolomics should be viewed as a robust and noninvasive source of biomarkers in specific pathophysiological scenarios.

Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing.

PubMed

Liu, Gary W; Livesay, Brynn R; Kacherovsky, Nataly A; Cieslewicz, Maryelise; Lutz, Emi; Waalkes, Adam; Jensen, Michael C; Salipante, Stephen J; Pun, Suzie H

2015-08-19

Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying "parasitic sequences" and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.

Validity of the mockwitness paradigm: testing the assumptions.

PubMed

McQuiston, Dawn E; Malpass, Roy S

2002-08-01

Mockwitness identifications are used to provide a quantitative measure of lineup fairness. Some theoretical and practical assumptions of this paradigm have not been studied in terms of mockwitnesses' decision processes and procedural variation (e.g., instructions, lineup presentation method), and the current experiment was conducted to empirically evaluate these assumptions. Four hundred and eighty mockwitnesses were given physical information about a culprit, received 1 of 4 variations of lineup instructions, and were asked to identify the culprit from either a fair or unfair sequential lineup containing 1 of 2 targets. Lineup bias estimates varied as a result of lineup fairness and the target presented. Mockwitnesses generally reported that the target's physical description was their main source of identifying information. Our findings support the use of mockwitness identifications as a useful technique for sequential lineup evaluation, but only for mockwitnesses who selected only 1 lineup member. Recommendations for the use of this evaluation procedure are discussed.

Indel-seq: a fast-forward genetics approach for identification of trait-associated putative candidate genomic regions and its application in pigeonpea (Cajanus cajan).

PubMed

Singh, Vikas K; Khan, Aamir W; Saxena, Rachit K; Sinha, Pallavi; Kale, Sandip M; Parupalli, Swathi; Kumar, Vinay; Chitikineni, Annapurna; Vechalapu, Suryanarayana; Sameer Kumar, Chanda Venkata; Sharma, Mamta; Ghanta, Anuradha; Yamini, Kalinati Narasimhan; Muniswamy, Sonnappa; Varshney, Rajeev K

2017-07-01

Identification of candidate genomic regions associated with target traits using conventional mapping methods is challenging and time-consuming. In recent years, a number of single nucleotide polymorphism (SNP)-based mapping approaches have been developed and used for identification of candidate/putative genomic regions. However, in the majority of these studies, insertion-deletion (Indel) were largely ignored. For efficient use of Indels in mapping target traits, we propose Indel-seq approach, which is a combination of whole-genome resequencing (WGRS) and bulked segregant analysis (BSA) and relies on the Indel frequencies in extreme bulks. Deployment of Indel-seq approach for identification of candidate genomic regions associated with fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonpea has identified 16 Indels affecting 26 putative candidate genes. Of these 26 affected putative candidate genes, 24 genes showed effect in the upstream/downstream of the genic region and two genes showed effect in the genes. Validation of these 16 candidate Indels in other FW- and SMD-resistant and FW- and SMD-susceptible genotypes revealed a significant association of five Indels (three for FW and two for SMD resistance). Comparative analysis of Indel-seq with other genetic mapping approaches highlighted the importance of the approach in identification of significant genomic regions associated with target traits. Therefore, the Indel-seq approach can be used for quick and precise identification of candidate genomic regions for any target traits in any crop species. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

IQs Are Very Strong but Imperfect Indicators of Psychometric "g": Results from Joint Confirmatory Factor Analysis

ERIC Educational Resources Information Center

Farmer, Ryan L.; Floyd, Randy G.; Reynolds, Matthew R.; Kranzler, John H.

2014-01-01

The most global score yielded by intelligence tests, IQs, are supported by substantial validity evidence and have historically been central to the identification of intellectual disabilities, learning disabilities, and giftedness. This study examined the extent to which IQs measure the ability they target, psychometric "g." Data from…

Photoaffinity labeling in target- and binding-site identification

PubMed Central

Smith, Ewan; Collins, Ian

2015-01-01

Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas. PMID:25686004

First siRNA library screening in hard-to-transfect HUVEC cells

PubMed Central

Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole UE; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra BS; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

2010-01-01

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection. PMID:20628494

Real-Time Gas Identification by Analyzing the Transient Response of Capillary-Attached Conductive Gas Sensor

PubMed Central

Bahraminejad, Behzad; Basri, Shahnor; Isa, Maryam; Hambli, Zarida

2010-01-01

In this study, the ability of the Capillary-attached conductive gas sensor (CGS) in real-time gas identification was investigated. The structure of the prototype fabricated CGS is presented. Portions were selected from the beginning of the CGS transient response including the first 11 samples to the first 100 samples. Different feature extraction and classification methods were applied on the selected portions. Validation of methods was evaluated to study the ability of an early portion of the CGS transient response in target gas (TG) identification. Experimental results proved that applying extracted features from an early part of the CGS transient response along with a classifier can distinguish short-chain alcohols from each other perfectly. Decreasing time of exposition in the interaction between target gas and sensing element improved the reliability of the sensor. Classification rate was also improved and time of identification was decreased. Moreover, the results indicated the optimum interval of the early transient response of the CGS for selecting portions to achieve the best classification rates. PMID:22219666

Targeting Thromboxane A2 Receptor for Anti-Metastasis Therapy of Breast Cancer

DTIC Science & Technology

2011-09-01

of cell function by Rho GTPases." Drug News Perspect 14(7): 389-95. Erickson, J. W., R. A. Cerione, et al. (1997). "Identification of an actin...Focusing Tumor Microenvironment, Stem Cells and Metastasis 570 (MTOC) and Golgi apparatus to the front of the nucleus, oriented toward the direction of...define the function of TP in tumor cell motility and to validate TP as a target for anti-metastasis therapy of breast cancer. In the first aim, the

Dana-Farber Cancer Institute: Identification of Therapeutic Targets Across Cancer Types | Office of Cancer Genomics

Cancer.gov

The Dana Farber Cancer Institute CTD2 Center focuses on the use of high-throughput genetic and bioinformatic approaches to identify and credential oncogenes and co-dependencies in cancers. This Center aims to provide the cancer research community with information that will facilitate the prioritization of targets based on both genomic and functional evidence, inform the most appropriate genetic context for downstream mechanistic and validation studies, and enable the translation of this information into therapeutics and diagnostics.

TargetMiner: microRNA target prediction with systematic identification of tissue-specific negative examples.

PubMed

Bandyopadhyay, Sanghamitra; Mitra, Ramkrishna

2009-10-15

Prediction of microRNA (miRNA) target mRNAs using machine learning approaches is an important area of research. However, most of the methods suffer from either high false positive or false negative rates. One reason for this is the marked deficiency of negative examples or miRNA non-target pairs. Systematic identification of non-target mRNAs is still not addressed properly, and therefore, current machine learning approaches are compelled to rely on artificially generated negative examples for training. In this article, we have identified approximately 300 tissue-specific negative examples using a novel approach that involves expression profiling of both miRNAs and mRNAs, miRNA-mRNA structural interactions and seed-site conservation. The newly generated negative examples are validated with pSILAC dataset, which elucidate the fact that the identified non-targets are indeed non-targets.These high-throughput tissue-specific negative examples and a set of experimentally verified positive examples are then used to build a system called TargetMiner, a support vector machine (SVM)-based classifier. In addition to assessing the prediction accuracy on cross-validation experiments, TargetMiner has been validated with a completely independent experimental test dataset. Our method outperforms 10 existing target prediction algorithms and provides a good balance between sensitivity and specificity that is not reflected in the existing methods. We achieve a significantly higher sensitivity and specificity of 69% and 67.8% based on a pool of 90 feature set and 76.5% and 66.1% using a set of 30 selected feature set on the completely independent test dataset. In order to establish the effectiveness of the systematically generated negative examples, the SVM is trained using a different set of negative data generated using the method in Yousef et al. A significantly higher false positive rate (70.6%) is observed when tested on the independent set, while all other factors are kept the same. Again, when an existing method (NBmiRTar) is executed with the our proposed negative data, we observe an improvement in its performance. These clearly establish the effectiveness of the proposed approach of selecting the negative examples systematically. TargetMiner is now available as an online tool at www.isical.ac.in/ approximately bioinfo_miu

Computational-experimental approach to drug-target interaction mapping: A case study on kinase inhibitors

PubMed Central

Ravikumar, Balaguru; Parri, Elina; Timonen, Sanna; Airola, Antti; Wennerberg, Krister

2017-01-01

Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001) between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel-based modeling approach offers practical benefits for probing novel insights into the mode of action of investigational compounds, and for the identification of new target selectivities for drug repurposing applications. PMID:28787438

Simultaneous analysis of coccidiostats and sulfonamides in non-target feed by HPLC-MS/MS and validation following the Commission Decision 2002/657/EC.

PubMed

Gavilán, Rosa Elvira; Nebot, Carolina; Patyra, Ewelina; Miranda, Jose Manuel; Franco, Carlos Manuel; Cepeda, Alberto

2018-05-02

Taking into consideration the maximum level for coccidiostats included in the European Regulation 574/2011 and the fact that the presence of residues of sulfonamides in non-target feed is forbidden, the aim of this article is to present an analytical method based on HPLC-MS/MS for the identification and quantification of sulfonamides and coccidiostats in non-target feeds. The method was validated following Decision 2002/657/EC and recovery, repeatability, and reproducibility were within the limits stablished in the Decision. For coccidiostats, the decision limit and detection capability were calculated for the different species taking into account the maximum level allowed in Regulation 574/2011. The applicability of the method was investigated in 50 feed samples collected from dairy farms, 50 obtained from feed mills, and 10 interlaboratory feed samples.

Identification and Targeting of Candidate Pre-Existing Lurker Cells that Give Rise to Castration-Resistant Prostate Cancer

DTIC Science & Technology

2015-10-01

information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE October 2015 2...cells, inflammatory signaling and prostate cancer. Canary Center at Stanford for Cancer Early Detection, Palo Alto, CA. 9 Goldstein AS. (2014

A Global Genomic and Genetic Strategy to Identify, Validate and Use Gene Signatures of Xenobiotic-Responsive Transcription Factors in Prediction of Pathway Activation in the Mouse Liver

EPA Science Inventory

Many drugs and environmentally-relevant chemicals activate xenobiotic-responsive transcription factors. Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening as well as their involvement in disease states. ...

Identification of active sources inside cavities using the equivalent source method-based free-field recovery technique

NASA Astrophysics Data System (ADS)

Bi, Chuan-Xing; Hu, Ding-Yu; Zhang, Yong-Bin; Jing, Wen-Qian

2015-06-01

In previous studies, an equivalent source method (ESM)-based technique for recovering the free sound field in a noisy environment has been successfully applied to exterior problems. In order to evaluate its performance when applied to a more general noisy environment, that technique is used to identify active sources inside cavities where the sound field is composed of the field radiated by active sources and that reflected by walls. A patch approach with two semi-closed surfaces covering the target active sources is presented to perform the measurements, and the field that would be radiated by these target active sources into free space is extracted from the mixed field by using the proposed technique, which will be further used as the input of nearfield acoustic holography for source identification. Simulation and experimental results validate the effectiveness of the proposed technique for source identification in cavities, and show the feasibility of performing the measurements with a double layer planar array.

Evaluating the effect of database inflation in proteogenomic search on sensitive and reliable peptide identification.

PubMed

Li, Honglan; Joh, Yoon Sung; Kim, Hyunwoo; Paek, Eunok; Lee, Sang-Won; Hwang, Kyu-Baek

2016-12-22

Proteogenomics is a promising approach for various tasks ranging from gene annotation to cancer research. Databases for proteogenomic searches are often constructed by adding peptide sequences inferred from genomic or transcriptomic evidence to reference protein sequences. Such inflation of databases has potential of identifying novel peptides. However, it also raises concerns on sensitive and reliable peptide identification. Spurious peptides included in target databases may result in underestimated false discovery rate (FDR). On the other hand, inflation of decoy databases could decrease the sensitivity of peptide identification due to the increased number of high-scoring random hits. Although several studies have addressed these issues, widely applicable guidelines for sensitive and reliable proteogenomic search have hardly been available. To systematically evaluate the effect of database inflation in proteogenomic searches, we constructed a variety of real and simulated proteogenomic databases for yeast and human tandem mass spectrometry (MS/MS) data, respectively. Against these databases, we tested two popular database search tools with various approaches to search result validation: the target-decoy search strategy (with and without a refined scoring-metric) and a mixture model-based method. The effect of separate filtering of known and novel peptides was also examined. The results from real and simulated proteogenomic searches confirmed that separate filtering increases the sensitivity and reliability in proteogenomic search. However, no one method consistently identified the largest (or the smallest) number of novel peptides from real proteogenomic searches. We propose to use a set of search result validation methods with separate filtering, for sensitive and reliable identification of peptides in proteogenomic search.

Identification and Validation of Ifit1 as an Important Innate Immune Bottleneck

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDermott, Jason E.; Vartanian, Keri B.; Mitchell, Hugh D.

The innate immune system plays important roles in a number of disparate processes. Foremost, innate immunity is a first responder to invasion by pathogens and triggers early defensive responses and recruits the adaptive immune system. The innate immune system also responds to endogenous damage signals that arise from tissue injury. Recently it has been found that innate immunity plays an important role in neuroprotection against ischemic stroke through the activation of the primary innate immune receptors, Toll-like receptors (TLRs). Using several large-scale transcriptomic data sets from mouse and mouse macrophage studies we identified targets predicted to be important in controllingmore » innate immune processes initiated by TLR activation. Targets were identified as genes with high betweenness centrality, so-called bottlenecks, in networks inferred from statistical associations between gene expression patterns. A small set of putative bottlenecks were identified in each of the data sets investigated including interferon-stimulated genes (Ifit1, Ifi47, Tgtp and Oasl2) as well as genes uncharacterized in immune responses (Axud1 and Ppp1r15a). We further validated one of these targets, Ifit1, in mouse macrophages by showing that silencing it suppresses induction of predicted downstream genes by lipopolysaccharide (LPS)-mediated TLR4 activation through an unknown direct or indirect mechanism. Our study demonstrates the utility of network analysis for identification of interesting targets related to innate immune function, and highlights that Ifit1 can exert a positive regulatory effect on downstream genes.« less

Advances towards the design and development of personalized non-small-cell lung cancer drug therapy.

PubMed

Vari, Sabrina; Pilotto, Sara; Maugeri-Saccà, Marcello; Ciuffreda, Ludovica; Cesta Incani, Ursula; Falcone, Italia; Del Curatolo, Anais; Ceribelli, Anna; Gelibter, Alain; De Maria, Ruggero; Tortora, Giampaolo; Cognetti, Francesco; Bria, Emilio; Milella, Michele

2013-11-01

Non-small-cell lung cancer (NSCLC) subtypes are driven by specific genetic aberrations. For reasons such as this, there is a call for treatment personalization. The ability to instigate NSCLC fragmentation poses new methodological problems, and new 'driver' molecular aberrations are being discovered at an unprecedented pace. This article describes the clinical development of epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKIs) and crizotinib for EGFR-mutant and anaplastic lymphoma kinase (ALK)-rearranged NSCLC. Further, the authors briefly describe the emerging molecular targets in NSCLC, in terms of both rationale for therapeutic targeting and strategies, for clinical development. Target identification and validation in NSCLC still requires considerable effort, as not all of the molecular alterations are clear 'drivers' nor can they be efficiently targeted with available drugs. However, 50% of the NSCLC cases are without clear-defined molecular aberrations. Clinical trial methodology will need to develop novel paradigms for targeted drug development, aiming at the validation of an ideal 'biology-to-trial' approach. Despite significant challenges, a truly 'personalized' approach to NSCLC therapy appears to be within our reach.

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

PubMed

Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

2013-08-01

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

Approaches to the identification of new nonhormonal targets for male contraception.

PubMed

Kopf, Gregory S

2008-10-01

Fertility control is a global health issue with major personal and societal impact. Although, currently, there are several different options for contraception, the technologies behind these are antiquated, and the options for male-based contraception (i.e., withdrawal, condom and vasectomy) are inadequate. The genomic, proteomic and bioinformatic revolutions have provided new tools and new targets for contraceptive development, and the results of such approaches have identified gene products that play critical roles in male reproduction, thus expanding the array of potential targets for novel and innovative male-based contraceptives. This article will review the types of targets being considered in the development of nonhormonal male contraceptives and the technologies used to identify and validate these targets.

Biophysics: for HTS hit validation, chemical lead optimization, and beyond.

PubMed

Genick, Christine C; Wright, S Kirk

2017-09-01

There are many challenges to the drug discovery process, including the complexity of the target, its interactions, and how these factors play a role in causing the disease. Traditionally, biophysics has been used for hit validation and chemical lead optimization. With its increased throughput and sensitivity, biophysics is now being applied earlier in this process to empower target characterization and hit finding. Areas covered: In this article, the authors provide an overview of how biophysics can be utilized to assess the quality of the reagents used in screening assays, to validate potential tool compounds, to test the integrity of screening assays, and to create follow-up strategies for compound characterization. They also briefly discuss the utilization of different biophysical methods in hit validation to help avoid the resource consuming pitfalls caused by the lack of hit overlap between biophysical methods. Expert opinion: The use of biophysics early on in the drug discovery process has proven crucial to identifying and characterizing targets of complex nature. It also has enabled the identification and classification of small molecules which interact in an allosteric or covalent manner with the target. By applying biophysics in this manner and at the early stages of this process, the chances of finding chemical leads with novel mechanisms of action are increased. In the future, focused screens with biophysics as a primary readout will become increasingly common.

In silico analysis and experimental validation of azelastine hydrochloride (N4) targeting sodium taurocholate co-transporting polypeptide (NTCP) in HBV therapy.

PubMed

Fu, L-L; Liu, J; Chen, Y; Wang, F-T; Wen, X; Liu, H-Q; Wang, M-Y; Ouyang, L; Huang, J; Bao, J-K; Wei, Y-Q

2014-08-01

The aim of this study was to explore sodium taurocholate co-transporting polypeptide (NTCP) exerting its function with hepatitis B virus (HBV) and its targeted candidate compounds, in HBV therapy. Identification of NTCP as a novel HBV target for screening candidate small molecules, was used by phylogenetic analysis, network construction, molecular modelling, molecular docking and molecular dynamics (MD) simulation. In vitro virological examination, q-PCR, western blotting and cytotoxicity studies were used for validating efficacy of the candidate compound. We used the phylogenetic analysis of NTCP and constructed its protein-protein network. Also, we screened compounds from Drugbank and ZINC, among which five were validated for their authentication in HepG 2.2.15 cells. Then, we selected compound N4 (azelastine hydrochloride) as the most potent of them. This showed good inhibitory activity against HBsAg (IC50 = 7.5 μm) and HBeAg (IC50 = 3.7 μm), as well as high SI value (SI = 4.68). Further MD simulation results supported good interaction between compound N4 and NTCP. In silico analysis and experimental validation together demonstrated that compound N4 can target NTCP in HepG2.2.15 cells, which may shed light on exploring it as a potential anti-HBV drug. © 2014 John Wiley & Sons Ltd.

A Statistics-based Platform for Quantitative N-terminome Analysis and Identification of Protease Cleavage Products*

PubMed Central

auf dem Keller, Ulrich; Prudova, Anna; Gioia, Magda; Butler, Georgina S.; Overall, Christopher M.

2010-01-01

Terminal amine isotopic labeling of substrates (TAILS), our recently introduced platform for quantitative N-terminome analysis, enables wide dynamic range identification of original mature protein N-termini and protease cleavage products. Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode. The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF). Furthermore, the isoform assignment score (IAS) is introduced, a new scoring system for the evaluation of single peptide-to-protein assignments based on high confidence protein identifications in the same sample prior to negative selection enrichment of N-terminal peptides. By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed. PMID:20305283

Improved False Discovery Rate Estimation Procedure for Shotgun Proteomics.

PubMed

Keich, Uri; Kertesz-Farkas, Attila; Noble, William Stafford

2015-08-07

Interpreting the potentially vast number of hypotheses generated by a shotgun proteomics experiment requires a valid and accurate procedure for assigning statistical confidence estimates to identified tandem mass spectra. Despite the crucial role such procedures play in most high-throughput proteomics experiments, the scientific literature has not reached a consensus about the best confidence estimation methodology. In this work, we evaluate, using theoretical and empirical analysis, four previously proposed protocols for estimating the false discovery rate (FDR) associated with a set of identified tandem mass spectra: two variants of the target-decoy competition protocol (TDC) of Elias and Gygi and two variants of the separate target-decoy search protocol of Käll et al. Our analysis reveals significant biases in the two separate target-decoy search protocols. Moreover, the one TDC protocol that provides an unbiased FDR estimate among the target PSMs does so at the cost of forfeiting a random subset of high-scoring spectrum identifications. We therefore propose the mix-max procedure to provide unbiased, accurate FDR estimates in the presence of well-calibrated scores. The method avoids biases associated with the two separate target-decoy search protocols and also avoids the propensity for target-decoy competition to discard a random subset of high-scoring target identifications.

Improved False Discovery Rate Estimation Procedure for Shotgun Proteomics

PubMed Central

2016-01-01

Interpreting the potentially vast number of hypotheses generated by a shotgun proteomics experiment requires a valid and accurate procedure for assigning statistical confidence estimates to identified tandem mass spectra. Despite the crucial role such procedures play in most high-throughput proteomics experiments, the scientific literature has not reached a consensus about the best confidence estimation methodology. In this work, we evaluate, using theoretical and empirical analysis, four previously proposed protocols for estimating the false discovery rate (FDR) associated with a set of identified tandem mass spectra: two variants of the target-decoy competition protocol (TDC) of Elias and Gygi and two variants of the separate target-decoy search protocol of Käll et al. Our analysis reveals significant biases in the two separate target-decoy search protocols. Moreover, the one TDC protocol that provides an unbiased FDR estimate among the target PSMs does so at the cost of forfeiting a random subset of high-scoring spectrum identifications. We therefore propose the mix-max procedure to provide unbiased, accurate FDR estimates in the presence of well-calibrated scores. The method avoids biases associated with the two separate target-decoy search protocols and also avoids the propensity for target-decoy competition to discard a random subset of high-scoring target identifications. PMID:26152888

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

PubMed Central

Mondal, Milon; Radeva, Nedyalka; Fanlo‐Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard

2016-01-01

Abstract Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization. PMID:27400756

Dana-Farber Cancer Institute: Identification of Therapeutic Targets in KRAS Driven Lung Cancer | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at Dana Farber Cancer Institute focuses on the use of high-throughput genetic and bioinformatic approaches to identify and credential oncogenes and co-dependencies in cancers. This Center aims to provide the cancer research community with information that will facilitate the prioritization of targets based on both genomic and functional evidence, inform the most appropriate genetic context for downstream mechanistic and validation studies, and enable the translation of this information into therapeutics and diagnostics.

Evaluation of matrix-assisted laser desorption/ionization time of flight mass spectrometry for the identification of ceratopogonid and culicid larvae.

PubMed

Steinmann, I C; Pflüger, V; Schaffner, F; Mathis, A; Kaufmann, C

2013-03-01

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was evaluated for the rapid identification of ceratopogonid larvae. Optimal sample preparation as evaluated with laboratory-reared biting midges Culicoides nubeculosus was the homogenization of gut-less larvae in 10% formic acid, and analysis of 0.2 mg/ml crude protein homogenate mixed with SA matrix at a ratio of 1:1.5. Using 5 larvae each of 4 ceratopogonid species (C. nubeculosus, C. obsoletus, C. decor, and Dasyhelea sp.) and of 2 culicid species (Aedes aegypti, Ae. japonicus), biomarker mass sets between 27 and 33 masses were determined. In a validation study, 67 larvae belonging to the target species were correctly identified by automated database-based identification (91%) or manual full comparison (9%). Four specimens of non-target species did not yield identification. As anticipated for holometabolous insects, the biomarker mass sets of adults cannot be used for the identification of larvae, and vice versa, because they share only very few similar masses as shown for C. nubeculosus, C. obsoletus, and Ae. japonicus. Thus, protein profiling by MALDI-TOF as a quick, inexpensive and accurate alternative tool is applicable to identify insect larvae of vector species collected in the field.

Using MALDI-IMS and MRM to stablish a pipeline for discovery and validation of tumor neovasculature biomarker candidates. — EDRN Public Portal

Cancer.gov

In an effort to circumvent the limitations associated with biomarker discovery workflows involving cell lines and cell cultures, histology-directed MALDI protein profiling and imaging mass spectrometry will be used for identification of vascular endothelial biomarkers suitable for early prostate cancer detection by CEUS targeted molecular imaging

Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

USDA-ARS?s Scientific Manuscript database

Gene expression analysis requires the use of reference genes in the target species. The long yellow daylily is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asia. However, reference genes for use in RT-qPCR in this ...

Genome-Scale Screening of Drug-Target Associations Relevant to Ki Using a Chemogenomics Approach

PubMed Central

Cao, Dong-Sheng; Liang, Yi-Zeng; Deng, Zhe; Hu, Qian-Nan; He, Min; Xu, Qing-Song; Zhou, Guang-Hua; Zhang, Liu-Xia; Deng, Zi-xin; Liu, Shao

2013-01-01

The identification of interactions between drugs and target proteins plays a key role in genomic drug discovery. In the present study, the quantitative binding affinities of drug-target pairs are differentiated as a measurement to define whether a drug interacts with a protein or not, and then a chemogenomics framework using an unbiased set of general integrated features and random forest (RF) is employed to construct a predictive model which can accurately classify drug-target pairs. The predictability of the model is further investigated and validated by several independent validation sets. The built model is used to predict drug-target associations, some of which were confirmed by comparing experimental data from public biological resources. A drug-target interaction network with high confidence drug-target pairs was also reconstructed. This network provides further insight for the action of drugs and targets. Finally, a web-based server called PreDPI-Ki was developed to predict drug-target interactions for drug discovery. In addition to providing a high-confidence list of drug-target associations for subsequent experimental investigation guidance, these results also contribute to the understanding of drug-target interactions. We can also see that quantitative information of drug-target associations could greatly promote the development of more accurate models. The PreDPI-Ki server is freely available via: http://sdd.whu.edu.cn/dpiki. PMID:23577055

Identification of anti-cancer targets of eco-friendly waste Punica granatum peel by dual reverse virtual screening and binding analysis.

PubMed

Usha, Talambedu; Goyal, Arvind Kumar; Lubna, Syed; Prashanth, Hp; Mohan, T Madhan; Pande, Veena; Middha, Sushil Kumar

2014-01-01

Punica granatum (family: Lythraceae) is mainly found in Iran, which is considered to be its primary centre of origin. Studies on pomegranate peel have revealed antioxidant, anti-inflammatory, anti- angiogenesis activities, with prevention of premature aging and reducing inflammation. In addition to this it is also useful in treating various diseases like diabetes, maintaining blood pressure and treatment of neoplasms such as prostate and breast cancer. In this study we identified anti-cancer targets of active compounds like corilagin (tannins), quercetin (flavonoids) and pseudopelletierine (alkaloids) present in pomegranate peel by employing dual reverse screening and binding analysis. The potent targets of the pomegranate peel were annotated by the PharmMapper and ReverseScreen 3D, then compared with targets identified from different Bioassay databases (NPACT and HIT's). Docking was then further employed using AutoDock pyrx and validated through discovery studio for studying molecular interactions. A number of potent anti-cancerous targets were attained from the PharmMapper server according to their fit score and from ReverseScreen 3D server according to decreasing 3D scores. The identified targets now need to be further validated through in vitro and in vivo studies.

Identification of human microRNA targets from isolated argonaute protein complexes.

PubMed

Beitzinger, Michaela; Peters, Lasse; Zhu, Jia Yun; Kremmer, Elisabeth; Meister, Gunter

2007-06-01

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3' untranslated region (UTR) of specific target mRNAs. Computer algorithms based on factors such as free binding energy or sequence conservation have been used to predict miRNA target mRNAs. Based on such predictions, up to one third of all mammalian mRNAs seem to be under miRNA regulation. However, due to the low degree of complementarity between the miRNA and its target, such computer programs are often imprecise and therefore not very reliable. Here we report the first biochemical identification approach of miRNA targets from human cells. Using highly specific monoclonal antibodies against members of the Ago protein family, we co-immunoprecipitate Ago-bound mRNAs and identify them by cloning. Interestingly, most of the identified targets are also predicted by different computer programs. Moreover, we randomly analyzed six different target candidates and were able to experimentally validate five as miRNA targets. Our data clearly indicate that miRNA targets can be experimentally identified from Ago complexes and therefore provide a new tool to directly analyze miRNA function.

Identification of microRNA-mRNA modules using microarray data.

PubMed

Jayaswal, Vivek; Lutherborrow, Mark; Ma, David D F; Yang, Yee H

2011-03-06

MicroRNAs (miRNAs) are post-transcriptional regulators of mRNA expression and are involved in numerous cellular processes. Consequently, miRNAs are an important component of gene regulatory networks and an improved understanding of miRNAs will further our knowledge of these networks. There is a many-to-many relationship between miRNAs and mRNAs because a single miRNA targets multiple mRNAs and a single mRNA is targeted by multiple miRNAs. However, most of the current methods for the identification of regulatory miRNAs and their target mRNAs ignore this biological observation and focus on miRNA-mRNA pairs. We propose a two-step method for the identification of many-to-many relationships between miRNAs and mRNAs. In the first step, we obtain miRNA and mRNA clusters using a combination of miRNA-target mRNA prediction algorithms and microarray expression data. In the second step, we determine the associations between miRNA clusters and mRNA clusters based on changes in miRNA and mRNA expression profiles. We consider the miRNA-mRNA clusters with statistically significant associations to be potentially regulatory and, therefore, of biological interest. Our method reduces the interactions between several hundred miRNAs and several thousand mRNAs to a few miRNA-mRNA groups, thereby facilitating a more meaningful biological analysis and a more targeted experimental validation.

Measuring Implicit European and Mediterranean Landscape Identity: A Tool Proposal.

PubMed

Fornara, Ferdinando; Dentale, Francesco; Troffa, Renato; Piras, Simona

2016-01-01

This study presents a tool - the Landscape Identity Implicit Association Test (LI-IAT) - devoted to measure the implicit identification with European and Mediterranean landscapes. To this aim, a series of prototypical landscapes was selected as stimulus, following an accurate multi-step procedure. Participants (N = 174), recruited in two Italian cities, performed two LI-IATs devoted to assess their identification with European vs. Not-European and Mediterranean vs. Not-Mediterranean prototypical landscapes. Psychometric properties and criterion validity of these measures were investigated. Two self-report measures, assessing, respectively, European and Mediterranean place identity and pleasantness of the target landscapes, were also administered. Results showed: (1) an adequate level of internal consistency for both LI-IATs; (2) a higher identification with European and Mediterranean landscapes than, respectively, with Not-European and Not-Mediterranean ones; and (3) a significant positive relationship between the European and Mediterranean LI-IATs and the corresponding place identity scores, also when pleasantness of landscapes was controlled for. Overall, these findings provide a first evidence supporting the reliability and criterion validity of the European and Mediterranean LI-IATs.

Measuring Implicit European and Mediterranean Landscape Identity: A Tool Proposal

PubMed Central

Fornara, Ferdinando; Dentale, Francesco; Troffa, Renato; Piras, Simona

2016-01-01

This study presents a tool – the Landscape Identity Implicit Association Test (LI-IAT) – devoted to measure the implicit identification with European and Mediterranean landscapes. To this aim, a series of prototypical landscapes was selected as stimulus, following an accurate multi-step procedure. Participants (N = 174), recruited in two Italian cities, performed two LI-IATs devoted to assess their identification with European vs. Not-European and Mediterranean vs. Not-Mediterranean prototypical landscapes. Psychometric properties and criterion validity of these measures were investigated. Two self-report measures, assessing, respectively, European and Mediterranean place identity and pleasantness of the target landscapes, were also administered. Results showed: (1) an adequate level of internal consistency for both LI-IATs; (2) a higher identification with European and Mediterranean landscapes than, respectively, with Not-European and Not-Mediterranean ones; and (3) a significant positive relationship between the European and Mediterranean LI-IATs and the corresponding place identity scores, also when pleasantness of landscapes was controlled for. Overall, these findings provide a first evidence supporting the reliability and criterion validity of the European and Mediterranean LI-IATs. PMID:27642284

From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data.

PubMed

Bottini, Silvia; Hamouda-Tekaya, Nedra; Tanasa, Bogdan; Zaragosi, Laure-Emmanuelle; Grandjean, Valerie; Repetto, Emanuela; Trabucchi, Michele

2017-05-19

Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data

PubMed Central

Bottini, Silvia; Hamouda-Tekaya, Nedra; Tanasa, Bogdan; Zaragosi, Laure-Emmanuelle; Grandjean, Valerie; Repetto, Emanuela

2017-01-01

Abstract Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification. PMID:28108660

Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants

PubMed Central

Ojini, Irene; Gammie, Alison

2015-01-01

Resistance to cancer therapy is a major obstacle in the long-term treatment of cancer. A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring. Here, we exploit the mutator phenotype of mismatch repair defective yeast cells combined with whole genome sequencing to identify drug resistance mutations in key pathways involved in the development of chemoresistance. The utility of this approach was demonstrated via the identification of the known CAN1 and TOP1 resistance targets for two compounds, canavanine and camptothecin, respectively. We have also experimentally validated the plasma membrane transporter HNM1 as the primary drug resistance target of mechlorethamine. Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis. In the case of bactobolin, a promising anticancer drug, the endocytosis pathway was identified as the drug resistance target responsible for conferring resistance. Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance. The rapid and robust nature of these techniques, using Saccharomyces cerevisiae as a model organism, should accelerate the identification of drug resistance targets and guide the development of novel therapeutic combination strategies to prevent the development of chemoresistance in various cancers. PMID:26199284

Precise Geolocation Of Persistent Scatterers Aided And Validated By Lidar DSM

NASA Astrophysics Data System (ADS)

Chang, Ling; Dheenathayalan, Prabu; Hanessen, Ramon

2013-12-01

Persistent Scatterers (PS) interferometry results in the de- formation history of time-coherent scatterers. Although several applications focus on smooth, spatially correlated signals, we aim for the detection, identification and analysis of single anomalies. These targets can be indicative of, e.g., strain in structures, potentially leading to the failure of such structures. For the identification and analysis it is of the greatest importance to know the exact position of the effective scattering center, to avoid an improper interpretation of the driving mechanism. Here we present an approach to optimize the geolocation of important scatterers, when necessary aided by an a priori Lidar-derived DSM (AHN-1 data) with 15cm and 5m resolution in vertical and horizontal directions, respectively. The DSM is also used to validate the geocoding. We implement our approach on a near-collapse event of a shopping mall in Heerlen, the Netherlands, to generate the precise geolocation of local PS points.

Animal models of addiction

PubMed Central

Spanagel, Rainer

2017-01-01

In recent years, animal models in psychiatric research have been criticized for their limited translational value to the clinical situation. Failures in clinical trials have thus often been attributed to the lack of predictive power of preclinical animal models. Here, I argue that animal models of voluntary drug intake—under nonoperant and operant conditions—and addiction models based on the Diagnostic and Statistical Manual of Mental Disorders are crucial and informative tools for the identification of pathological mechanisms, target identification, and drug development. These models provide excellent face validity, and it is assumed that the neurochemical and neuroanatomical substrates involved in drug-intake behavior are similar in laboratory rodents and humans. Consequently, animal models of drug consumption and addiction provide predictive validity. This predictive power is best illustrated in alcohol research, in which three approved medications—acamprosate, naltrexone, and nalmefene—were developed by means of animal models and then successfully translated into the clinical situation. PMID:29302222

Comparative genomics identification of a novel set of temporally regulated hedgehog target genes in the retina.

PubMed

McNeill, Brian; Perez-Iratxeta, Carol; Mazerolle, Chantal; Furimsky, Marosh; Mishina, Yuji; Andrade-Navarro, Miguel A; Wallace, Valerie A

2012-03-01

The hedgehog (Hh) signaling pathway is involved in numerous developmental and adult processes with many links to cancer. In vertebrates, the activity of the Hh pathway is mediated primarily through three Gli transcription factors (Gli1, 2 and 3) that can serve as transcriptional activators or repressors. The identification of Gli target genes is essential for the understanding of the Hh-mediated processes. We used a comparative genomics approach using the mouse and human genomes to identify 390 genes that contained conserved Gli binding sites. RT-qPCR validation of 46 target genes in E14.5 and P0.5 retinal explants revealed that Hh pathway activation resulted in the modulation of 30 of these targets, 25 of which demonstrated a temporal regulation. Further validation revealed that the expression of Bok, FoxA1, Sox8 and Wnt7a was dependent upon Sonic Hh (Shh) signaling in the retina and their regulation is under positive and negative controls by Gli2 and Gli3, respectively. We also show using chromatin immunoprecipitation that Gli2 binds to the Sox8 promoter, suggesting that Sox8 is an Hh-dependent direct target of Gli2. Finally, we demonstrate that the Hh pathway also modulates the expression of Sox9 and Sox10, which together with Sox8 make up the SoxE group. Previously, it has been shown that Hh and SoxE group genes promote Müller glial cell development in the retina. Our data are consistent with the possibility for a role of SoxE group genes downstream of Hh signaling on Müller cell development. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Automatic target validation based on neuroscientific literature mining for tractography

PubMed Central

Vasques, Xavier; Richardet, Renaud; Hill, Sean L.; Slater, David; Chappelier, Jean-Cedric; Pralong, Etienne; Bloch, Jocelyne; Draganski, Bogdan; Cif, Laura

2015-01-01

Target identification for tractography studies requires solid anatomical knowledge validated by an extensive literature review across species for each seed structure to be studied. Manual literature review to identify targets for a given seed region is tedious and potentially subjective. Therefore, complementary approaches would be useful. We propose to use text-mining models to automatically suggest potential targets from the neuroscientific literature, full-text articles and abstracts, so that they can be used for anatomical connection studies and more specifically for tractography. We applied text-mining models to three structures: two well-studied structures, since validated deep brain stimulation targets, the internal globus pallidus and the subthalamic nucleus and, the nucleus accumbens, an exploratory target for treating psychiatric disorders. We performed a systematic review of the literature to document the projections of the three selected structures and compared it with the targets proposed by text-mining models, both in rat and primate (including human). We ran probabilistic tractography on the nucleus accumbens and compared the output with the results of the text-mining models and literature review. Overall, text-mining the literature could find three times as many targets as two man-weeks of curation could. The overall efficiency of the text-mining against literature review in our study was 98% recall (at 36% precision), meaning that over all the targets for the three selected seeds, only one target has been missed by text-mining. We demonstrate that connectivity for a structure of interest can be extracted from a very large amount of publications and abstracts. We believe this tool will be useful in helping the neuroscience community to facilitate connectivity studies of particular brain regions. The text mining tools used for the study are part of the HBP Neuroinformatics Platform, publicly available at http://connectivity-brainer.rhcloud.com/. PMID:26074781

Evaluation of the Seeplex® Meningitis ACE Detection kit for the detection of 12 common bacterial and viral pathogens of acute meningitis.

PubMed

Shin, So Youn; Kwon, Kye Chul; Park, Jong Woo; Kim, Ji Myung; Shin, So Young; Koo, Sun Hoe

2012-01-01

Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. The lower detection limits ranged from 10(1) copies/µL to 5×10(1) copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens.

Evaluation of the Seeplex® Meningitis ACE Detection Kit for the Detection of 12 Common Bacterial and Viral Pathogens of Acute Meningitis

PubMed Central

Shin, So Youn; Kwon, Kye Chul; Park, Jong Woo; Kim, Ji Myung; Shin, So Young

2012-01-01

Background Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. Methods Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. Results The lower detection limits ranged from 101 copies/µL to 5×101 copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. Conclusions The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens. PMID:22259778

RUCS: rapid identification of PCR primers for unique core sequences.

PubMed

Thomsen, Martin Christen Frølund; Hasman, Henrik; Westh, Henrik; Kaya, Hülya; Lund, Ole

2017-12-15

Designing PCR primers to target a specific selection of whole genome sequenced strains can be a long, arduous and sometimes impractical task. Such tasks would benefit greatly from an automated tool to both identify unique targets, and to validate the vast number of potential primer pairs for the targets in silico. Here we present RUCS, a program that will find PCR primer pairs and probes for the unique core sequences of a positive genome dataset complement to a negative genome dataset. The resulting primer pairs and probes are in addition to simple selection also validated through a complex in silico PCR simulation. We compared our method, which identifies the unique core sequences, against an existing tool called ssGeneFinder, and found that our method was 6.5-20 times more sensitive. We used RUCS to design primer pairs that would target a set of genomes known to contain the mcr-1 colistin resistance gene. Three of the predicted pairs were chosen for experimental validation using PCR and gel electrophoresis. All three pairs successfully produced an amplicon with the target length for the samples containing mcr-1 and no amplification products were produced for the negative samples. The novel methods presented in this manuscript can reduce the time needed to identify target sequences, and provide a quick virtual PCR validation to eliminate time wasted on ambiguously binding primers. Source code is freely available on https://bitbucket.org/genomicepidemiology/rucs. Web service is freely available on https://cge.cbs.dtu.dk/services/RUCS. mcft@cbs.dtu.dk. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Performance Characteristics of Compact Mobile LIFS (Laser-Induced Fluorescence Spectrum) Lidar

NASA Astrophysics Data System (ADS)

Tomida, Takayuki; Nishizawa, Naoto; Sakurai, Kosuke; Suganumata, Hikaru; Tsukada, Shodai; Song, Sung-Moo; Park, Ho-Dong; Saito, Yasunori

2016-06-01

We developed a compact but versatile laser-induced fluorescence spectrum (LIFS) lidar that has potential use for material or aerosol identification outside experimental rooms. The compactness and mobility of the LIFS lidar means observations can be more freely conducted at any place and any time. Its performance characteristics were validated by threedimensional fluorescence imaging of targets and remote detection of quasi bio/organic aerosols.

Molecular Targets in Advanced Therapeutics of Cancers: The Role of Pharmacogenetics.

PubMed

Abubakar, Murtala B; Gan, Siew Hua

2016-01-01

The advent of advanced molecular targeted therapy has resulted in improved prognoses for patients with advanced malignancies. However, despite the significant success and specificity of this advocated targeted therapy, significant on- and off-target adverse effects and inter-individual variability in treatment responses have been reported. The interpatient variability in drug response has been suggested to be partly due to variations in patient genomes. Therefore, the identification of genetic biomarkers by conducting pharmacogenetics studies can help predict patient responses to targeted therapy and may serve as a basis for individualized treatment. In this review, both clinically established and potential molecular targets are highlighted. Overall, current literature suggests that individualization of targeted therapy is promising; however, integrating the clinical benefits of identified biomarkers into clinical practice for personalized medicine remains a major challenge, and further studies to validate these markers and identify novel therapeutic approaches are needed. © 2016 S. Karger AG, Basel.

Identification of a New Isoindole-2-yl Scaffold as a Qo and Qi Dual Inhibitor of Cytochrome bc 1 Complex: Virtual Screening, Synthesis, and Biochemical Assay.

PubMed

Azizian, Homa; Bagherzadeh, Kowsar; Shahbazi, Sophia; Sharifi, Niusha; Amanlou, Massoud

2017-09-18

Respiratory chain ubiquinol-cytochrome (cyt) c oxidoreductase (cyt bc 1 or complex III) has been demonstrated as a promising target for numerous antibiotics and fungicide applications. In this study, a virtual screening of NCI diversity database was carried out in order to find novel Qo/Qi cyt bc 1 complex inhibitors. Structure-based virtual screening and molecular docking methodology were employed to further screen compounds with inhibition activity against cyt bc 1 complex after extensive reliability validation protocol with cross-docking method and identification of the best score functions. Subsequently, the application of rational filtering procedure over the target database resulted in the elucidation of a novel class of cyt bc 1 complex potent inhibitors with comparable binding energies and biological activities to those of the standard inhibitor, antimycin.

Identification and characterization of microRNAs and their target genes from Nile tilapia (Oreochromis niloticus).

PubMed

Huang, Yong; Ma, Xiu Ying; Yang, You Bing; Ren, Hong Tao; Sun, Xi Hong; Wang, Li Rui

MicroRNAs (miRNAs) are a class of small single-stranded, endogenous 21-22 nt non-coding RNAs that regulate their target mRNA levels by causing either inactivation or degradation of the mRNAs. In recent years, miRNA genes have been identified from mammals, insects, worms, plants, and viruses. In this research, bioinformatics approaches were used to predict potential miRNAs and their targets in Nile tilapia from the expressed sequence tag (EST) and genomic survey sequence (GSS) database, respectively, based on the conservation of miRNAs in many animal species. A total of 19 potential miRNAs were detected following a range of strict filtering criteria. To test the validity of the bioinformatics method, seven predicted Nile tilapia miRNA genes were selected for further biological validation, and their mature miRNA transcripts were successfully detected by stem-loop RT-PCR experiments. Using these potential miRNAs, we found 56 potential targets in this species. Most of the target mRNAs appear to be involved in development, metabolism, signal transduction, transcription regulation and stress responses. Overall, our findings will provide an important foundation for further research on miRNAs function in the Nile tilapia.

Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

PubMed Central

Butts, Arielle; DeJarnette, Christian; Peters, Tracy L.; Parker, Josie E.; Kerns, Morgan E.; Eberle, Karen E.; Kelly, Steve L.

2017-01-01

ABSTRACT Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound. PMID:28989971

Identification of regulatory targets of tissue-specific transcription factors: application to retina-specific gene regulation

PubMed Central

Qian, Jiang; Esumi, Noriko; Chen, Yangjian; Wang, Qingliang; Chowers, Itay; Zack, Donald J.

2005-01-01

Identification of tissue-specific gene regulatory networks can yield insights into the molecular basis of a tissue's development, function and pathology. Here, we present a computational approach designed to identify potential regulatory target genes of photoreceptor cell-specific transcription factors (TFs). The approach is based on the hypothesis that genes related to the retina in terms of expression, disease and/or function are more likely to be the targets of retina-specific TFs than other genes. A list of genes that are preferentially expressed in retina was obtained by integrating expressed sequence tag, SAGE and microarray datasets. The regulatory targets of retina-specific TFs are enriched in this set of retina-related genes. A Bayesian approach was employed to integrate information about binding site location relative to a gene's transcription start site. Our method was applied to three retina-specific TFs, CRX, NRL and NR2E3, and a number of potential targets were predicted. To experimentally assess the validity of the bioinformatic predictions, mobility shift, transient transfection and chromatin immunoprecipitation assays were performed with five predicted CRX targets, and the results were suggestive of CRX regulation in 5/5, 3/5 and 4/5 cases, respectively. Together, these experiments strongly suggest that RP1, GUCY2D, ABCA4 are novel targets of CRX. PMID:15967807

Modeling human target acquisition in ground-to-air weapon systems

NASA Technical Reports Server (NTRS)

Phatak, A. V.; Mohr, R. L.; Vikmanis, M.; Wei, K. C.

1982-01-01

The problems associated with formulating and validating mathematical models for describing and predicting human target acquisition response are considered. In particular, the extension of the human observer model to include the acquisition phase as well as the tracking segment is presented. Relationship of the Observer model structure to the more complex Standard Optimal Control model formulation and to the simpler Transfer Function/Noise representation is discussed. Problems pertinent to structural identifiability and the form of the parameterization are elucidated. A systematic approach toward the identification of the observer acquisition model parameters from ensemble tracking error data is presented.

Focus on flaviviruses: current and future drug targets.

PubMed

Geiss, Brian J; Stahla, Hillary; Hannah, Amanda M; Gari, Amanda M; Keenan, Susan M

2009-05-01

Infection by mosquito-borne flaviviruses (family Flaviviridae) is increasing in prevalence worldwide. The vast global, social and economic impact due to the morbidity and mortality associated with the diseases caused by these viruses necessitates therapeutic intervention. There is currently no effective clinical treatment for any flaviviral infection. Therefore, there is a great need for the identification of novel inhibitors to target the virus life cycle. In this article, we discuss structural and nonstructural viral proteins that are the focus of current target validation and drug discovery efforts. Both inhibition of essential enzymatic activities and disruption of necessary protein–protein interactions are considered. In addition, we address promising new targets for future research. As our molecular and biochemical understanding of the flavivirus life cycle increases, the number of targets for antiviral therapeutic discovery grows and the possibility for novel drug discovery continues to strengthen.

In Vitro Evaluation of Molecular Tumor Targets in Nuclear Medicine: Immunohistochemistry Is One Option, but Under Which Conditions?

PubMed

Reubi, Jean Claude

2017-12-01

The identification of new molecular targets for diagnostic and therapeutic applications using in vitro methods is an important challenge in nuclear medicine. One such method is immunohistochemistry, increasingly popular because it is easy to perform. This review presents the case for conducting receptor immunohistochemistry to evaluate potential molecular targets in human tumor tissue sections. The focus is on the immunohistochemistry of G-protein-coupled receptors, one of the largest families of cell surface proteins, representing a major class of drug targets and thus playing an important role in nuclear medicine. This review identifies common pitfalls and challenges and provides guidelines on performing such immunohistochemical studies. An appropriate validation of the target is a prerequisite for developing robust and informative new molecular probes. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma.

PubMed

Arora, Shilpi; Gonzales, Irma M; Hagelstrom, R Tanner; Beaudry, Christian; Choudhary, Ashish; Sima, Chao; Tibes, Raoul; Mousses, Spyro; Azorsa, David O

2010-08-18

Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.

Systems genetics for drug target discovery

PubMed Central

Penrod, Nadia M.; Cowper-Sal_lari, Richard; Moore, Jason H.

2011-01-01

The collection and analysis of genomic data has the potential to reveal novel druggable targets by providing insight into the genetic basis of disease. However, the number of drugs, targeting new molecular entities, approved by the US Food and Drug Administration (FDA) has not increased in the years since the collection of genomic data has become commonplace. The paucity of translatable results can be partly attributed to conventional analysis methods that test one gene at a time in an effort to identify disease-associated factors as candidate drug targets. By disengaging genetic factors from their position within the genetic regulatory system, much of the information stored within the genomic data set is lost. Here we discuss how genomic data is used to identify disease-associated genes or genomic regions, how disease-associated regions are validated as functional targets, and the role network analysis can play in bridging the gap between data generation and effective drug target identification. PMID:21862141

Structure guided design of a series of selective pyrrolopyrimidinone MARK inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katz, Jason D.; Haidle, Andrew; Childers, Kaleen K.

2017-01-01

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimer’s disease.

Cloud computing approaches to accelerate drug discovery value chain.

PubMed

Garg, Vibhav; Arora, Suchir; Gupta, Chitra

2011-12-01

Continued advancements in the area of technology have helped high throughput screening (HTS) evolve from a linear to parallel approach by performing system level screening. Advanced experimental methods used for HTS at various steps of drug discovery (i.e. target identification, target validation, lead identification and lead validation) can generate data of the order of terabytes. As a consequence, there is pressing need to store, manage, mine and analyze this data to identify informational tags. This need is again posing challenges to computer scientists to offer the matching hardware and software infrastructure, while managing the varying degree of desired computational power. Therefore, the potential of "On-Demand Hardware" and "Software as a Service (SAAS)" delivery mechanisms cannot be denied. This on-demand computing, largely referred to as Cloud Computing, is now transforming the drug discovery research. Also, integration of Cloud computing with parallel computing is certainly expanding its footprint in the life sciences community. The speed, efficiency and cost effectiveness have made cloud computing a 'good to have tool' for researchers, providing them significant flexibility, allowing them to focus on the 'what' of science and not the 'how'. Once reached to its maturity, Discovery-Cloud would fit best to manage drug discovery and clinical development data, generated using advanced HTS techniques, hence supporting the vision of personalized medicine.

Antibody therapeutics for treating prostate cancer: where are we now and what comes next?

PubMed

Vlachostergios, Panagiotis J; Galletti, Giuseppe; Palmer, Jessica; Lam, Linda; Karir, Beerinder S; Tagawa, Scott T

2017-02-01

Progress in the understanding of molecular events of carcinogenesis and cancer evolution as well as the identification of tumor antigens has led to the development of different targeted therapeutic approaches, including the use of monoclonal antibodies (mAbs). Prostate cancer (PC) is highly amenable to mAb targeting given the existence of prostate-specific targets and the natural history and localization of metastatic disease. Areas covered: Several aspects of the PC phenotype, including growth factors, angiogenesis mediators, bone microenvironment signals, and immune evasion pathways, have become areas of ongoing investigation in terms of mAb targeting. These are reviewed. The greatest success so far has been the development of mAbs against prostate-specific tumor antigen (PSMA), which opened an opportunity to improve diagnostic accuracy and simultaneously target metastatic disease. Expert opinion: As mAb use in PC continues to evolve, more accurate imaging of the extent of disease and more effective mAb therapies (naked or conjugated with drugs, toxins or radioactive molecules) are emerging. In addition, the combination of mAbs with other treatment modalities is expected to further improve responses and overall survival. Identification of validated biomarkers is necessary for better recognition of patient subgroups who will derive the greatest benefit from mAb therapy.

Specific detection and identification of [Actinobacillus] muris by PCR using primers targeting the 16S-23S rRNA internal transcribed spacer regions.

PubMed

Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Sager, Martin

2013-08-01

[Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S-23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

PubMed

Tu, Yu-Hsuan; Ho, Yu-Hsuan; Chuang, Ying-Chih; Chen, Po-Chung; Chen, Chien-Sheng

2011-01-01

Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

Identification of Lactoferricin B Intracellular Targets Using an Escherichia coli Proteome Chip

PubMed Central

Chen, Po-Chung; Chen, Chien-Sheng

2011-01-01

Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach. PMID:22164243

Membrane protease degradomics: proteomic identification and quantification of cell surface protease substrates.

PubMed

Butler, Georgina S; Dean, Richard A; Smith, Derek; Overall, Christopher M

2009-01-01

The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry.

PubMed

Mondal, Milon; Radeva, Nedyalka; Fanlo-Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard; Hirsch, Anna K H

2016-08-01

Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Mathematical modeling for novel cancer drug discovery and development.

PubMed

Zhang, Ping; Brusic, Vladimir

2014-10-01

Mathematical modeling enables: the in silico classification of cancers, the prediction of disease outcomes, optimization of therapy, identification of promising drug targets and prediction of resistance to anticancer drugs. In silico pre-screened drug targets can be validated by a small number of carefully selected experiments. This review discusses the basics of mathematical modeling in cancer drug discovery and development. The topics include in silico discovery of novel molecular drug targets, optimization of immunotherapies, personalized medicine and guiding preclinical and clinical trials. Breast cancer has been used to demonstrate the applications of mathematical modeling in cancer diagnostics, the identification of high-risk population, cancer screening strategies, prediction of tumor growth and guiding cancer treatment. Mathematical models are the key components of the toolkit used in the fight against cancer. The combinatorial complexity of new drugs discovery is enormous, making systematic drug discovery, by experimentation, alone difficult if not impossible. The biggest challenges include seamless integration of growing data, information and knowledge, and making them available for a multiplicity of analyses. Mathematical models are essential for bringing cancer drug discovery into the era of Omics, Big Data and personalized medicine.

Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

PubMed Central

Pavelin, Jon; Reynolds, Natalie; Chiweshe, Stephen; Wu, Guanming; Tiribassi, Rebecca; Grey, Finn

2013-01-01

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions. PMID:24385903

In silico prediction of novel therapeutic targets using gene-disease association data.

PubMed

Ferrero, Enrico; Dunham, Ian; Sanseau, Philippe

2017-08-29

Target identification and validation is a pressing challenge in the pharmaceutical industry, with many of the programmes that fail for efficacy reasons showing poor association between the drug target and the disease. Computational prediction of successful targets could have a considerable impact on attrition rates in the drug discovery pipeline by significantly reducing the initial search space. Here, we explore whether gene-disease association data from the Open Targets platform is sufficient to predict therapeutic targets that are actively being pursued by pharmaceutical companies or are already on the market. To test our hypothesis, we train four different classifiers (a random forest, a support vector machine, a neural network and a gradient boosting machine) on partially labelled data and evaluate their performance using nested cross-validation and testing on an independent set. We then select the best performing model and use it to make predictions on more than 15,000 genes. Finally, we validate our predictions by mining the scientific literature for proposed therapeutic targets. We observe that the data types with the best predictive power are animal models showing a disease-relevant phenotype, differential expression in diseased tissue and genetic association with the disease under investigation. On a test set, the neural network classifier achieves over 71% accuracy with an AUC of 0.76 when predicting therapeutic targets in a semi-supervised learning setting. We use this model to gain insights into current and failed programmes and to predict 1431 novel targets, of which a highly significant proportion has been independently proposed in the literature. Our in silico approach shows that data linking genes and diseases is sufficient to predict novel therapeutic targets effectively and confirms that this type of evidence is essential for formulating or strengthening hypotheses in the target discovery process. Ultimately, more rapid and automated target prioritisation holds the potential to reduce both the costs and the development times associated with bringing new medicines to patients.

Active imaging system performance model for target acquisition

NASA Astrophysics Data System (ADS)

Espinola, Richard L.; Teaney, Brian; Nguyen, Quang; Jacobs, Eddie L.; Halford, Carl E.; Tofsted, David H.

2007-04-01

The U.S. Army RDECOM CERDEC Night Vision & Electronic Sensors Directorate has developed a laser-range-gated imaging system performance model for the detection, recognition, and identification of vehicle targets. The model is based on the established US Army RDECOM CERDEC NVESD sensor performance models of the human system response through an imaging system. The Java-based model, called NVLRG, accounts for the effect of active illumination, atmospheric attenuation, and turbulence effects relevant to LRG imagers, such as speckle and scintillation, and for the critical sensor and display components. This model can be used to assess the performance of recently proposed active SWIR systems through various trade studies. This paper will describe the NVLRG model in detail, discuss the validation of recent model components, present initial trade study results, and outline plans to validate and calibrate the end-to-end model with field data through human perception testing.

Target identification, lead optimization and antitumor evaluation of some new 1,2,4-triazines as c-Met kinase inhibitors.

PubMed

El-Wakil, Marwa H; Ashour, Hayam M; Saudi, Manal N; Hassan, Ahmed M; Labouta, Ibrahim M

2017-08-01

In silico target fishing approach using PharmMapper server identified c-Met kinase as the selective target for our previously synthesized compound NCI 748494/1. This approach was validated by in vitro kinase assay which showed that NCI 748494/1 possessed promising inhibitory activity against c-Met kinase (IC 50 =31.70μM). Assessment of ADMET profiling, drug-likeness, drug score as well as docking simulation for the binding pose of that compound in the active site of c-Met kinase domain revealed that NCI 748494/1 could be considered as a promising drug lead. Based on target identification and validation, it was observed that there is structure similarity between NCI 748494/1 and the reported type II c-Met kinase inhibitor BMS-777607. Optimization of our lead NCI 748494/1 furnished newly synthesized 1,2,4-triazine derivatives based on well-established structure-activity relationships, whereas three compounds namely; 4d, 7a and 8c displayed excellent in vitro cytotoxicity against three c-Met addicted cancer cell lines; A549 (lung adenocarcinoma), HT-29 (colon cancer) and MKN-45 (gastric carcinoma); with IC 50 values in the range 0.01-1.86µM. In vitro c-Met kinase assay showed 8c to possess the highest c-Met kinase inhibition profile (IC 50 =4.31µM). Docking of the active compounds in c-Met kinase active site revealed strong binding interactions comparable to the lead NCI 748494/1 and BMS-777607, suggesting that c-Met inhibition is very likely to be the mechanism of the antitumor effect of these derivatives. Copyright © 2017 Elsevier Inc. All rights reserved.

Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction

PubMed Central

Jalal, Hamid; Stephen, Hannah; Curran, Martin D.; Burton, Janet; Bradley, Michelle; Carne, Christopher

2006-01-01

A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload. PMID:16390971

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond.

PubMed

Zhang, Hongkang; Cohen, Adam E

2017-07-01

Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, in particular tools for all-optical electrophysiology, now provide a means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline - from target identification and validation, to target-based and phenotypic screens, to clinical trials. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Scalable Approach for Protein False Discovery Rate Estimation in Large Proteomic Data Sets.

PubMed

Savitski, Mikhail M; Wilhelm, Mathias; Hahne, Hannes; Kuster, Bernhard; Bantscheff, Marcus

2015-09-01

Calculating the number of confidently identified proteins and estimating false discovery rate (FDR) is a challenge when analyzing very large proteomic data sets such as entire human proteomes. Biological and technical heterogeneity in proteomic experiments further add to the challenge and there are strong differences in opinion regarding the conceptual validity of a protein FDR and no consensus regarding the methodology for protein FDR determination. There are also limitations inherent to the widely used classic target-decoy strategy that particularly show when analyzing very large data sets and that lead to a strong over-representation of decoy identifications. In this study, we investigated the merits of the classic, as well as a novel target-decoy-based protein FDR estimation approach, taking advantage of a heterogeneous data collection comprised of ∼19,000 LC-MS/MS runs deposited in ProteomicsDB (https://www.proteomicsdb.org). The "picked" protein FDR approach treats target and decoy sequences of the same protein as a pair rather than as individual entities and chooses either the target or the decoy sequence depending on which receives the highest score. We investigated the performance of this approach in combination with q-value based peptide scoring to normalize sample-, instrument-, and search engine-specific differences. The "picked" target-decoy strategy performed best when protein scoring was based on the best peptide q-value for each protein yielding a stable number of true positive protein identifications over a wide range of q-value thresholds. We show that this simple and unbiased strategy eliminates a conceptual issue in the commonly used "classic" protein FDR approach that causes overprediction of false-positive protein identification in large data sets. The approach scales from small to very large data sets without losing performance, consistently increases the number of true-positive protein identifications and is readily implemented in proteomics analysis software. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Shielded Heavy-Ion Environment Linear Detector (SHIELD): an experiment for the Radiation and Technology Demonstration (RTD) Mission.

PubMed

Shavers, M R; Cucinotta, F A; Miller, J; Zeitlin, C; Heilbronn, L; Wilson, J W; Singleterry, R C

2001-01-01

Radiological assessment of the many cosmic ion species of widely distributed energies requires the use of theoretical transport models to accurately describe diverse physical processes related to nuclear reactions in spacecraft structures, planetary atmospheres and surfaces, and tissues. Heavy-ion transport models that were designed to characterize shielded radiation fields have been validated through comparison with data from thick-target irradiation experiments at particle accelerators. With the RTD Mission comes a unique opportunity to validate existing radiation transport models and guide the development of tools for shield design. For the first time, transport properties will be measured in free-space to characterize the shielding effectiveness of materials that are likely to be aboard interplanetary space missions. Target materials composed of aluminum, advanced composite spacecraft structure and other shielding materials, helium (a propellant) and tissue equivalent matrices will be evaluated. Large solid state detectors will provide kinetic energy and charge identification for incident heavy-ions and for secondary ions created in the target material. Transport calculations using the HZETRN model suggest that 8 g cm -2 thick targets would be adequate to evaluate the shielding effectiveness during solar minimum activity conditions for a period of 30 days or more.

Applications of LC-MS in PET Radioligand Development and Metabolic Elucidation

PubMed Central

Ma, Ying; Kiesewetter, Dale O.; Lang, Lixin; Gu, Dongyu; Chen, Xiaoyuan

2013-01-01

Positron emission tomography (PET) is a very sensitive molecular imaging technique that when employed with an appropriate radioligand has the ability to quantititate physiological processes in a non-invasive manner. Since the imaging technique detects all radioactive emissions in the field of view, the presence and biological activity of radiolabeled metabolites must be determined for each radioligand in order to validate the utility of the radiotracer for measuring the desired physiological process. Thus, the identification of metabolic profiles of radiolabeled compounds is an important aspect of design, development, and validation of new radiopharmaceuticals and their applications in drug development and molecular imaging. Metabolite identification for different chemical classes of radiopharmaceuticals allows rational design to minimize the formation and accumulation of metabolites in the target tissue, either through enhanced excretion or minimized metabolism. This review will discuss methods for identifying and quantitating metabolites during the pre-clinical development of radiopharmaceuticals with special emphasis on the application of LC/MS. PMID:20540692

Patterns of Cognitive Strengths and Weaknesses: Identification Rates, Agreement, and Validity for Learning Disabilities Identification

PubMed Central

Miciak, Jeremy; Fletcher, Jack M.; Stuebing, Karla; Vaughn, Sharon; Tolar, Tammy D.

2014-01-01

Purpose Few empirical investigations have evaluated LD identification methods based on a pattern of cognitive strengths and weaknesses (PSW). This study investigated the reliability and validity of two proposed PSW methods: the concordance/discordance method (C/DM) and cross battery assessment (XBA) method. Methods Cognitive assessment data for 139 adolescents demonstrating inadequate response to intervention was utilized to empirically classify participants as meeting or not meeting PSW LD identification criteria using the two approaches, permitting an analysis of: (1) LD identification rates; (2) agreement between methods; and (3) external validity. Results LD identification rates varied between the two methods depending upon the cut point for low achievement, with low agreement for LD identification decisions. Comparisons of groups that met and did not meet LD identification criteria on external academic variables were largely null, raising questions of external validity. Conclusions This study found low agreement and little evidence of validity for LD identification decisions based on PSW methods. An alternative may be to use multiple measures of academic achievement to guide intervention. PMID:24274155

A pan-European ring trial to validate an International Standard for detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in seafoods.

PubMed

Hartnell, R E; Stockley, L; Keay, W; Rosec, J-P; Hervio-Heath, D; Van den Berg, H; Leoni, F; Ottaviani, D; Henigman, U; Denayer, S; Serbruyns, B; Georgsson, F; Krumova-Valcheva, G; Gyurova, E; Blanco, C; Copin, S; Strauch, E; Wieczorek, K; Lopatek, M; Britova, A; Hardouin, G; Lombard, B; In't Veld, P; Leclercq, A; Baker-Austin, C

2018-02-10

Globally, vibrios represent an important and well-established group of bacterial foodborne pathogens. The European Commission (EC) mandated the Comite de European Normalisation (CEN) to undertake work to provide validation data for 15 methods in microbiology to support EC legislation. As part of this mandated work programme, merging of ISO/TS 21872-1:2007, which specifies a horizontal method for the detection of V. parahaemolyticus and V. cholerae, and ISO/TS 21872-2:2007, a similar horizontal method for the detection of potentially pathogenic vibrios other than V. cholerae and V. parahaemolyticus was proposed. Both parts of ISO/TS 21872 utilized classical culture-based isolation techniques coupled with biochemical confirmation steps. The work also considered simplification of the biochemical confirmation steps. In addition, because of advances in molecular based methods for identification of human pathogenic Vibrio spp. classical and real-time PCR options were also included within the scope of the validation. These considerations formed the basis of a multi-laboratory validation study with the aim of improving the precision of this ISO technical specification and providing a single ISO standard method to enable detection of these important foodborne Vibrio spp.. To achieve this aim, an international validation study involving 13 laboratories from 9 countries in Europe was conducted in 2013. The results of this validation have enabled integration of the two existing technical specifications targeting the detection of the major foodborne Vibrio spp., simplification of the suite of recommended biochemical identification tests and the introduction of molecular procedures that provide both species level identification and discrimination of putatively pathogenic strains of V. parahaemolyticus by the determination of the presence of theromostable direct and direct related haemolysins. The method performance characteristics generated in this have been included in revised international standard, ISO 21872:2017, published in July 2017. Copyright © 2018. Published by Elsevier B.V.

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

PubMed

Kim, Hyun-Kyoung; Park, Won Cheol; Lee, Kwang Man; Hwang, Hai-Li; Park, Seong-Yeol; Sorn, Sungbin; Chandra, Vishal; Kim, Kwang Gi; Yoon, Woong-Bae; Bae, Joon Seol; Shin, Hyoung Doo; Shin, Jong-Yeon; Seoh, Ju-Young; Kim, Jong-Il; Hong, Kyeong-Man

2014-01-01

The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends. As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA. Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation. Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.

Application of chemical biology in target identification and drug discovery.

PubMed

Zhu, Yue; Xiao, Ting; Lei, Saifei; Zhou, Fulai; Wang, Ming-Wei

2015-09-01

Drug discovery and development is vital to the well-being of mankind and sustainability of the pharmaceutical industry. Using chemical biology approaches to discover drug leads has become a widely accepted path partially because of the completion of the Human Genome Project. Chemical biology mainly solves biological problems through searching previously unknown targets for pharmacologically active small molecules or finding ligands for well-defined drug targets. It is a powerful tool to study how these small molecules interact with their respective targets, as well as their roles in signal transduction, molecular recognition and cell functions. There have been an increasing number of new therapeutic targets being identified and subsequently validated as a result of advances in functional genomics, which in turn led to the discovery of numerous active small molecules via a variety of high-throughput screening initiatives. In this review, we highlight some applications of chemical biology in the context of drug discovery.

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

PubMed Central

2010-01-01

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research. PMID:21406066

Two-dimensional hidden semantic information model for target saliency detection and eyetracking identification

NASA Astrophysics Data System (ADS)

Wan, Weibing; Yuan, Lingfeng; Zhao, Qunfei; Fang, Tao

2018-01-01

Saliency detection has been applied to the target acquisition case. This paper proposes a two-dimensional hidden Markov model (2D-HMM) that exploits the hidden semantic information of an image to detect its salient regions. A spatial pyramid histogram of oriented gradient descriptors is used to extract features. After encoding the image by a learned dictionary, the 2D-Viterbi algorithm is applied to infer the saliency map. This model can predict fixation of the targets and further creates robust and effective depictions of the targets' change in posture and viewpoint. To validate the model with a human visual search mechanism, two eyetrack experiments are employed to train our model directly from eye movement data. The results show that our model achieves better performance than visual attention. Moreover, it indicates the plausibility of utilizing visual track data to identify targets.

Translational research in addiction: toward a framework for the development of novel therapeutics.

PubMed

Paterson, Neil E

2011-06-15

The development of novel substance use disorder (SUD) therapeutics is insufficient to meet the medical needs of a growing SUD patient population. The identification of translatable SUD models and tests is a crucial step in establishing a framework for SUD therapeutic development programs. The present review begins by identifying the clinical features of SUDs and highlights the narrow regulatory end-point required for approval of a novel SUD therapeutic. A conceptual overview of dependence is provided, followed by identification of potential intervention targets in the addiction cycle. The main components of the addiction cycle provide the framework for a discussion of preclinical models and their clinical analogs, all of which are focused on isolated behavioral end-points thought to be relevant to the persistence of compulsive drug use. Thus, the greatest obstacle to successful development is the gap between the multiplicity of preclinical and early clinical end-points and the regulatory end-point of sustained abstinence. This review proposes two pathways to bridging this gap: further development and validation of the preclinical extended access self-administration model; inclusion of secondary end-points comprising all of the measures highlighted in the present discussion in Phase 3 trials. Further, completion of the postdictive validation of analogous preclinical and clinical assays is of high priority. Ultimately, demonstration of the relevance and validity of a variety of end-points to the ultimate goal of abstinence will allow researchers to identify truly relevant therapeutic mechanisms and intervention targets, and establish a framework for SUD therapeutic development that allows optimal decision-making and resource allocation. 2011 Elsevier Inc. All rights reserved.

Identification of the Transcriptional Targets of FOXP2, a Gene Linked to Speech and Language, in Developing Human Brain

PubMed Central

Spiteri, Elizabeth ; Konopka, Genevieve ; Coppola, Giovanni ; Bomar, Jamee ; Oldham, Michael ; Ou, Jing ; Vernes, Sonja C. ; Fisher, Simon E. ; Ren, Bing ; Geschwind, Daniel H. 

2007-01-01

Mutations in FOXP2, a member of the forkhead family of transcription factor genes, are the only known cause of developmental speech and language disorders in humans. To date, there are no known targets of human FOXP2 in the nervous system. The identification of FOXP2 targets in the developing human brain, therefore, provides a unique tool with which to explore the development of human language and speech. Here, we define FOXP2 targets in human basal ganglia (BG) and inferior frontal cortex (IFC) by use of chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and validate the functional regulation of targets in vitro. ChIP-chip identified 285 FOXP2 targets in fetal human brain; statistically significant overlap of targets in BG and IFC indicates a core set of 34 transcriptional targets of FOXP2. We identified targets specific to IFC or BG that were not observed in lung, suggesting important regional and tissue differences in FOXP2 activity. Many target genes are known to play critical roles in specific aspects of central nervous system patterning or development, such as neurite outgrowth, as well as plasticity. Subsets of the FOXP2 transcriptional targets are either under positive selection in humans or differentially expressed between human and chimpanzee brain. This is the first ChIP-chip study to use human brain tissue, making the FOXP2-target genes identified in these studies important to understanding the pathways regulating speech and language in the developing human brain. These data provide the first insight into the functional network of genes directly regulated by FOXP2 in human brain and by evolutionary comparisons, highlighting genes likely to be involved in the development of human higher-order cognitive processes. PMID:17999357

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis.

PubMed

Camps, Montserrat; Rückle, Thomas; Ji, Hong; Ardissone, Vittoria; Rintelen, Felix; Shaw, Jeffrey; Ferrandi, Chiara; Chabert, Christian; Gillieron, Corine; Françon, Bernard; Martin, Thierry; Gretener, Denise; Perrin, Dominique; Leroy, Didier; Vitte, Pierre-Alain; Hirsch, Emilio; Wymann, Matthias P; Cirillo, Rocco; Schwarz, Matthias K; Rommel, Christian

2005-09-01

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.

Improved prediction of drug-target interactions using regularized least squares integrating with kernel fusion technique.

PubMed

Hao, Ming; Wang, Yanli; Bryant, Stephen H

2016-02-25

Identification of drug-target interactions (DTI) is a central task in drug discovery processes. In this work, a simple but effective regularized least squares integrating with nonlinear kernel fusion (RLS-KF) algorithm is proposed to perform DTI predictions. Using benchmark DTI datasets, our proposed algorithm achieves the state-of-the-art results with area under precision-recall curve (AUPR) of 0.915, 0.925, 0.853 and 0.909 for enzymes, ion channels (IC), G protein-coupled receptors (GPCR) and nuclear receptors (NR) based on 10 fold cross-validation. The performance can further be improved by using a recalculated kernel matrix, especially for the small set of nuclear receptors with AUPR of 0.945. Importantly, most of the top ranked interaction predictions can be validated by experimental data reported in the literature, bioassay results in the PubChem BioAssay database, as well as other previous studies. Our analysis suggests that the proposed RLS-KF is helpful for studying DTI, drug repositioning as well as polypharmacology, and may help to accelerate drug discovery by identifying novel drug targets. Published by Elsevier B.V.

Visualization and Analysis of MiRNA-Targets Interactions Networks.

PubMed

León, Luis E; Calligaris, Sebastián D

2017-01-01

MicroRNAs are a class of small, noncoding RNA molecules of 21-25 nucleotides in length that regulate the gene expression by base-pairing with the target mRNAs, mainly leading to down-regulation or repression of the target genes. MicroRNAs are involved in diverse regulatory pathways in normal and pathological conditions. In this context, it is highly important to identify the targets of specific microRNA in order to understand the mechanism of its regulation and consequently its involvement in disease. However, the microRNA target identification is experimentally laborious and time-consuming. The in silico prediction of microRNA targets is an extremely useful approach because you can identify potential mRNA targets, reduce the number of possibilities and then, validate a few microRNA-mRNA interactions in an in vitro experimental model. In this chapter, we describe, in a simple way, bioinformatics guidelines to use miRWalk database and Cytoscape software for analyzing microRNA-mRNA interactions through their visualization as a network.

Customizable PCR-microplate array for differential identification of multiple pathogens

PubMed Central

Woubit, Abdela; Yehualaeshet, Teshome; Roberts, Sherrelle; Graham, Martha; Kim, Moonil; Samuel, Temesgen

2014-01-01

Customizable PCR-microplate arrays were developed for the rapid identification of Francisella tularensis subsp. tularensis, Salmonella Typhi, Shigella dysenteriae, Yersinia pestis, Vibrio cholerae Escherichia coli O157:H7, Salmonella Typhimurium, Salmonella Saintpaul, Francisella tularensis subsp. novicida, Vibrio parahaemolyticus, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of the pathogens above. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers. A mixed aliquot of genomic DNA from 38 different strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Results show specific amplifications on all the three custom plates. In a preliminary test to evaluate the sensitivity of these assays in laboratory-inoculated samples, detection limits as low as 9 cfu/g/ml S. Typhimurium were obtained from beef hot dog, and 78 cfu/ml from milk. Such microplate arrays could serve as valuable tools for initial identification or secondary confirmation of these pathogens. PMID:24215700

Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2.

PubMed

Cao, Jijuan; Xu, Junyi; Zhao, Tongtong; Cao, Dongmei; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2014-01-01

The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2.

33 CFR 101.514 - TWIC Requirement.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Document (MMD); (2) A valid Merchant Mariner License and a valid photo identification; or (3) A valid Certificate of Registry and a valid photo identification. [USCG-2006-24196, 72 FR 3578, Jan. 25, 2007, as...

33 CFR 101.514 - TWIC Requirement.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Document (MMD); (2) A valid Merchant Mariner License and a valid photo identification; or (3) A valid Certificate of Registry and a valid photo identification. [USCG-2006-24196, 72 FR 3578, Jan. 25, 2007, as...

33 CFR 101.514 - TWIC Requirement.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Document (MMD); (2) A valid Merchant Mariner License and a valid photo identification; or (3) A valid Certificate of Registry and a valid photo identification. [USCG-2006-24196, 72 FR 3578, Jan. 25, 2007, as...

33 CFR 101.514 - TWIC Requirement.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Document (MMD); (2) A valid Merchant Mariner License and a valid photo identification; or (3) A valid Certificate of Registry and a valid photo identification. [USCG-2006-24196, 72 FR 3578, Jan. 25, 2007, as...

33 CFR 101.514 - TWIC Requirement.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Document (MMD); (2) A valid Merchant Mariner License and a valid photo identification; or (3) A valid Certificate of Registry and a valid photo identification. [USCG-2006-24196, 72 FR 3578, Jan. 25, 2007, as...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Webb-Robertson, Bobbie-Jo M.

Accurate identification of peptides is a current challenge in mass spectrometry (MS) based proteomics. The standard approach uses a search routine to compare tandem mass spectra to a database of peptides associated with the target organism. These database search routines yield multiple metrics associated with the quality of the mapping of the experimental spectrum to the theoretical spectrum of a peptide. The structure of these results make separating correct from false identifications difficult and has created a false identification problem. Statistical confidence scores are an approach to battle this false positive problem that has led to significant improvements in peptidemore » identification. We have shown that machine learning, specifically support vector machine (SVM), is an effective approach to separating true peptide identifications from false ones. The SVM-based peptide statistical scoring method transforms a peptide into a vector representation based on database search metrics to train and validate the SVM. In practice, following the database search routine, a peptides is denoted in its vector representation and the SVM generates a single statistical score that is then used to classify presence or absence in the sample« less

Chemical proteomics for target discovery of head-to-tail cyclized mini-proteins

NASA Astrophysics Data System (ADS)

Hellinger, Roland; Thell, Kathrin; Vasileva, Mina; Muhammad, Taj; Gunasekera, Sunithi; Kümmel, Daniel; Göransson, Ulf; Becker, Christian W.; Gruber, Christian W.

2017-10-01

Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomal synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target of the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

PubMed

Arduino, Daniela M; Wettmarshausen, Jennifer; Vais, Horia; Navas-Navarro, Paloma; Cheng, Yiming; Leimpek, Anja; Ma, Zhongming; Delrio-Lorenzo, Alba; Giordano, Andrea; Garcia-Perez, Cecilia; Médard, Guillaume; Kuster, Bernhard; García-Sancho, Javier; Mokranjac, Dejana; Foskett, J Kevin; Alonso, M Teresa; Perocchi, Fabiana

2017-08-17

The mitochondrial calcium uniporter complex is essential for calcium (Ca 2+ ) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca 2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed.

PubMed

Bahrdt, C; Krech, A B; Wurz, A; Wulff, D

2010-03-01

For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD(abs)) < or = ten target copies was proven in hexaplex format. A sensitivity < or = ten target copies of each GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency.

Opportunities and challenges of disease biomarkers: a new section in the Journal of Translational Medicine.

PubMed

Wang, Xiangdong; Ward, Peter A

2012-12-05

Disease biomarkers are defined to diagnose various phases of diseases, monitor severities of diseases and responses to therapies, or predict prognosis of patients. Disease-specific biomarkers should benefit drug discovery and development, integrate multidisciplinary sciences, be validated by molecular imaging. The opportunities and challenges in biomarker development are emphasized and considered. The Journal of Translational Medicine opens a new Section of Disease Biomarkers to bridge identification and validation of gene or protein-based biomarkers, network biomarkers, dynamic network biomarkers in human diseases, patient phenotypes, and clinical applications. Disease biomarkers are also important for determining drug effects, target specificities and binding, dynamic metabolism and pharmacological kinetics, or toxicity profiles.

Functional validation of the oncogenic cooperativity and targeting potential of tuberous sclerosis mutation in medulloblastoma using a MYC-amplified model cell line.

PubMed

Henderson, Jacob J; Wagner, Jacob P; Hofmann, Nicolle E; Eide, Christopher A; Cho, Yoon-Jae; Druker, Brian J; Davare, Monika A

2017-10-01

Medulloblastoma is the most common malignant brain tumor of childhood. To identify targetable vulnerabilities, we employed inhibitor screening that revealed mTOR inhibitor hypersensitivity in the MYC-overexpressing medulloblastoma cell line, D341. Concomitant exome sequencing unveiled an uncharacterized missense mutation, TSC2 A415V , in these cells. We biochemically demonstrate that the TSC2 A415V mutation is functionally deleterious, leading to shortened half-life and proteasome-mediated protein degradation. These data suggest that MYC cooperates with activated kinase pathways, enabling pharmacologic intervention in these treatment refractory tumors. We propose that identification of activated kinase pathways may allow for tailoring targeted therapy to improve survival and treatment-related morbidity in medulloblastoma. © 2017 Wiley Periodicals, Inc.

Drug Target Protein-Protein Interaction Networks: A Systematic Perspective

PubMed Central

2017-01-01

The identification and validation of drug targets are crucial in biomedical research and many studies have been conducted on analyzing drug target features for getting a better understanding on principles of their mechanisms. But most of them are based on either strong biological hypotheses or the chemical and physical properties of those targets separately. In this paper, we investigated three main ways to understand the functional biomolecules based on the topological features of drug targets. There are no significant differences between targets and common proteins in the protein-protein interactions network, indicating the drug targets are neither hub proteins which are dominant nor the bridge proteins. According to some special topological structures of the drug targets, there are significant differences between known targets and other proteins. Furthermore, the drug targets mainly belong to three typical communities based on their modularity. These topological features are helpful to understand how the drug targets work in the PPI network. Particularly, it is an alternative way to predict potential targets or extract nontargets to test a new drug target efficiently and economically. By this way, a drug target's homologue set containing 102 potential target proteins is predicted in the paper. PMID:28691014

Common pitfalls in preclinical cancer target validation.

PubMed

Kaelin, William G

2017-07-01

An alarming number of papers from laboratories nominating new cancer drug targets contain findings that cannot be reproduced by others or are simply not robust enough to justify drug discovery efforts. This problem probably has many causes, including an underappreciation of the danger of being misled by off-target effects when using pharmacological or genetic perturbants in complex biological assays. This danger is particularly acute when, as is often the case in cancer pharmacology, the biological phenotype being measured is a 'down' readout (such as decreased proliferation, decreased viability or decreased tumour growth) that could simply reflect a nonspecific loss of cellular fitness. These problems are compounded by multiple hypothesis testing, such as when candidate targets emerge from high-throughput screens that interrogate multiple targets in parallel, and by a publication and promotion system that preferentially rewards positive findings. In this Perspective, I outline some of the common pitfalls in preclinical cancer target identification and some potential approaches to mitigate them.

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Gene expression complex networks: synthesis, identification, and analysis.

PubMed

Lopes, Fabrício M; Cesar, Roberto M; Costa, Luciano Da F

2011-10-01

Thanks to recent advances in molecular biology, allied to an ever increasing amount of experimental data, the functional state of thousands of genes can now be extracted simultaneously by using methods such as cDNA microarrays and RNA-Seq. Particularly important related investigations are the modeling and identification of gene regulatory networks from expression data sets. Such a knowledge is fundamental for many applications, such as disease treatment, therapeutic intervention strategies and drugs design, as well as for planning high-throughput new experiments. Methods have been developed for gene networks modeling and identification from expression profiles. However, an important open problem regards how to validate such approaches and its results. This work presents an objective approach for validation of gene network modeling and identification which comprises the following three main aspects: (1) Artificial Gene Networks (AGNs) model generation through theoretical models of complex networks, which is used to simulate temporal expression data; (2) a computational method for gene network identification from the simulated data, which is founded on a feature selection approach where a target gene is fixed and the expression profile is observed for all other genes in order to identify a relevant subset of predictors; and (3) validation of the identified AGN-based network through comparison with the original network. The proposed framework allows several types of AGNs to be generated and used in order to simulate temporal expression data. The results of the network identification method can then be compared to the original network in order to estimate its properties and accuracy. Some of the most important theoretical models of complex networks have been assessed: the uniformly-random Erdös-Rényi (ER), the small-world Watts-Strogatz (WS), the scale-free Barabási-Albert (BA), and geographical networks (GG). The experimental results indicate that the inference method was sensitive to average degree variation, decreasing its network recovery rate with the increase of . The signal size was important for the inference method to get better accuracy in the network identification rate, presenting very good results with small expression profiles. However, the adopted inference method was not sensible to recognize distinct structures of interaction among genes, presenting a similar behavior when applied to different network topologies. In summary, the proposed framework, though simple, was adequate for the validation of the inferred networks by identifying some properties of the evaluated method, which can be extended to other inference methods.

TipMT: Identification of PCR-based taxon-specific markers.

PubMed

Rodrigues-Luiz, Gabriela F; Cardoso, Mariana S; Valdivia, Hugo O; Ayala, Edward V; Gontijo, Célia M F; Rodrigues, Thiago de S; Fujiwara, Ricardo T; Lopes, Robson S; Bartholomeu, Daniella C

2017-02-11

Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity. Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species. The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/ .

Life in the fast lane: high-throughput chemistry for lead generation and optimisation.

PubMed

Hunter, D

2001-01-01

The pharmaceutical industry has come under increasing pressure due to regulatory restrictions on the marketing and pricing of drugs, competition, and the escalating costs of developing new drugs. These forces can be addressed by the identification of novel targets, reductions in the development time of new drugs, and increased productivity. Emphasis has been placed on identifying and validating new targets and on lead generation: the response from industry has been very evident in genomics and high throughput screening, where new technologies have been applied, usually coupled with a high degree of automation. The combination of numerous new potential biological targets and the ability to screen large numbers of compounds against many of these targets has generated the need for large diverse compound collections. To address this requirement, high-throughput chemistry has become an integral part of the drug discovery process. Copyright 2002 Wiley-Liss, Inc.

Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.

PubMed

Bosse, Kristopher R; Raman, Pichai; Zhu, Zhongyu; Lane, Maria; Martinez, Daniel; Heitzeneder, Sabine; Rathi, Komal S; Kendsersky, Nathan M; Randall, Michael; Donovan, Laura; Morrissy, Sorana; Sussman, Robyn T; Zhelev, Doncho V; Feng, Yang; Wang, Yanping; Hwang, Jennifer; Lopez, Gonzalo; Harenza, Jo Lynne; Wei, Jun S; Pawel, Bruce; Bhatti, Tricia; Santi, Mariarita; Ganguly, Arupa; Khan, Javed; Marra, Marco A; Taylor, Michael D; Dimitrov, Dimiter S; Mackall, Crystal L; Maris, John M

2017-09-11

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target. Copyright © 2017 Elsevier Inc. All rights reserved.

Ships and Maritime Targets Observation Campaigns Using Available C- and X-Band SAR Satellite

NASA Astrophysics Data System (ADS)

Velotto, Domenico; Bentes, Carlos; Lehner, Susanne

2015-04-01

Obviously, radar resolution and swath width are two very important factors when it comes to synthetic aperture radar (SAR) maritime targets detections. The dilemma of using single polarization SAR imagery with higher resolution and coverage or quad- (or dual- polarimetric) imagery with its richness of information, is still unsolved when it comes to this application.In the framework of ESA project MARISS and EU project DOLPHIN, in situ campaigns aimed at solving this dilemma have been carried out. Single and multi- polarimetric SAR data acquired by TerraSAR-X, RADARSAT-2 and COSMO-SkyMed have been acquired with close time gaps and partial coverage overlap. In this way several moving and non-moving maritime targets have been imaged with different polarization, geometry and working frequency. Available ground truth reports provided by Automatic Identification System (AIS) data, nautical chart and wind farm location are used to validate the different types of maritime targets.

Identification and characterization of potential druggable targets among hypothetical proteins of extensively drug resistant Mycobacterium tuberculosis (XDR KZN 605) through subtractive genomics approach.

PubMed

Uddin, Reaz; Siddiqui, Quratulain Nehal; Azam, Syed Sikander; Saima, Bibi; Wadood, Abdul

2018-03-01

Among the resistant isolates of tuberculosis (TB), the multidrug resistance tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) are the areas of growing concern for which the front-line antibiotics are no more effective. As a result, the search of new therapeutic targets against TB is an imperative need of time. On the other hand, the target identification is an a priori step in drug discovery based research. Furthermore, the availability of the complete proteomic data of extensively drug resistant Mycobacterium tuberculosis (XDR-MTB) made it possible to carry out in silico analysis for the discovery of new drug targets. In the current study, we aimed to prioritize the potential drug targets among the hypothetical proteins of XDR-TB via subtractive genomics approach. In the subtractive genomics, we stepwise reduced the complete proteome of XDR-MTB to only two hypothetical proteins and evidently proposed them as new therapeutic targets. The 3D structure of one of the two target proteins was predicted via homology modeling and later on, validated by various analysis tools. Our study suggested that the domains identified and the motif hits found in the sequences of the shortlisted drug targets are crucial for the survival of the XDR-MTB. To the best of our knowledge, the current study is the first attempt in which the complete proteomic data of XDR-MTB was subjected to the computational subtractive genomics approach and therefore, would provide an opportunity to identify the unique therapeutic targets against deadly XDR-MTB. Copyright © 2017 Elsevier B.V. All rights reserved.

Biocomputational identification and validation of novel microRNAs predicted from bubaline whole genome shotgun sequences.

PubMed

Manku, H K; Dhanoa, J K; Kaur, S; Arora, J S; Mukhopadhyay, C S

2017-10-01

MicroRNAs (miRNAs) are small (19-25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo. Copyright © 2017 Elsevier Ltd. All rights reserved.

Objective, Quantitative, Data-Driven Assessment of Chemical Probes.

PubMed

Antolin, Albert A; Tym, Joseph E; Komianou, Angeliki; Collins, Ian; Workman, Paul; Al-Lazikani, Bissan

2018-02-15

Chemical probes are essential tools for understanding biological systems and for target validation, yet selecting probes for biomedical research is rarely based on objective assessment of all potential compounds. Here, we describe the Probe Miner: Chemical Probes Objective Assessment resource, capitalizing on the plethora of public medicinal chemistry data to empower quantitative, objective, data-driven evaluation of chemical probes. We assess >1.8 million compounds for their suitability as chemical tools against 2,220 human targets and dissect the biases and limitations encountered. Probe Miner represents a valuable resource to aid the identification of potential chemical probes, particularly when used alongside expert curation. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

PubMed

Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing

2008-02-01

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.

High-throughput identification and dendritic cell-based functional validation of MHC class I-restricted Mycobacterium tuberculosis epitopes

PubMed Central

Nair, Smita K.; Tomaras, Georgia D.; Sales, Ana Paula; Boczkowski, David; Chan, Cliburn; Plonk, Kelly; Cai, Yongting; Dannull, Jens; Kepler, Thomas B.; Pruitt, Scott K.; Weinhold, Kent J.

2014-01-01

Emergence of drug-resistant strains of the pathogen Mycobacterium tuberculosis (Mtb) and the ineffectiveness of BCG in curtailing Mtb infection makes vaccine development for tuberculosis an important objective. Identifying immunogenic CD8+ T cell peptide epitopes is necessary for peptide-based vaccine strategies. We present a three-tiered strategy for identifying and validating immunogenic peptides: first, identify peptides that form stable complexes with class I MHC molecules; second, determine whether cytotoxic T lymphocytes (CTLs) raised against the whole protein antigen recognize and lyse target cells pulsed with peptides that passed step 1; third, determine whether peptides that passed step 2, when administered in vivo as a vaccine in HLA-A2 transgenic mice, elicit CTLs that lyse target cells expressing the whole protein antigen. Our innovative approach uses dendritic cells transfected with Mtb antigen-encoding mRNA to drive antigen expression. Using this strategy, we have identified five novel peptide epitopes from the Mtb proteins Apa, Mtb8.4 and Mtb19. PMID:24755960

Measuring health-related problem solving among African Americans with multiple chronic conditions: application of Rasch analysis.

PubMed

Fitzpatrick, Stephanie L; Hill-Briggs, Felicia

2015-10-01

Identification of patients with poor chronic disease self-management skills can facilitate treatment planning, determine effectiveness of interventions, and reduce disease complications. This paper describes the use of a Rasch model, the Rating Scale Model, to examine psychometric properties of the 50-item Health Problem-Solving Scale (HPSS) among 320 African American patients with high risk for cardiovascular disease. Items on the positive/effective HPSS subscales targeted patients at low, moderate, and high levels of positive/effective problem solving, whereas items on the negative/ineffective problem solving subscales mostly targeted those at moderate or high levels of ineffective problem solving. Validity was examined by correlating factor scores on the measure with clinical and behavioral measures. Items on the HPSS show promise in the ability to assess health-related problem solving among high risk patients. However, further revisions of the scale are needed to increase its usability and validity with large, diverse patient populations in the future.

The topoisomerase II-Hsp90 complex: a new chemotherapeutic target?

PubMed

Barker, Catherine R; Hamlett, Jane; Pennington, Stephen R; Burrows, Francis; Lundgren, Karen; Lough, Rachel; Watson, Alastair J M; Jenkins, John R

2006-06-01

The modulation of DNA topology by topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and has thus become a highly attractive target for chemotherapeutic drugs. However, these drugs are highly toxic, and so new approaches are required. One such strategy is to target topoisomerase II-interacting proteins. Here we report the identification of potential topoisomerase II-associated proteins using immunoprecipitation, followed by 1-D and 2-D gel electrophoresis and MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIalpha-associated proteins by coimmunoprecipitation and Western blot. Six of the interacting proteins were cellular chaperones, including 3 members of the heat shock protein-90 (Hsp90) family, and so the effect of Hsp90 modulation on the antitumor activity of topoisomerase II drugs was tested using the sulforhodamine B assay, clonogenic assays and a xenograft model. The Hsp90 inhibitors geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and radicicol significantly enhanced the activity of the topoisomerase II poisons etoposide and mitoxantrone in vitro and in vivo. Thus, our method of identifying topoisomerase II-interacting proteins appears to be effective, and at least 1 novel topoisomerase IIalpha-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy.

Spectral algorithm for non-destructive damage localisation: Application to an ancient masonry arch model

NASA Astrophysics Data System (ADS)

Masciotta, Maria-Giovanna; Ramos, Luís F.; Lourenço, Paulo B.; Vasta, Marcello

2017-02-01

Structural monitoring and vibration-based damage identification methods are fundamental tools for condition assessment and early-stage damage identification, especially when dealing with the conservation of historical constructions and the maintenance of strategic civil structures. However, although the substantial advances in the field, several issues must still be addressed to broaden the application range of such tools and to assert their reliability. This study deals with the experimental validation of a novel method for non-destructive damage identification purposes. This method is based on the use of spectral output signals and has been recently validated by the authors through a numerical simulation. After a brief insight into the basic principles of the proposed approach, the spectral-based technique is applied to identify the experimental damage induced on a masonry arch through statically increasing loading. Once the direct and cross spectral density functions of the nodal response processes are estimated, the system's output power spectrum matrix is built and decomposed in eigenvalues and eigenvectors. The present study points out how the extracted spectral eigenparameters contribute to the damage analysis allowing to detect the occurrence of damage and to locate the target points where the cracks appear during the experimental tests. The sensitivity of the spectral formulation to the level of noise in the modal data is investigated and discussed. As a final evaluation criterion, the results from the spectrum-driven method are compared with the ones obtained from existing non-model based damage identification methods.

Genome-wide identification of conserved and novel microRNAs in one bud and two tender leaves of tea plant (Camellia sinensis) by small RNA sequencing, microarray-based hybridization and genome survey scaffold sequences.

PubMed

Jeyaraj, Anburaj; Zhang, Xiao; Hou, Yan; Shangguan, Mingzhu; Gajjeraman, Prabu; Li, Yeyun; Wei, Chaoling

2017-11-21

MicroRNAs (miRNAs) are important for plant growth and responses to environmental stresses via post-transcriptional regulation of gene expression. Tea, which is primarily produced from one bud and two tender leaves of the tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages worldwide owing to its abundance of secondary metabolites. A large number of miRNAs have been identified in various plants, including non-model species. However, due to the lack of reference genome sequences and/or information of tea plant genome survey scaffold sequences, discovery of miRNAs has been limited in C. sinensis. Using small RNA sequencing, combined with our recently obtained genome survey data, we have identified and analyzed 175 conserved and 83 novel miRNAs mainly in one bud and two tender leaves of the tea plant. Among these, 93 conserved and 18 novel miRNAs were validated using miRNA microarray hybridization. In addition, the expression pattern of 11 conserved and 8 novel miRNAs were validated by stem-loop-qRT-PCR. A total of 716 potential target genes of identified miRNAs were predicted. Further, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the target genes were primarily involved in stress response and enzymes related to phenylpropanoid biosynthesis. The predicted targets of 4 conserved miRNAs were further validated by 5'RLM-RACE. A negative correlation between expression profiles of 3 out of 4 conserved miRNAs (csn-miR160a-5p, csn-miR164a, csn-miR828 and csn-miR858a) and their targets (ARF17, NAC100, WER and MYB12 transcription factor) were observed. In summary, the present study is one of few such studies on miRNA detection and identification in the tea plant. The predicted target genes of majority of miRNAs encoded enzymes, transcription factors, and functional proteins. The miRNA-target transcription factor gene interactions may provide important clues about the regulatory mechanism of these miRNAs in the tea plant. The data reported in this study will make a huge contribution to knowledge on the potential miRNA regulators of the secondary metabolism pathway and other important biological processes in C. sinensis.

Identification and validation of vesicant therapeutic targets using a high, throughput siRNA screening approach

DTIC Science & Technology

2014-12-24

toxlet.2011.04.007 Rogers JV, Choi YW, Kiser RC et al (2004) Microarray analysis of gene expression in murine skin exposed to sulfur mustard. J Bio...Chemotactic factors released in culture by intact developing and healing skin lesions produced in rabbits by the irritant sulfur mustard. Inflam- mation 21(2...Project ID Number CBM.CUTOC.04.10. RC 00114. ABSTRACT See reprint. 15. SUBJECT TERMS sulfur mustard, cutaneous injury, siRNA, high-throughput screening

Identification of miRNA Signatures Associated with Epithelial Ovarian Cancer Chemoresistance with Further Biological and Functional Validation of Identified Key miRNAs

DTIC Science & Technology

2015-02-01

OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE 2. REPORT TYPE Annual 3 . DATES COVERED (From - To) 1/30...specimens from women diagnosed with stage III, grade 3 , papillary serous adenocarcinoma all treated with platinum-based chemotherapy. Furthermore, we...and reveal that targeting this miRNA as a novel therapeutic option in ovarian cancer (Task 3 ). 15. SUBJECT TERMS microRNA, ovarian cancer, platinum

Nematode Species Identification-Current Status, Challenges and Future Perspectives for Cyathostomins.

PubMed

Bredtmann, Christina M; Krücken, Jürgen; Murugaiyan, Jayaseelan; Kuzmina, Tetiana; von Samson-Himmelstjerna, Georg

2017-01-01

Human and animal health is globally affected by a variety of parasitic helminths. The impact of co-infections and development of anthelmintic resistance requires improved diagnostic tools, especially for parasitic nematodes e.g., to identify resistant species or attribute pathological effects to individual species or particular species combinations. In horses, co-infection with cyathostomins is rather a rule than an exception with typically 5 to 15 species (out of more than 40 described) per individual host. In cyathostomins, reliable morphological species differentiation is currently limited to adults and requires highly specialized expertize while precise morphological identification of eggs and early stage larvae is impossible. The situation is further complicated by a questionable validity of some cyathostomins while others might actually represent cryptic species complexes. Several molecular methods using different target sequences were established to overcome these limitations. For adult worms, PCR followed by sequencing of mitochondrial genes or external or internal ribosomal RNA spacers is suitable to genetically confirm morphological identifications. The most commonly used method to differentiate eggs or larvae is the reverse-line-blot hybridization assay. However, both methods suffer from the fact that target sequences are not available for many species or even that GenBank® entries are unreliable regarding the cyathostomin species. Recent advances in proteomic tools for identification of metazoans including insects and nematodes of the genus Trichinella will be evaluated for suitability to diagnose cyathostomins. Future research should focus on the comparative analysis of morphological, molecular and proteomic data from the same cyathostomin specimen to optimize tools for species-specific identification.

Proteomics meets blood banking: identification of protein targets for the improvement of platelet quality.

PubMed

Schubert, Peter; Devine, Dana V

2010-01-03

Proteomics has brought new perspectives to the fields of hematology and transfusion medicine in the last decade. The steady improvement of proteomic technology is propelling novel discoveries of molecular mechanisms by studying protein expression, post-translational modifications and protein interactions. This review article focuses on the application of proteomics to the identification of molecular mechanisms leading to the deterioration of blood platelets during storage - a critical aspect in the provision of platelet transfusion products. Several proteomic approaches have been employed to analyse changes in the platelet protein profile during storage and the obtained data now need to be translated into platelet biochemistry in order to connect the results to platelet function. Targeted biochemical applications then allow the identification of points for intervention in signal transduction pathways. Once validated and placed in a transfusion context, these data will provide further understanding of the underlying molecular mechanisms leading to platelet storage lesion. Future aspects of proteomics in blood banking will aim to make use of protein markers identified for platelet storage lesion development to monitor proteome changes when alterations such as the use of additive solutions or pathogen reduction strategies are put in place in order to improve platelet quality for patients. (c) 2009 Elsevier B.V. All rights reserved.

Systematic Identification of Molecular Subtype-Selective Vulnerabilities in Non Small Cell Lung Cancer

PubMed Central

Kim, Hyun Seok; Mendiratta, Saurabh; Kim, Jiyeon; Pecot, Chad Victor; Larsen, Jill E.; Zubovych, Iryna; Seo, Bo Yeun; Kim, Jimi; Eskiocak, Banu; Chung, Hannah; McMillan, Elizabeth; Wu, Sherry; De Brabander, Jef; Komurov, Kakajan; Toombs, Jason E.; Wei, Shuguang; Peyton, Michael; Williams, Noelle; Gazdar, Adi F.; Posner, Bruce A.; Brekken, Rolf; Sood, Anil K.; Deberardinis, Ralph J.; Roth, Michael G.; Minna, John D.; White, Michael A.

2013-01-01

SUMMARY Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6–16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occuring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a 7-gene expression signature. Target efficacies were validated in vivo, and mechanism of action studies uncovered new cancer cell biology. PMID:24243015

Systematic identification of molecular subtype-selective vulnerabilities in non-small-cell lung cancer.

PubMed

Kim, Hyun Seok; Mendiratta, Saurabh; Kim, Jiyeon; Pecot, Chad Victor; Larsen, Jill E; Zubovych, Iryna; Seo, Bo Yeun; Kim, Jimi; Eskiocak, Banu; Chung, Hannah; McMillan, Elizabeth; Wu, Sherry; De Brabander, Jef; Komurov, Kakajan; Toombs, Jason E; Wei, Shuguang; Peyton, Michael; Williams, Noelle; Gazdar, Adi F; Posner, Bruce A; Brekken, Rolf A; Sood, Anil K; Deberardinis, Ralph J; Roth, Michael G; Minna, John D; White, Michael A

2013-10-24

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of KasA as the cellular target of an anti-tubercular scaffold

PubMed Central

Abrahams, Katherine A.; Chung, Chun-wa; Ghidelli-Disse, Sonja; Rullas, Joaquín; Rebollo-López, María José; Gurcha, Sudagar S.; Cox, Jonathan A. G.; Mendoza, Alfonso; Jiménez-Navarro, Elena; Martínez-Martínez, María Santos; Neu, Margarete; Shillings, Anthony; Homes, Paul; Argyrou, Argyrides; Casanueva, Ruth; Loman, Nicholas J.; Moynihan, Patrick J.; Lelièvre, Joël; Selenski, Carolyn; Axtman, Matthew; Kremer, Laurent; Bantscheff, Marcus; Angulo-Barturen, Iñigo; Izquierdo, Mónica Cacho; Cammack, Nicholas C.; Drewes, Gerard; Ballell, Lluis; Barros, David; Besra, Gurdyal S.; Bates, Robert H.

2016-01-01

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis. PMID:27581223

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

PubMed

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

2014-04-15

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Efforts Aimed To Reduce Attrition in Antimalarial Drug Discovery: A Systematic Evaluation of the Current Antimalarial Targets Portfolio.

PubMed

Chaparro, María Jesús; Calderón, Félix; Castañeda, Pablo; Fernández-Alvaro, Elena; Gabarró, Raquel; Gamo, Francisco Javier; Gómez-Lorenzo, María G; Martín, Julio; Fernández, Esther

2018-04-13

Malaria remains a major global health problem. In 2015 alone, more than 200 million cases of malaria were reported, and more than 400,000 deaths occurred. Since 2010, emerging resistance to current front-line ACTs (artemisinin combination therapies) has been detected in endemic countries. Therefore, there is an urgency for new therapies based on novel modes of action, able to relieve symptoms as fast as the artemisinins and/or block malaria transmission. During the past few years, the antimalarial community has focused their efforts on phenotypic screening as a pragmatic approach to identify new hits. Optimization efforts on several chemical series have been successful, and clinical candidates have been identified. In addition, recent advances in genetics and proteomics have led to the target deconvolution of phenotypic clinical candidates. New mechanisms of action will also be critical to overcome resistance and reduce attrition. Therefore, a complementary strategy focused on identifying well-validated targets to start hit identification programs is essential to reinforce the clinical pipeline. Leveraging published data, we have assessed the status quo of the current antimalarial target portfolio with a focus on the blood stage clinical disease. From an extensive list of reported Plasmodium targets, we have defined triage criteria. These criteria consider genetic, pharmacological, and chemical validation, as well as tractability/doability, and safety implications. These criteria have provided a quantitative score that has led us to prioritize those targets with the highest probability to deliver successful and differentiated new drugs.

Loss of Cytoplasmic CDK1 Predicts Poor Survival in Human Lung Cancer and Confers Chemotherapeutic Resistance

PubMed Central

Zhang, Chunyu; Elkahloun, Abdel G.; Robertson, Matthew; Gills, Joell J.; Tsurutani, Junji; Shih, Joanna H.; Fukuoka, Junya; Hollander, M. Christine; Harris, Curtis C.; Travis, William D.; Jen, Jin; Dennis, Phillip A.

2011-01-01

The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery. PMID:21887332

Lineup identification by children: effects of clothing bias.

PubMed

Freire, Alejo; Lee, Kang; Williamson, Karen S; Stuart, Sarah J E; Lindsay, R C L

2004-06-01

This study examined effects of clothing cues on children's identification accuracy from lineups. Four- to 14-year-olds (n = 228) saw 12 video clips of individuals, each wearing a distinctly colored shirt. After watching each clip children were presented with a target-present or target-absent photo lineup. Three clothing conditions were included. In 2 conditions all lineup members wore the same colored shirt; in the third, biased condition, the shirt color of only one individual matched that seen in the preceding clip (the target in target-present trials and the replacement in target-absent trials). Correct identifications of the target in target-present trials were most frequent in the biased condition, whereas in target-absent trials the biased condition led to more false identifications of the target replacement. Older children were more accurate than younger children, both in choosing the target from target-present lineups and rejecting target-absent lineups. These findings suggest that a simple clothing cue such as shirt color can have a significant impact on children's lineup identification accuracy.

Lineup Identification by Children: Effects of Clothing Bias

PubMed Central

Freire, Alejo; Lee, Kang; Williamson, Karen S.; Stuart, Sarah J. E.; Lindsay, R. C. L.

2008-01-01

This study examined effects of clothing cues on children's identification accuracy from lineups. Four- to 14-year-olds (n = 228) saw 12 video clips of individuals, each wearing a distinctly colored shirt. After watching each clip children were presented with a target-present or target-absent photo lineup. Three clothing conditions were included. In 2 conditions all lineup members wore the same colored shirt; in the third, biased condition, the shirt color of only one individual matched that seen in the preceding clip (the target in target-present trials and the replacement in target-absent trials). Correct identifications of the target in target-present trials were most frequent in the biased condition, whereas in target-absent trials the biased condition led to more false identifications of the target replacement. Older children were more accurate than younger children, both in choosing the target from target-present lineups and rejecting target-absent lineups. These findings suggest that a simple clothing cue such as shirt color can have a significant impact on children's lineup identification accuracy. PMID:15264450

An integrated miRNA functional screening and target validation method for organ morphogenesis.

PubMed

Rebustini, Ivan T; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A; Maas, Richard L

2016-03-16

The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs.

Application of Fragment Based Drug Discovery to Membrane Proteins: Biophysical Identification of Ligands of the Integral Membrane Enzyme DsbB

PubMed Central

Früh, Virginie; Zhou, Yunpeng; Chen, Dan; Loch, Caroline; Eiso, AB; Grinkova, Yelena N.; Verheij, Herman; Sligar, Stephen G; Bushweller, John H.; Siegal, Gregg

2014-01-01

Summary Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The E. coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1,071 drug fragments for binding using Target Immobilized NMR Screening. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins. PMID:20797617

Pharmacokinetics of Chinese medicines: strategies and perspectives.

PubMed

Yan, Ru; Yang, Ying; Chen, Yijia

2018-01-01

The modernization and internationalization of Chinese medicines (CMs) are hampered by increasing concerns on the safety and the efficacy. Pharmacokinetic (PK) study is indispensable to establish concentration-activity/toxicity relationship and facilitate target identification and new drug discovery from CMs. To cope with tremendous challenges rooted from chemical complexity of CMs, the classic PK strategies have evolved rapidly from PK study focusing on marker/main drug components to PK-PD correlation study adopting metabolomics approaches to characterize associations between disposition of global drug-related components and host metabolic network shifts. However, the majority of PK studies of CMs have adopted the approaches tailored for western medicines and focused on the systemic exposures of drug-related components, most of which were found to be too low to account for the holistic benefits of CMs. With an area under concentration-time curve- or activity-weighted approach, integral PK attempts to understand the PK-PD relevance with the integrated PK profile of multiple co-existing structural analogs (prototyes/metabolites). Cellular PK-PD complements traditional PK-PD when drug targets localize inside the cells, instead of at the surface of cell membrane or extracellular space. Considering the validated clinical benefits of CMs, reverse pharmacology-based reverse PK strategy was proposed to facilitate target identification and new drug discovery. Recently, gut microbiota have demonstrated multifaceted roles in drug efficacy/toxicity. In traditional oral intake, the presystemic interactions of CMs with gut microbiota seem inevitable, which can contribute to the holistic benefits of CMs through biotransforming CMs components, acting as the peripheral target, and regulating host drug disposition. Hence, we propose a global PK-PD approach which includes the presystemic interaction of CMs with gut microbiota and combines omics with physiologically based pharmacokinetic modeling to offer a comprehensive understanding of the PK-PD relationship of CMs. Moreover, validated clinical benefits of CMs and poor translational potential of animal PK data urge more research efforts in human PK study.

Identification of reduced-order thermal therapy models using thermal MR images: theory and validation.

PubMed

Niu, Ran; Skliar, Mikhail

2012-07-01

In this paper, we develop and validate a method to identify computationally efficient site- and patient-specific models of ultrasound thermal therapies from MR thermal images. The models of the specific absorption rate of the transduced energy and the temperature response of the therapy target are identified in the reduced basis of proper orthogonal decomposition of thermal images, acquired in response to a mild thermal test excitation. The method permits dynamic reidentification of the treatment models during the therapy by recursively utilizing newly acquired images. Such adaptation is particularly important during high-temperature therapies, which are known to substantially and rapidly change tissue properties and blood perfusion. The developed theory was validated for the case of focused ultrasound heating of a tissue phantom. The experimental and computational results indicate that the developed approach produces accurate low-dimensional treatment models despite temporal and spatial noises in MR images and slow image acquisition rate.

Customizable PCR-microplate array for differential identification of multiple pathogens.

PubMed

Woubit, Abdela; Yehualaeshet, Teshome; Roberts, Sherrelle; Graham, Martha; Kim, Moonil; Samuel, Temesgen

2013-11-01

Customizable PCR-microplate arrays were developed for the rapid identification of Salmonella Typhimurium, Salmonella Saintpaul, Salmonella Typhi, Shigella dysenteriae, Escherichia coli O157:H7, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. novicida, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of these pathogens. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers identified. A mixed aliquot of genomic DNA from 38 strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Specific amplifications were obtained on all three custom plates. In preliminary tests performed to evaluate the sensitivity of these assays in samples inoculated in the laboratory with Salmonella Typhimurium, amplifications were obtained from 1 g of beef hot dog inoculated at as low as 9 CFU/ml or from milk inoculated at as low as 78 CFU/ml. Such microplate arrays could be valuable tools for initial identification or secondary confirmation of contamination by these pathogens.

Computer aided manual validation of mass spectrometry-based proteomic data.

PubMed

Curran, Timothy G; Bryson, Bryan D; Reigelhaupt, Michael; Johnson, Hannah; White, Forest M

2013-06-15

Advances in mass spectrometry-based proteomic technologies have increased the speed of analysis and the depth provided by a single analysis. Computational tools to evaluate the accuracy of peptide identifications from these high-throughput analyses have not kept pace with technological advances; currently the most common quality evaluation methods are based on statistical analysis of the likelihood of false positive identifications in large-scale data sets. While helpful, these calculations do not consider the accuracy of each identification, thus creating a precarious situation for biologists relying on the data to inform experimental design. Manual validation is the gold standard approach to confirm accuracy of database identifications, but is extremely time-intensive. To palliate the increasing time required to manually validate large proteomic datasets, we provide computer aided manual validation software (CAMV) to expedite the process. Relevant spectra are collected, catalogued, and pre-labeled, allowing users to efficiently judge the quality of each identification and summarize applicable quantitative information. CAMV significantly reduces the burden associated with manual validation and will hopefully encourage broader adoption of manual validation in mass spectrometry-based proteomics. Copyright © 2013 Elsevier Inc. All rights reserved.

Treponema pallidum Putative Novel Drug Target Identification and Validation: Rethinking Syphilis Therapeutics with Plant-Derived Terpenoids

PubMed Central

Tiwari, Sameeksha; Singh, Priyanka; Singh, Swati; Awasthi, Manika; Pandey, Veda P.

2015-01-01

Abstract Syphilis, a slow progressive and the third most common sexually transmitted disease found worldwide, is caused by a spirochete gram negative bacteria Treponema pallidum. Emergence of antibiotic resistant T. pallidum has led to a search for novel drugs and their targets. Subtractive genomics analyses of pathogen T. pallidum and host Homo sapiens resulted in identification of 126 proteins essential for survival and viability of the pathogen. Metabolic pathway analyses of these essential proteins led to discovery of nineteen proteins distributed among six metabolic pathways unique to T. pallidum. One hundred plant-derived terpenoids, as potential therapeutic molecules against T. pallidum, were screened for their drug likeness and ADMET (absorption, distribution, metabolism, and toxicity) properties. Subsequently the resulting nine terpenoids were docked with five unique T. pallidum targets through molecular modeling approaches. Out of five targets analyzed, D-alanine:D-alanine ligase was found to be the most promising target, while terpenoid salvicine was the most potent inhibitor. A comparison of the inhibitory potential of the best docked readily available natural compound, namely pomiferin (flavonoid) with that of the best docked terpenoid salvicine, revealed that salvicine was a more potent inhibitor than that of pomiferin. To the best of our knowledge, this is the first report of a terpenoid as a potential therapeutic molecule against T. pallidum with D-alanine:D-alanine ligase as a novel target. Further studies are warranted to evaluate and explore the potential clinical ramifications of these findings in relation to syphilis that has public health importance worldwide. PMID:25683888

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development*

PubMed Central

Litholdo, Celso G.; Parker, Benjamin L.; Eamens, Andrew L.; Larsen, Martin R.; Cordwell, Stuart J.; Waterhouse, Peter M.

2016-01-01

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. PMID:27067051

Tobacco Use Prevention for the Young (TUPY-S): Development, Validity and Reliability of an Interactive Multimedia Strategy from the Adolescents’ Perspective in Malaysia

PubMed Central

Zin, Faridah Mohd; Hillaluddin, Azlin Hilma; Mustaffa, Jamaludin

2017-01-01

Objective: This study aims to develop, validate and determine the reliability of an interactive multimedia strategy to prevent tobacco use among the young (TUPY-S) from an adolescents’ perspective. Methods: A descriptive study design was utilized. A modular instruction guideline by Russel (1974) was followed in the entire process, comprising a feasibility study, a review of existing modules, specification of the objectives, identification of the construct criterion items, learner analysis and entry behavior specification, establishment of the sequence instruction and media selection, a tryout with students and a field test. Result: Feasibility was agreed among the researchers and the school authorities. Culturally suitable rigorously developed tobacco use preventive strategies delivered using information technology (IT) are lacking in the literature. The objective of TUPY-S is to prevent tobacco use among adolescents living in Malaysia. Identified construct criterion items include knowledge, attitude, intention to use, self-efficacy, and refusal skill. The target population was early adolescents belonging to generation-Z. Content was developed from the adolescents’ perspective and delivered using IT in Malay language. Content validity, assessed by six experts in the field and module development, was good at 86%. The students’ tryout showed satisfactory face validity subjectively and objectively (85.5%) and high alpha Cronbach reliability (0.91). Conclusion: TUPY-S was confirmed to suit early adolescents of the current generation living in Malaysia. It demonstrated good content validity among the experts, satisfactory face validity and reliability among the target population. TUPY-S is ready to be evaluated for its effectiveness among early adolescents. PMID:28612599

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

PubMed

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis.

PubMed

Clancy, Eoin; Coughlan, Helena; Higgins, Owen; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Reddington, Kate; Barry, Thomas

2016-08-01

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification and Validation of ESP Teacher Competencies: A Research Design

ERIC Educational Resources Information Center

Venkatraman, G.; Prema, P.

2013-01-01

The paper presents the research design used for identifying and validating a set of competencies required of ESP (English for Specific Purposes) teachers. The identification of the competencies and the three-stage validation process are also discussed. The observation of classes of ESP teachers for field-testing the validated competencies and…

In silico target prediction for elucidating the mode of action of herbicides including prospective validation.

PubMed

Chiddarwar, Rucha K; Rohrer, Sebastian G; Wolf, Antje; Tresch, Stefan; Wollenhaupt, Sabrina; Bender, Andreas

2017-01-01

The rapid emergence of pesticide resistance has given rise to a demand for herbicides with new mode of action (MoA). In the agrochemical sector, with the availability of experimental high throughput screening (HTS) data, it is now possible to utilize in silico target prediction methods in the early discovery phase to suggest the MoA of a compound via data mining of bioactivity data. While having been established in the pharmaceutical context, in the agrochemical area this approach poses rather different challenges, as we have found in this work, partially due to different chemistry, but even more so due to different (usually smaller) amounts of data, and different ways of conducting HTS. With the aim to apply computational methods for facilitating herbicide target identification, 48,000 bioactivity data against 16 herbicide targets were processed to train Laplacian modified Naïve Bayesian (NB) classification models. The herbicide target prediction model ("HerbiMod") is an ensemble of 16 binary classification models which are evaluated by internal, external and prospective validation sets. In addition to the experimental inactives, 10,000 random agrochemical inactives were included in the training process, which showed to improve the overall balanced accuracy of our models up to 40%. For all the models, performance in terms of balanced accuracy of≥80% was achieved in five-fold cross validation. Ranking target predictions was addressed by means of z-scores which improved predictivity over using raw scores alone. An external testset of 247 compounds from ChEMBL and a prospective testset of 394 compounds from BASF SE tested against five well studied herbicide targets (ACC, ALS, HPPD, PDS and PROTOX) were used for further validation. Only 4% of the compounds in the external testset lied in the applicability domain and extrapolation (and correct prediction) was hence impossible, which on one hand was surprising, and on the other hand illustrated the utilization of using applicability domains in the first place. However, performance better than 60% in balanced accuracy was achieved on the prospective testset, where all the compounds fell within the applicability domain, and which hence underlines the possibility of using target prediction also in the area of agrochemicals. Copyright © 2016 Elsevier Inc. All rights reserved.

Organs-on-chips at the frontiers of drug discovery

PubMed Central

Esch, Eric W.; Bahinski, Anthony; Huh, Dongeun

2016-01-01

Improving the effectiveness of preclinical predictions of human drug responses is critical to reducing costly failures in clinical trials. Recent advances in cell biology, microfabrication and microfluidics have enabled the development of microengineered models of the functional units of human organs — known as organs-on-chips — that could provide the basis for preclinical assays with greater predictive power. Here, we examine the new opportunities for the application of organ-on-chip technologies in a range of areas in preclinical drug discovery, such as target identification and validation, target-based screening, and phenotypic screening. We also discuss emerging drug discovery opportunities enabled by organs-on-chips, as well as important challenges in realizing the full potential of this technology. PMID:25792263

Identification of the TFII-I family target genes in the vertebrate genome.

PubMed

Chimge, Nyam-Osor; Makeyev, Aleksandr V; Ruddle, Frank H; Bayarsaihan, Dashzeveg

2008-07-01

GTF2I and GTF2IRD1 encode members of the TFII-I transcription factor family and are prime candidates in the Williams syndrome, a complex neurodevelopmental disorder. Our previous expression microarray studies implicated TFII-I proteins in the regulation of a number of genes critical in various aspects of cell physiology. Here, we combined bioinformatics and microarray results to identify TFII-I downstream targets in the vertebrate genome. These results were validated by chromatin immunoprecipitation and siRNA analysis. The collected evidence revealed the complexity of TFII-I-mediated processes that involve distinct regulatory networks. Altogether, these results lead to a better understanding of specific molecular events, some of which may be responsible for the Williams syndrome phenotype.

Protein Neddylation: Beyond Cullin-RING Ligases

PubMed Central

Enchev, Radoslav I.; Schulman, Brenda A.; Peter, Matthias

2016-01-01

NEDD8 is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here we re-evaluate these studies in light of the current understanding of the neddylation pathway, and suggest criteria for the identification of genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting research prospect primed to reveal fundamental insights. PMID:25531226

Network-assisted target identification for haploinsufficiency and homozygous profiling screens

PubMed Central

Wang, Sheng

2017-01-01

Chemical genomic screens have recently emerged as a systematic approach to drug discovery on a genome-wide scale. Drug target identification and elucidation of the mechanism of action (MoA) of hits from these noisy high-throughput screens remain difficult. Here, we present GIT (Genetic Interaction Network-Assisted Target Identification), a network analysis method for drug target identification in haploinsufficiency profiling (HIP) and homozygous profiling (HOP) screens. With the drug-induced phenotypic fitness defect of the deletion of a gene, GIT also incorporates the fitness defects of the gene’s neighbors in the genetic interaction network. On three genome-scale yeast chemical genomic screens, GIT substantially outperforms previous scoring methods on target identification on HIP and HOP assays, respectively. Finally, we showed that by combining HIP and HOP assays, GIT further boosts target identification and reveals potential drug’s mechanism of action. PMID:28574983

Designing a definitive trial for adjuvant targeted therapy in genotype defined lung cancer: the ALCHEMIST trials.

PubMed

Alden, Ryan S; Mandrekar, Sumithra J; Oxnard, Geoffrey R

2015-09-01

Genotype-directed targeted therapies have revolutionized the treatment of metastatic non-small cell lung cancer (NSCLC) but they have not yet been comprehensively studied in the adjuvant setting. Previous trials of adjuvant targeted therapy in unselected early stage NSCLC patients showed no benefit versus placebo, however retrospective data suggests improved disease free survival (DFS) with epidermal growth factor receptor (EGFR) inhibitors in patients with appropriate molecular alterations. A definitive prospective, randomized, placebo-controlled trial of targeted therapies for NSCLC is needed to determine the efficacy of targeted therapy following surgical resection and standard adjuvant therapy. The principal challenges facing such a trial are (I) identification of actionable alterations in early stage patients; and (II) realization of sufficient enrollment to power definitive analyses. The ALCHEMIST trial (Adjuvant Lung Cancer Enrichment Marker Identification and Sequencing Trial) was designed to overcome these challenges. Using the national clinical trials network (NCTN) of the National Cancer Institute (NCI), several thousand patients with operable NSCLC will undergo tumor genotyping for EGFR mutations or rearrangement of anaplastic lymphoma kinase (ALK). Following resection and completion of standard adjuvant therapy, patients with EGFR-mutant NSCLC will be randomized to erlotinib versus placebo (1:1), those with ALK-rearranged NSCLC will be randomized to crizotinib versus placebo (1:1), while those not enrolled onto the adjuvant trials will continue to be followed on the screening trial. ALCHEMIST also provides for the collection of tissue at baseline and at recurrence (if available) to characterize mechanisms of recurrence and of resistance to targeted therapy. Thus, ALCHEMIST is a platform for validation of targeted therapy as part of curative care in NSCLC and creates an opportunity to advance our understanding of disease biology.

Identification of HMX1 target genes: A predictive promoter model approach

PubMed Central

Boulling, Arnaud; Wicht, Linda

2013-01-01

Purpose A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway’s function, we sought to identify the target genes. Methods We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. Results The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. Conclusions Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1. PMID:23946633

The confidence-accuracy relationship in eyewitness identification: effects of lineup instructions, foil similarity, and target-absent base rates.

PubMed

Brewer, Neil; Wells, Gary L

2006-03-01

Discriminating accurate from mistaken eyewitness identifications is a major issue facing criminal justice systems. This study examined whether eyewitness confidence assists such decisions under a variety of conditions using a confidence-accuracy (CA) calibration approach. Participants (N = 1,200) viewed a simulated crime and attempted 2 separate identifications from 8-person target-present or target-absent lineups. Confidence and accuracy were calibrated for choosers (but not nonchoosers) for both targets under all conditions. Lower overconfidence was associated with higher diagnosticity, lower target-absent base rates, and shorter identification latencies. Although researchers agree that courtroom expressions of confidence are uninformative, our findings indicate that confidence assessments obtained immediately after a positive identification can provide a useful guide for investigators about the likely accuracy of an identification.

Rapid screening and identification of chemical hazards in surface and drinking water using high resolution mass spectrometry and a case-control filter.

PubMed

Kaserzon, Sarit L; Heffernan, Amy L; Thompson, Kristie; Mueller, Jochen F; Gomez Ramos, Maria Jose

2017-09-01

Access to clean, safe drinking water poses a serious challenge to regulators, and requires analytical strategies capable of rapid screening and identification of potentially hazardous chemicals, specifically in situations when threats to water quality or security require rapid investigations and potential response. This study describes a fast and efficient chemical hazard screening strategy for characterising trace levels of polar organic contaminants in water matrices, based on liquid chromatography high resolution mass spectrometry with post-acquisition 'case-control' data processing. This method allowed for a rapid response time of less than 24 h for the screening of target, suspect and non-target unknown chemicals via direct injection analysis, and a second, more sensitive analysis option requiring sample pre-concentration. The method was validated by fortifying samples with a range of pesticides, pharmaceuticals and personal care products (n = 46); with >90% of target compounds positively screened in samples at 1 ng mL -1 , and 46% at 0.1 ng mL -1 when analysed via direct injection. To simulate a contamination event samples were fortified with compounds not present in the commercial library (designated 'non-target compounds'; fipronil and fenitrothion), tentatively identified at 0.2 and 1 ng mL -1 , respectively; and a compound not included in any known commercial library or public database (designated 'unknown' compounds; 8Cl - perfluorooctanesulfonic acid), at 0.8 ng mL -1 . The method was applied to two 'real-case' scenarios: (1) the assessment of drinking water safety during a high-profile event in Brisbane, Australia; and (2) to screen treated, re-circulated drinking water and pre-treated (raw) water. The validated workflow was effective for rapid prioritisation and screening of suspect and non-target potential hazards at trace levels, and could be applied to a wide range of matrices and investigations where comparison of organic contaminants between an affected and control site and or timeframe is warranted. Copyright © 2017 Elsevier Ltd. All rights reserved.

Repositioning the substrate activity screening (SAS) approach as a fragment-based method for identification of weak binders.

PubMed

Gladysz, Rafaela; Cleenewerck, Matthias; Joossens, Jurgen; Lambeir, Anne-Marie; Augustyns, Koen; Van der Veken, Pieter

2014-10-13

Fragment-based drug discovery (FBDD) has evolved into an established approach for "hit" identification. Typically, most applications of FBDD depend on specialised cost- and time-intensive biophysical techniques. The substrate activity screening (SAS) approach has been proposed as a relatively cheap and straightforward alternative for identification of fragments for enzyme inhibitors. We have investigated SAS for the discovery of inhibitors of oncology target urokinase (uPA). Although our results support the key hypotheses of SAS, we also encountered a number of unreported limitations. In response, we propose an efficient modified methodology: "MSAS" (modified substrate activity screening). MSAS circumvents the limitations of SAS and broadens its scope by providing additional fragments and more coherent SAR data. As well as presenting and validating MSAS, this study expands existing SAR knowledge for the S1 pocket of uPA and reports new reversible and irreversible uPA inhibitor scaffolds. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PepPat, a pattern-based oligopeptide homology search method and the identification of a novel tachykinin-like peptide.

PubMed

Jiang, Ying; Gao, Ge; Fang, Gang; Gustafson, Eric L; Laverty, Maureen; Yin, Yanbin; Zhang, Yong; Luo, Jingchu; Greene, Jonathan R; Bayne, Marvin L; Hedrick, Joseph A; Murgolo, Nicholas J

2003-05-01

PepPat, a hybrid method that combines pattern matching with similarity scoring, is described. We also report PepPat's application in the identification of a novel tachykinin-like peptide. PepPat takes as input a query peptide and a user-specified regular expression pattern within the peptide. It first performs a database pattern match and then ranks candidates on the basis of their similarity to the query peptide. PepPat calculates similarity over the pattern spanning region, enhancing PepPat's sensitivity for short query peptides. PepPat can also search for a user-specified number of occurrences of a repeated pattern within the target sequence. We illustrate PepPat's application in short peptide ligand mining. As a validation example, we report the identification of a novel tachykinin-like peptide, C14TKL-1, and show it is an NK1 (neuokinin receptor 1) agonist whose message is widely expressed in human periphery. PepPat is offered online at: http://peppat.cbi.pku.edu.cn.

Simultaneous Identification of Four "Legal High" Plant Species in a Multiplex PCR High-Resolution Melt Assay.

PubMed

Elkins, Kelly M; Perez, Anjelica C U; Quinn, Alicia A

2017-05-01

The international prevalence of "legal high" drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real-time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high-resolution melt using LCGreen Plus ® . The PCR assay was evaluated based on the following: (i) specificity and selectivity-primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity-serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability-sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging "legal high" species. © 2016 American Academy of Forensic Sciences.

Maui-VIA: A User-Friendly Software for Visual Identification, Alignment, Correction, and Quantification of Gas Chromatography–Mass Spectrometry Data

PubMed Central

Kuich, P. Henning J. L.; Hoffmann, Nils; Kempa, Stefan

2015-01-01

A current bottleneck in GC–MS metabolomics is the processing of raw machine data into a final datamatrix that contains the quantities of identified metabolites in each sample. While there are many bioinformatics tools available to aid the initial steps of the process, their use requires both significant technical expertise and a subsequent manual validation of identifications and alignments if high data quality is desired. The manual validation is tedious and time consuming, becoming prohibitively so as sample numbers increase. We have, therefore, developed Maui-VIA, a solution based on a visual interface that allows experts and non-experts to simultaneously and quickly process, inspect, and correct large numbers of GC–MS samples. It allows for the visual inspection of identifications and alignments, facilitating a unique and, due to its visualization and keyboard shortcuts, very fast interaction with the data. Therefore, Maui-Via fills an important niche by (1) providing functionality that optimizes the component of data processing that is currently most labor intensive to save time and (2) lowering the threshold of expertise required to process GC–MS data. Maui-VIA projects are initiated with baseline-corrected raw data, peaklists, and a database of metabolite spectra and retention indices used for identification. It provides functionality for retention index calculation, a targeted library search, the visual annotation, alignment, correction interface, and metabolite quantification, as well as the export of the final datamatrix. The high quality of data produced by Maui-VIA is illustrated by its comparison to data attained manually by an expert using vendor software on a previously published dataset concerning the response of Chlamydomonas reinhardtii to salt stress. In conclusion, Maui-VIA provides the opportunity for fast, confident, and high-quality data processing validation of large numbers of GC–MS samples by non-experts. PMID:25654076

Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

PubMed

Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

2018-01-01

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

Identification of STAT1 and STAT3 Specific Inhibitors Using Comparative Virtual Screening and Docking Validation

PubMed Central

Szelag, Malgorzata; Czerwoniec, Anna; Wesoly, Joanna; Bluyssen, Hans A. R.

2015-01-01

Signal transducers and activators of transcription (STATs) facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr)-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (h)STATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the ‘STAT-comparative binding affinity value’ and ‘ligand binding pose variation’ as selection criteria. In silico screening of a multi-million clean leads (CL) compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our understanding of the functional role of these STATs in different diseases and benefit the clinical need for more drugable STAT inhibitors with high specificity, potency and excellent bioavailability. PMID:25710482

Characterization of Aspergillus species on Brazil nut from the Brazilian Amazonian region and development of a PCR assay for identification at the genus level

PubMed Central

2014-01-01

Background Brazil nut is a protein-rich extractivist tree crop in the Amazon region. Fungal contamination of shells and kernel material frequently includes the presence of aflatoxigenic Aspergillus species from the section Flavi. Aflatoxins are polyketide secondary metabolites, which are hepatotoxic carcinogens in mammals. The objectives of this study were to identify Aspergillus species occurring on Brazil nut grown in different states in the Brazilian Amazon region and develop a specific PCR method for collective identification of member species of the genus Aspergillus. Results Polyphasic identification of 137 Aspergillus strains isolated from Brazil nut shell material from cooperatives across the Brazilian Amazon states of Acre, Amapá and Amazonas revealed five species, with Aspergillus section Flavi species A. nomius and A. flavus the most abundant. PCR primers ASP_GEN_MTSSU_F1 and ASP_GEN_MTSSU_R1 were designed for the genus Aspergillus, targeting a portion of the mitochondrial small subunit ribosomal RNA gene. Primer specificity was validated through both electronic PCR against target gene sequences at Genbank and in PCR reactions against DNA from Aspergillus species and other fungal genera common on Brazil nut. Collective differentiation of the observed section Flavi species A. flavus, A. nomius and A. tamarii from other Aspergillus species was possible on the basis of RFLP polymorphism. Conclusions Given the abundance of Aspergillus section Flavi species A. nomius and A. flavus observed on Brazil nut, and associated risk of mycotoxin accumulation, simple identification methods for such mycotoxigenic species are of importance for Hazard Analysis Critical Control Point system implementation. The assay for the genus Aspergillus represents progress towards specific PCR identification and detection of mycotoxigenic species. PMID:24885088

Identification of Forensically Important Calliphoridae and Sarcophagidae Species Collected in Korea Using SNaPshot Multiplex System Targeting the Cytochrome c Oxidase Subunit I Gene

PubMed Central

Park, Ji Hye

2018-01-01

Estimation of postmortem interval (PMI) is paramount in modern forensic investigation. After the disappearance of the early postmortem phenomena conventionally used to estimate PMI, entomologic evidence provides important indicators for PMI estimation. The age of the oldest fly larvae or pupae can be estimated to pinpoint the time of oviposition, which is considered the minimum PMI (PMImin). The development rate of insects is usually temperature dependent and species specific. Therefore, species identification is mandatory for PMImin estimation using entomological evidence. The classical morphological identification method cannot be applied when specimens are damaged or have not yet matured. To overcome this limitation, some investigators employ molecular identification using mitochondrial cytochrome c oxidase subunit I (COI) nucleotide sequences. The molecular identification method commonly uses Sanger's nucleotide sequencing and molecular phylogeny, which are complex and time consuming and constitute another obstacle for forensic investigators. In this study, instead of using conventional Sanger's nucleotide sequencing, single-nucleotide polymorphisms (SNPs) in the COI gene region, which are unique between fly species, were selected and targeted for single-base extension (SBE) technology. These SNPs were genotyped using a SNaPshot® kit. Eleven Calliphoridae and seven Sarcophagidae species were covered. To validate this genotyping, fly DNA samples (103 adults, 84 larvae, and 4 pupae) previously confirmed by DNA barcoding were used. This method worked quickly with minimal DNA, providing a potential alternative to conventional DNA barcoding. Consisting of only a few simple electropherogram peaks, the results were more straightforward compared with those of the conventional DNA barcoding produced by Sanger's nucleotide sequencing. PMID:29682531

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

PubMed

Li, Wenbin; Teixeira, Diva C; Hartung, John S; Huang, Qi; Duan, Yongping; Zhou, Lijuan; Chen, Jianchi; Lin, Hong; Lopes, Silvio; Ayres, A Juliano; Levy, Laurene

2013-01-01

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the specific identification of X. fastidiosa strains that cause citrus variegated chlorosis. Published by Elsevier B.V.

Open Access High Throughput Drug Discovery in the Public Domain: A Mount Everest in the Making

PubMed Central

Roy, Anuradha; McDonald, Peter R.; Sittampalam, Sitta; Chaguturu, Rathnam

2013-01-01

High throughput screening (HTS) facilitates screening large numbers of compounds against a biochemical target of interest using validated biological or biophysical assays. In recent years, a significant number of drugs in clinical trails originated from HTS campaigns, validating HTS as a bona fide mechanism for hit finding. In the current drug discovery landscape, the pharmaceutical industry is embracing open innovation strategies with academia to maximize their research capabilities and to feed their drug discovery pipeline. The goals of academic research have therefore expanded from target identification and validation to probe discovery, chemical genomics, and compound library screening. This trend is reflected in the emergence of HTS centers in the public domain over the past decade, ranging in size from modestly equipped academic screening centers to well endowed Molecular Libraries Probe Centers Network (MLPCN) centers funded by the NIH Roadmap initiative. These centers facilitate a comprehensive approach to probe discovery in academia and utilize both classical and cutting-edge assay technologies for executing primary and secondary screening campaigns. The various facets of academic HTS centers as well as their implications on technology transfer and drug discovery are discussed, and a roadmap for successful drug discovery in the public domain is presented. New lead discovery against therapeutic targets, especially those involving the rare and neglected diseases, is indeed a Mount Everestonian size task, and requires diligent implementation of pharmaceutical industry’s best practices for a successful outcome. PMID:20809896

Evaluating lineup fairness: Variations across methods and measures.

PubMed

Mansour, Jamal K; Beaudry, Jennifer L; Kalmet, Natalie; Bertrand, Michelle I; Lindsay, R C L

2017-02-01

Triers of fact sometimes consider lineup fairness when determining the suggestiveness of an identification procedure. Likewise, researchers often consider lineup fairness when comparing results across studies. Despite their importance, lineup fairness measures have received scant empirical attention and researchers inconsistently conduct and report mock-witness tasks and lineup fairness measures. We conducted a large-scale, online experiment (N = 1,010) to examine how lineup fairness measures varied with mock-witness task methodologies as well as to explore the validity and reliability of the measures. In comparison to descriptions compiled from multiple witnesses, when individual descriptions were presented in the mock-witness task, lineup fairness measures indicated a higher number of plausible lineup members but more bias toward the suspect. Target-absent lineups were consistently estimated to be fairer than target-present lineups-which is problematic because it suggests that lineups containing innocent suspects are less likely to be challenged in court than lineups containing guilty suspects. Correlations within lineup size measures and within some lineup bias measures indicated convergent validity and the correlations across the lineup size and lineup bias measures demonstrated discriminant validity. The reliability of lineup fairness measures across different descriptions was low and reliability across different sets of mock witnesses was moderate to high, depending on the measure. Researchers reporting lineup fairness measures should specify the type of description presented, the amount of detail in the description, and whether the mock witnesses viewed target-present and/or -absent lineups. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Identification and validation of sRNAs in Edwardsiella tarda S08.

PubMed

Sun, Yuying; Zhang, Jiquan; Qin, Lei; Yan, Cui; Zhang, Xiaojun; Liu, Dandan

2017-01-01

Bacterial small non-coding RNAs (sRNAs) are known as novel regulators involved in virulence, stress responsibility, and so on. Recently, a lot of new researches have highlighted the critical roles of sRNAs in fine-tune gene regulation in both prokaryotes and eukaryotes. Edwardsiella tarda (E. tarda) is a gram-negative, intracellular pathogen that causes edwardsiellosis in fish. Thus far, no sRNA has been reported in E. tarda. The present study represents the first attempt to identify sRNAs in E. tarda S08. Ten sRNAs were validated by RNA sequencing and quantitative PCR (qPCR). ET_sRNA_1 and ET_sRNA_2 were homolous to tmRNA and GcvB, respectively. However, the other candidate sRNAs have not been reported till now. The cellular abundance of 10 validated sRNA was detected by qPCR at different growth phases to monitor their biosynthesis. Nine candidate sRNAs were expressed in the late-stage of exponential growth and stationary stages of growth (36~60 h). And the expression of the nine sRNAs was growth phase-dependent. But ET_sRNA_10 was almost expressed all the time and reached the highest peak at 48 h. Their targets were predicted by TargetRNA2 and each sRNA target contains some genes that directly or indirectly relate to virulence. These results preliminary showed that sRNAs probably play a regulatory role of virulence in E. tarda.

AlzhCPI: A knowledge base for predicting chemical-protein interactions towards Alzheimer's disease.

PubMed

Fang, Jiansong; Wang, Ling; Li, Yecheng; Lian, Wenwen; Pang, Xiaocong; Wang, Hong; Yuan, Dongsheng; Wang, Qi; Liu, Ai-Lin; Du, Guan-Hua

2017-01-01

Alzheimer's disease (AD) is a complicated progressive neurodegeneration disorder. To confront AD, scientists are searching for multi-target-directed ligands (MTDLs) to delay disease progression. The in silico prediction of chemical-protein interactions (CPI) can accelerate target identification and drug discovery. Previously, we developed 100 binary classifiers to predict the CPI for 25 key targets against AD using the multi-target quantitative structure-activity relationship (mt-QSAR) method. In this investigation, we aimed to apply the mt-QSAR method to enlarge the model library to predict CPI towards AD. Another 104 binary classifiers were further constructed to predict the CPI for 26 preclinical AD targets based on the naive Bayesian (NB) and recursive partitioning (RP) algorithms. The internal 5-fold cross-validation and external test set validation were applied to evaluate the performance of the training sets and test set, respectively. The area under the receiver operating characteristic curve (ROC) for the test sets ranged from 0.629 to 1.0, with an average of 0.903. In addition, we developed a web server named AlzhCPI to integrate the comprehensive information of approximately 204 binary classifiers, which has potential applications in network pharmacology and drug repositioning. AlzhCPI is available online at http://rcidm.org/AlzhCPI/index.html. To illustrate the applicability of AlzhCPI, the developed system was employed for the systems pharmacology-based investigation of shichangpu against AD to enhance the understanding of the mechanisms of action of shichangpu from a holistic perspective.

Protein lipoxidation: Detection strategies and challenges

PubMed Central

Aldini, Giancarlo; Domingues, M. Rosário; Spickett, Corinne M.; Domingues, Pedro; Altomare, Alessandra; Sánchez-Gómez, Francisco J.; Oeste, Clara L.; Pérez-Sala, Dolores

2015-01-01

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets. PMID:26072467

Tools for in silico target fishing.

PubMed

Cereto-Massagué, Adrià; Ojeda, María José; Valls, Cristina; Mulero, Miquel; Pujadas, Gerard; Garcia-Vallve, Santiago

2015-01-01

Computational target fishing methods are designed to identify the most probable target of a query molecule. This process may allow the prediction of the bioactivity of a compound, the identification of the mode of action of known drugs, the detection of drug polypharmacology, drug repositioning or the prediction of the adverse effects of a compound. The large amount of information regarding the bioactivity of thousands of small molecules now allows the development of these types of methods. In recent years, we have witnessed the emergence of many methods for in silico target fishing. Most of these methods are based on the similarity principle, i.e., that similar molecules might bind to the same targets and have similar bioactivities. However, the difficult validation of target fishing methods hinders comparisons of the performance of each method. In this review, we describe the different methods developed for target prediction, the bioactivity databases most frequently used by these methods, and the publicly available programs and servers that enable non-specialist users to obtain these types of predictions. It is expected that target prediction will have a large impact on drug development and on the functional food industry. Copyright © 2014 Elsevier Inc. All rights reserved.

Preparation of Conjugates of Cytotoxic Lupane Triterpenes with Biotin.

PubMed

Soural, Miroslav; Hodon, Jiri; Dickinson, Niall J; Sidova, Veronika; Gurska, Sona; Dzubak, Petr; Hajduch, Marian; Sarek, Jan; Urban, Milan

2015-12-16

To better understand the mechanism of action of antitumor triterpenes, we are developing methods to identify their molecular targets. A promising method is based on combination of quantitative proteomics with SILAC and uses active compounds anchored to magnetic beads via biotin-streptavidin interaction. We developed a simple and fast solid-phase synthetic technique to connect terpenes to biotin through a linker. Betulinic acid was biotinylated from three different conjugation sites for use as a standard validation tool since many molecular targets of this triterpene are already known. Then, a set of four other cytotoxic triterpenoids was biotinylated. Biotinylated terpenes were similarly cytotoxic to their nonbiotinylated parents, which suggests that the target identification should not be influenced by linker or biotin. The developed solid-phase synthetic approach is the first attempt to use solid-phase synthesis to connect active triterpenes to biotin and is applicable as a general procedure for routine conjugation of triterpenes with other molecules of choice.

Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer

PubMed Central

Kim, Yunee; Jeon, Jouhyun; Mejia, Salvador; Yao, Cindy Q; Ignatchenko, Vladimir; Nyalwidhe, Julius O; Gramolini, Anthony O; Lance, Raymond S; Troyer, Dean A; Drake, Richard R; Boutros, Paul C; Semmes, O. John; Kislinger, Thomas

2016-01-01

Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers. PMID:27350604

Present and future breast cancer management--bench to bedside and back: a positioning paper of academia, regulatory authorities and pharmaceutical industry.

PubMed

Bartsch, R; Frings, S; Marty, M; Awada, A; Berghoff, A S; Conte, P; Dickin, S; Enzmann, H; Gnant, M; Hasmann, M; Hendriks, H R; Llombart, A; Massacesi, C; von Minckwitz, G; Penault-Llorca, F; Scaltriti, M; Yarden, Y; Zwierzina, H; Zielinski, C C

2014-04-01

Insights into tumour biology of breast cancer have led the path towards the introduction of targeted treatment approaches; still, breast cancer-related mortality remains relatively high. Efforts in the field of basic research revealed new druggable targets which now await validation within the context of clinical trials. Therefore, questions concerning the optimal design of future studies are becoming even more pertinent. Aspects such as the ideal end point, availability of predictive markers to identify the optimal cohort for drug testing, or potential mechanisms of resistance need to be resolved. An expert panel representing the academic community, the pharmaceutical industry, as well as European Regulatory Authorities met in Vienna, Austria, in November 2012, in order to discuss breast cancer biology, identification of novel biological targets and optimal drug development with the aim of treatment individualization. This article summarizes statements and perspectives provided by the meeting participants.

Discovery of novel drugs for promising targets.

PubMed

Martell, Robert E; Brooks, David G; Wang, Yan; Wilcoxen, Keith

2013-09-01

Once a promising drug target is identified, the steps to actually discover and optimize a drug are diverse and challenging. The goal of this study was to provide a road map to navigate drug discovery. Review general steps for drug discovery and provide illustrating references. A number of approaches are available to enhance and accelerate target identification and validation. Consideration of a variety of potential mechanisms of action of potential drugs can guide discovery efforts. The hit to lead stage may involve techniques such as high-throughput screening, fragment-based screening, and structure-based design, with informatics playing an ever-increasing role. Biologically relevant screening models are discussed, including cell lines, 3-dimensional culture, and in vivo screening. The process of enabling human studies for an investigational drug is also discussed. Drug discovery is a complex process that has significantly evolved in recent years. © 2013 Elsevier HS Journals, Inc. All rights reserved.

Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring.

PubMed

Lange, Vinzenz; Malmström, Johan A; Didion, John; King, Nichole L; Johansson, Björn P; Schäfer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W; Brusniak, Mi-Youn; Bühlmann, Peter; Björck, Lars; Domon, Bruno; Aebersold, Ruedi

2008-08-01

In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.

Fruitful research: drug target discovery for neurodegenerative diseases in Drosophila.

PubMed

Konsolaki, Mary

2013-12-01

Although vertebrate model systems have obvious advantages in the study of human disease, invertebrate organisms have contributed enormously to this field as well. The conservation of genome structure and physiology among organisms poses unexpected peculiarities, and the redundancy in certain gene families or the presence of polymorphisms that can slightly alter gene expression can, in certain instances, bring invertebrate systems, such as Drosophila, closer to humans than mice and vice versa. This necessitates the analysis of disease pathways in multiple model organisms. The author highlights findings from Drosophila models of neurodegenerative diseases that have occurred in the past few years. She also highlights and discusses various molecular, genetic and genomic tools used in flies, as well as methods for generating disease models. Finally, the author describes Drosophila models of Alzheimer's, Parkinson's tri-nucleotide repeat diseases, and Fragile X syndrome and summarizes insights in disease mechanisms that have been discovered directly in fly models. Full genome genetic screens in Drosophila can lead to the rapid identification of drug target candidates that can be subsequently validated in a vertebrate system. In addition, the Drosophila models of neurodegeneration may often show disease phenotypes that are absent in equivalent mouse models. The author believes that the extensive contribution of Drosophila to both new disease drug target discovery, in addition to target validation, makes them indispensible to drug discovery and development.

A Systematic Evaluation of Analogs for the Read-across ...

EPA Pesticide Factsheets

Read-across is a data gap filling technique widely used within category and analog approaches to predict a biological property for a target data-poor chemical using known information from similar (source analog) chemical(s). Potential source analogs are typically identified based on structural similarity. Although much guidance has been published for read-across, practical guiding principles for the identification and evaluation of the scientific validity of source analogs, which is a critical step in deriving a robust read-across prediction, remains largely lacking.This case study explores the extent to which 3 structure descriptor sets (Pubchem, Chemotyper and MoSS) and their combinations are able to identify valid analogs for reading across Estrogen Receptor (ER) activity for a specific class of chemicals: hindered phenols. For each target chemical, two sets of analogs (hindered and non-hindered phenols) were selected using each descriptor set with two cut-offs: (1). Minimum Tanimoto similarity (range 0.1 - 0.9), and (2). Closest N analogs (range 1 - 10). Each target-analog pair was then evaluated for its agreement with measured ER binding and agonism. Subsequently, the analogs were filtered using physchem properties (LogKow & Molecular Volume) and the resultant agreement between each target-analog pair was evaluated. The data set comprised 462 hindered phenols and 296 non-hindered phenols. The results demonstrate that: (1). The concordance in ER activity r

Identification of TMEM208 and PQLC2 as reference genes for normalizing mRNA expression in colorectal cancer treated with aspirin

PubMed Central

Zhu, Yuanyuan; Yang, Chao; Weng, Mingjiao; Zhang, Yan; Yang, Chunhui; Jin, Yinji; Yang, Weiwei; He, Yan; Wu, Yiqi; Zhang, Yuhua; Wang, Guangyu; RajkumarEzakiel Redpath, Riju James; Zhang, Lei; Jin, Xiaoming; Liu, Ying; Sun, Yuchun; Ning, Ning; Qiao, Yu; Zhang, Fengmin; Li, Zhiwei; Wang, Tianzhen; Zhang, Yanqiao; Li, Xiaobo

2017-01-01

Numerous evidences indicate that aspirin usage causes a significant reduction in colorectal cancer. However, the molecular mechanisms about aspirin preventing colon cancer are largely unknown. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a most frequently used method to identify the target molecules regulated by certain compound. However, this method needs stable internal reference genes to analyze the expression change of the targets. In this study, the transcriptional stabilities of several traditional reference genes were evaluated in colon cancer cells treated with aspirin, and also, the suitable internal reference genes were screened by using a microarray and were further identified by using the geNorm and NormFinder softwares, and then were validated in more cell lines and xenografts. We have showed that three traditional internal reference genes, β-actin, GAPDH and α-tubulin, are not suitable for studying gene transcription in colon cancer cells treated with aspirin, and we have identified and validated TMEM208 and PQLC2 as the ideal internal reference genes for detecting the molecular targets of aspirin in colon cancer in vitro and in vivo. This study reveals stable internal reference genes for studying the target genes of aspirin in colon cancer, which will contribute to identify the molecular mechanism behind aspirin preventing colon cancer. PMID:28184026

Identification of TMEM208 and PQLC2 as reference genes for normalizing mRNA expression in colorectal cancer treated with aspirin.

PubMed

Zhu, Yuanyuan; Yang, Chao; Weng, Mingjiao; Zhang, Yan; Yang, Chunhui; Jin, Yinji; Yang, Weiwei; He, Yan; Wu, Yiqi; Zhang, Yuhua; Wang, Guangyu; RajkumarEzakiel Redpath, Riju James; Zhang, Lei; Jin, Xiaoming; Liu, Ying; Sun, Yuchun; Ning, Ning; Qiao, Yu; Zhang, Fengmin; Li, Zhiwei; Wang, Tianzhen; Zhang, Yanqiao; Li, Xiaobo

2017-04-04

Numerous evidences indicate that aspirin usage causes a significant reduction in colorectal cancer. However, the molecular mechanisms about aspirin preventing colon cancer are largely unknown. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a most frequently used method to identify the target molecules regulated by certain compound. However, this method needs stable internal reference genes to analyze the expression change of the targets. In this study, the transcriptional stabilities of several traditional reference genes were evaluated in colon cancer cells treated with aspirin, and also, the suitable internal reference genes were screened by using a microarray and were further identified by using the geNorm and NormFinder softwares, and then were validated in more cell lines and xenografts. We have showed that three traditional internal reference genes, β-actin, GAPDH and α-tubulin, are not suitable for studying gene transcription in colon cancer cells treated with aspirin, and we have identified and validated TMEM208 and PQLC2 as the ideal internal reference genes for detecting the molecular targets of aspirin in colon cancer in vitro and in vivo. This study reveals stable internal reference genes for studying the target genes of aspirin in colon cancer, which will contribute to identify the molecular mechanism behind aspirin preventing colon cancer.

NMR screening in fragment-based drug design: a practical guide.

PubMed

Kim, Hai-Young; Wyss, Daniel F

2015-01-01

Fragment-based drug design (FBDD) comprises both fragment-based screening (FBS) to find hits and elaboration of these hits to lead compounds. Typical fragment hits have lower molecular weight (<300-350 Da) and lower initial potency but higher ligand efficiency when compared to those from high-throughput screening. NMR spectroscopy has been widely used for FBDD since it identifies and localizes the binding site of weakly interacting hits on the target protein. Here we describe ligand-based NMR methods for hit identification from fragment libraries and for functional cross-validation of primary hits.

Comparison of conventional ultrasonography and ultrasonography-computed tomography fusion imaging for target identification using digital/real hybrid phantoms: a preliminary study.

PubMed

Soyama, Takeshi; Sakuhara, Yusuke; Kudo, Kohsuke; Abo, Daisuke; Wang, Jeff; Ito, Yoichi M; Hasegawa, Yu; Shirato, Hiroki

2016-07-01

This preliminary study compared ultrasonography-computed tomography (US-CT) fusion imaging and conventional ultrasonography (US) for accuracy and time required for target identification using a combination of real phantoms and sets of digitally modified computed tomography (CT) images (digital/real hybrid phantoms). In this randomized prospective study, 27 spheres visible on B-mode US were placed at depths of 3.5, 8.5, and 13.5 cm (nine spheres each). All 27 spheres were digitally erased from the CT images, and a radiopaque sphere was digitally placed at each of the 27 locations to create 27 different sets of CT images. Twenty clinicians were instructed to identify the sphere target using US alone and fusion imaging. The accuracy of target identification of the two methods was compared using McNemar's test. The mean time required for target identification and error distances were compared using paired t tests. At all three depths, target identification was more accurate and the mean time required for target identification was significantly less with US-CT fusion imaging than with US alone, and the mean error distances were also shorter with US-CT fusion imaging. US-CT fusion imaging was superior to US alone in terms of accurate and rapid identification of target lesions.

Qualitative screening of undesirable compounds from feeds to fish by liquid chromatography coupled to mass spectrometry.

PubMed

Nácher-Mestre, Jaime; Ibáñez, María; Serrano, Roque; Pérez-Sánchez, Jaume; Hernández, Félix

2013-03-06

This paper describes the development, validation, and application of a rapid screening method for the detection and identification of undesirable organic compounds in aquaculture products. A generic sample treatment was applied without any purification or preconcentration step. After extraction of the samples with acetonitrile/water 80:20 (0.1% formic acid), the extracts were centrifuged and directly injected in the LC-HRMS system, consisting of ultra-high performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). A qualitative validation was carried out for over 70 representative compounds, including antibiotics, pesticides, and mycotoxins, in fish feed and fish fillets spiked at 20 and 100 μg/kg. At the highest level, the great majority of compounds were detected (using the most abundant ion, typically the protonated molecule) and unequivocally identified (on the basis of the presence of two accurate-mass measured ions). At the 20 μg/kg level, many contaminants could already be detected, although identification using two ions was not fully reached for some of them, mainly in fish feed due to the complexity of this matrix. Subsequent application of this screening methodology to aquaculture samples made it possible to find several compounds from the target list, such as the antibiotic ciprofloxacin, the insecticide pirimiphos-methyl, and the mycotoxins fumonisin B2 and zearalenone. A retrospective analysis of accurate-mass full-spectrum acquisition data provided by QTOF MS was also made, without either reprocessing or injecting the samples. This allowed the detection and tentative identification of other organic undesirables different from those included in the validated list.

Cultural adaptation of the Tuberculosis-related stigma scale to Brazil.

PubMed

Crispim, Juliane de Almeida; Touso, Michelle Mosna; Yamamura, Mellina; Popolin, Marcela Paschoal; Garcia, Maria Concebida da Cunha; Santos, Cláudia Benedita Dos; Palha, Pedro Fredemir; Arcêncio, Ricardo Alexandre

2016-06-01

The process of stigmatization associated with TB has been undervalued in national research as this social aspect is important in the control of the disease, especially in marginalized populations. This paper introduces the stages of the process of cultural adaptation in Brazil of the Tuberculosis-related stigma scale for TB patients. It is a methodological study in which the items of the scale were translated and back-translated with semantic validation with 15 individuals of the target population. After translation, the reconciled back-translated version was compared with the original version by the project coordinator in Southern Thailand, who approved the final version in Brazilian Portuguese. The results of the semantic validation conducted with TB patients enable the identification that, in general, the scale was well accepted and easily understood by the participants.

Drug Target Mining and Analysis of the Chinese Tree Shrew for Pharmacological Testing

PubMed Central

Liu, Jie; Lee, Wen-hui; Zhang, Yun

2014-01-01

The discovery of new drugs requires the development of improved animal models for drug testing. The Chinese tree shrew is considered to be a realistic candidate model. To assess the potential of the Chinese tree shrew for pharmacological testing, we performed drug target prediction and analysis on genomic and transcriptomic scales. Using our pipeline, 3,482 proteins were predicted to be drug targets. Of these predicted targets, 446 and 1,049 proteins with the highest rank and total scores, respectively, included homologs of targets for cancer chemotherapy, depression, age-related decline and cardiovascular disease. Based on comparative analyses, more than half of drug target proteins identified from the tree shrew genome were shown to be higher similarity to human targets than in the mouse. Target validation also demonstrated that the constitutive expression of the proteinase-activated receptors of tree shrew platelets is similar to that of human platelets but differs from that of mouse platelets. We developed an effective pipeline and search strategy for drug target prediction and the evaluation of model-based target identification for drug testing. This work provides useful information for future studies of the Chinese tree shrew as a source of novel targets for drug discovery research. PMID:25105297

49 CFR 236.905 - Railroad Safety Program Plan (RSPP).

Code of Federal Regulations, 2012 CFR

2012-10-01

... to be used in the verification and validation process, consistent with appendix C to this part. The...; and (iv) The identification of the safety assessment process. (2) Design for verification and validation. The RSPP must require the identification of verification and validation methods for the...

49 CFR 236.905 - Railroad Safety Program Plan (RSPP).

Code of Federal Regulations, 2014 CFR

2014-10-01

... to be used in the verification and validation process, consistent with appendix C to this part. The...; and (iv) The identification of the safety assessment process. (2) Design for verification and validation. The RSPP must require the identification of verification and validation methods for the...

49 CFR 236.905 - Railroad Safety Program Plan (RSPP).

Code of Federal Regulations, 2013 CFR

2013-10-01

... to be used in the verification and validation process, consistent with appendix C to this part. The...; and (iv) The identification of the safety assessment process. (2) Design for verification and validation. The RSPP must require the identification of verification and validation methods for the...

49 CFR 236.905 - Railroad Safety Program Plan (RSPP).

Code of Federal Regulations, 2011 CFR

2011-10-01

... to be used in the verification and validation process, consistent with appendix C to this part. The...; and (iv) The identification of the safety assessment process. (2) Design for verification and validation. The RSPP must require the identification of verification and validation methods for the...

Potentials of single-cell biology in identification and validation of disease biomarkers.

PubMed

Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

2016-09-01

Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Inhibition of 3-Hydroxy-3-Methylglutaryl–Coenzyme A Reductase and Application of Statins as a Novel Effective Therapeutic Approach against Acanthamoeba Infections

PubMed Central

Lorenzo-Morales, Jacob; Machin, Rubén P.; López-Arencibia, Atteneri; García-Castellano, José Manuel; de Fuentes, Isabel; Loftus, Brendan; Maciver, Sutherland K.; Valladares, Basilio; Piñero, José E.

2013-01-01

Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease. PMID:23114753

Automatic identification of bird targets with radar via patterns produced by wing flapping.

PubMed

Zaugg, Serge; Saporta, Gilbert; van Loon, Emiel; Schmaljohann, Heiko; Liechti, Felix

2008-09-06

Bird identification with radar is important for bird migration research, environmental impact assessments (e.g. wind farms), aircraft security and radar meteorology. In a study on bird migration, radar signals from birds, insects and ground clutter were recorded. Signals from birds show a typical pattern due to wing flapping. The data were labelled by experts into the four classes BIRD, INSECT, CLUTTER and UFO (unidentifiable signals). We present a classification algorithm aimed at automatic recognition of bird targets. Variables related to signal intensity and wing flapping pattern were extracted (via continuous wavelet transform). We used support vector classifiers to build predictive models. We estimated classification performance via cross validation on four datasets. When data from the same dataset were used for training and testing the classifier, the classification performance was extremely to moderately high. When data from one dataset were used for training and the three remaining datasets were used as test sets, the performance was lower but still extremely to moderately high. This shows that the method generalizes well across different locations or times. Our method provides a substantial gain of time when birds must be identified in large collections of radar signals and it represents the first substantial step in developing a real time bird identification radar system. We provide some guidelines and ideas for future research.

Collaborative identification method for sea battlefield target based on deep convolutional neural networks

NASA Astrophysics Data System (ADS)

Zheng, Guangdi; Pan, Mingbo; Liu, Wei; Wu, Xuetong

2018-03-01

The target identification of the sea battlefield is the prerequisite for the judgment of the enemy in the modern naval battle. In this paper, a collaborative identification method based on convolution neural network is proposed to identify the typical targets of sea battlefields. Different from the traditional single-input/single-output identification method, the proposed method constructs a multi-input/single-output co-identification architecture based on optimized convolution neural network and weighted D-S evidence theory. The simulation results show that

Nuclear Security: Target Analysis-rev

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Surinder Paul; Gibbs, Philip W.; Bultz, Garl A.

2014-03-01

The objectives of this presentation are to understand target identification, including roll-up and protracted theft; evaluate target identification in the SNRI; recognize the target characteristics and consequence levels; and understand graded safeguards.

Leveraging 3D chemical similarity, target and phenotypic data in the identification of drug-protein and drug-adverse effect associations.

PubMed

Vilar, Santiago; Hripcsak, George

2016-01-01

Drug-target identification is crucial to discover novel applications for existing drugs and provide more insights about mechanisms of biological actions, such as adverse drug effects (ADEs). Computational methods along with the integration of current big data sources provide a useful framework for drug-target and drug-adverse effect discovery. In this article, we propose a method based on the integration of 3D chemical similarity, target and adverse effect data to generate a drug-target-adverse effect predictor along with a simple leveraging system to improve identification of drug-targets and drug-adverse effects. In the first step, we generated a system for multiple drug-target identification based on the application of 3D drug similarity into a large target dataset extracted from the ChEMBL. Next, we developed a target-adverse effect predictor combining targets from ChEMBL with phenotypic information provided by SIDER data source. Both modules were linked to generate a final predictor that establishes hypothesis about new drug-target-adverse effect candidates. Additionally, we showed that leveraging drug-target candidates with phenotypic data is very useful to improve the identification of drug-targets. The integration of phenotypic data into drug-target candidates yielded up to twofold precision improvement. In the opposite direction, leveraging drug-phenotype candidates with target data also yielded a significant enhancement in the performance. The modeling described in the current study is simple and efficient and has applications at large scale in drug repurposing and drug safety through the identification of mechanism of action of biological effects.

The Development and Validation of a Life Experience Inventory for the Identification of Creative Electrical Engineers.

ERIC Educational Resources Information Center

Michael, William B.; Colson, Kenneth R.

1979-01-01

The construction and validation of the Life Experience Inventory (LEI) for the identification of creative electrical engineers are described. Using the number of patents held or pending as a criterion measure, the LEI was found to have high concurrent validity. (JKS)

Multi-look fusion identification: a paradigm shift from quality to quantity in data samples

NASA Astrophysics Data System (ADS)

Wong, S.

2009-05-01

A multi-look identification method known as score-level fusion is found to be capable of achieving very high identification accuracy, even when low quality target signatures are used. Analysis using measured ground vehicle radar signatures has shown that a 97% correct identification rate can be achieved using this multi-look fusion method; in contrast, only a 37% accuracy rate is obtained when single target signature input is used. The results suggest that quantity can be used to replace quality of the target data in improving identification accuracy. With the advent of sensor technology, a large amount of target signatures of marginal quality can be captured routinely. This quantity over quality approach allows maximum exploitation of the available data to improve the target identification performance and this could have the potential of being developed into a disruptive technology.

Botany, ethnomedicines, phytochemistry and pharmacology of Himalayan paeony (Paeonia emodi Royle.).

PubMed

Ahmad, Mushtaq; Malik, Khafsa; Tariq, Akash; Zhang, Guolin; Yaseen, Ghulam; Rashid, Neelam; Sultana, Shazia; Zafar, Muhammad; Ullah, Kifayat; Khan, Muhammad Pukhtoon Zada

2018-06-28

Himalayan paeony (Paeonia emodi Royle.) is an important species used to treat various diseases. This study aimed to compile the detailed traditional medicinal uses, phytochemistry, pharmacology and toxicological investigations on P. emodi. This study also highlights taxonomic validity, quality of experimental designs and shortcomings in previously reported information on Himalayan paeony. The data was extracted from unpublished theses (Pakistan, China, India and Nepal), and different published research articles confined to pharmacology, phytochemistry and antimicrobial activities using different databases through specific keywords. The relevant information regarding medicinal uses, taxonomic/common names, part used, collection and identification source, authentication, voucher specimen number, plant extracts and their characterization, isolation and identification of phytochemicals, methods of study in silico, in vivo or in vitro, model organism used, dose and duration, minimal active concentration, zone of inhibition (antimicrobial study), bioactive compound(s), mechanism of action on single or multiple targets, and toxicological information. P. emodi is reported for diverse medicinal uses with pharmacological properties like antioxidant, nephroprotective, lipoxygenase inhibitory, cognition and oxidative stress release, cytotoxic, anti-inflammatory, antiepileptic, anticonvulsant, haemaglutination, alpha-chymotrypsin inhibitory, hepatoprotective, hepatic chromes and pharmacokinetics of carbamazepine expression, β-glucuronidase inhibitory, spasmolytic and spasmogenic, and airway relaxant. Data confined to its taxonomic validity, shows 10% studies with correct taxonomic name while 90% studies with incorrect taxonomic, pharmacopeial and common names. The literature reviewed, shows lack of collection source (11 reports), without proper source of identification (15 reports), 33 studies without voucher specimen number, 26 reports lack information on authentic herbarium submission and most of the studies (90%) without validation of taxonomic names using recognized databases. In reported methods, 67% studies without characterization of extracts, 25% lack proper dose, 40% without duration and 31% reports lack information on proper controls. Similarly, only 18% studies reports active compound(s) responsible for pharmacological activities, 14% studies show minimal active concentration, only 2.5% studies report mechanism of action on target while none of the reports mentioned in silico approach. P. emodi is endemic to Himalayan region (Pakistan, China, India and Nepal) with diverse traditional therapeutic uses. Majority of reviewed studies showed confusion in its taxonomic validity, incomplete methodologies and ambiguous findings. Keeping in view the immense uses of P. emodi in various traditional medicinal systems, holistic pharmacological approaches in combination with reverse pharmacology, system biology, and "omics" technologies are recommended to improve the quality of research which leads to natural drug discovery development at global perspectives. Copyright © 2018 Elsevier B.V. All rights reserved.

Nematode Species Identification—Current Status, Challenges and Future Perspectives for Cyathostomins

PubMed Central

Bredtmann, Christina M.; Krücken, Jürgen; Murugaiyan, Jayaseelan; Kuzmina, Tetiana; von Samson-Himmelstjerna, Georg

2017-01-01

Human and animal health is globally affected by a variety of parasitic helminths. The impact of co-infections and development of anthelmintic resistance requires improved diagnostic tools, especially for parasitic nematodes e.g., to identify resistant species or attribute pathological effects to individual species or particular species combinations. In horses, co-infection with cyathostomins is rather a rule than an exception with typically 5 to 15 species (out of more than 40 described) per individual host. In cyathostomins, reliable morphological species differentiation is currently limited to adults and requires highly specialized expertize while precise morphological identification of eggs and early stage larvae is impossible. The situation is further complicated by a questionable validity of some cyathostomins while others might actually represent cryptic species complexes. Several molecular methods using different target sequences were established to overcome these limitations. For adult worms, PCR followed by sequencing of mitochondrial genes or external or internal ribosomal RNA spacers is suitable to genetically confirm morphological identifications. The most commonly used method to differentiate eggs or larvae is the reverse-line-blot hybridization assay. However, both methods suffer from the fact that target sequences are not available for many species or even that GenBank® entries are unreliable regarding the cyathostomin species. Recent advances in proteomic tools for identification of metazoans including insects and nematodes of the genus Trichinella will be evaluated for suitability to diagnose cyathostomins. Future research should focus on the comparative analysis of morphological, molecular and proteomic data from the same cyathostomin specimen to optimize tools for species-specific identification. PMID:28702376

Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

PubMed

Nongonierma, Alice B; FitzGerald, Richard J

2018-06-01

Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development.

PubMed

Litholdo, Celso G; Parker, Benjamin L; Eamens, Andrew L; Larsen, Martin R; Cordwell, Stuart J; Waterhouse, Peter M

2016-06-01

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Signal-Noise Identification of Magnetotelluric Signals Using Fractal-Entropy and Clustering Algorithm for Targeted De-Noising

NASA Astrophysics Data System (ADS)

Li, Jin; Zhang, Xian; Gong, Jinzhe; Tang, Jingtian; Ren, Zhengyong; Li, Guang; Deng, Yanli; Cai, Jin

A new technique is proposed for signal-noise identification and targeted de-noising of Magnetotelluric (MT) signals. This method is based on fractal-entropy and clustering algorithm, which automatically identifies signal sections corrupted by common interference (square, triangle and pulse waves), enabling targeted de-noising and preventing the loss of useful information in filtering. To implement the technique, four characteristic parameters — fractal box dimension (FBD), higuchi fractal dimension (HFD), fuzzy entropy (FuEn) and approximate entropy (ApEn) — are extracted from MT time-series. The fuzzy c-means (FCM) clustering technique is used to analyze the characteristic parameters and automatically distinguish signals with strong interference from the rest. The wavelet threshold (WT) de-noising method is used only to suppress the identified strong interference in selected signal sections. The technique is validated through signal samples with known interference, before being applied to a set of field measured MT/Audio Magnetotelluric (AMT) data. Compared with the conventional de-noising strategy that blindly applies the filter to the overall dataset, the proposed method can automatically identify and purposefully suppress the intermittent interference in the MT/AMT signal. The resulted apparent resistivity-phase curve is more continuous and smooth, and the slow-change trend in the low-frequency range is more precisely reserved. Moreover, the characteristic of the target-filtered MT/AMT signal is close to the essential characteristic of the natural field, and the result more accurately reflects the inherent electrical structure information of the measured site.

Identification, characterization and expression analysis of pigeonpea miRNAs in response to Fusarium wilt.

PubMed

Hussain, Khalid; Mungikar, Kanak; Kulkarni, Abhijeet; Kamble, Avinash

2018-05-05

Upon confrontation with unfavourable conditions, plants invoke a very complex set of biochemical and physiological reactions and alter gene expression patterns to combat the situations. MicroRNAs (miRNAs), a class of small non-coding RNA, contribute extensively in regulation of gene expression through translation inhibition or degradation of their target mRNAs during such conditions. Therefore, identification of miRNAs and their targets holds importance in understanding the regulatory networks triggered during stress. Structure and sequence similarity based in silico prediction of miRNAs in Cajanus cajan L. (Pigeonpea) draft genome sequence has been carried out earlier. These annotations also appear in related GenBank genome sequence entries. However, there are no reports available on context dependent miRNA expression and their targets in pigeonpea. Therefore, in the present study we addressed these questions computationally, using pigeonpea EST sequence information. We identified five novel pigeonpea miRNA precursors, their mature forms and targets. Interestingly, only one of these miRNAs (miR169i-3p) was identified earlier in draft genome sequence. We then validated expression of these miRNAs, experimentally. It was also observed that these miRNAs show differential expression patterns in response to Fusarium inoculation indicating their biotic stress responsive nature. Overall these results will help towards better understanding the regulatory network of defense during pigeonpea -pathogen interactions and role of miRNAs in the process. Copyright © 2018 Elsevier B.V. All rights reserved.

Reliability and validity of a talent identification test battery for seated and standing Paralympic throws.

PubMed

Spathis, Jemima Grace; Connick, Mark James; Beckman, Emma Maree; Newcombe, Peter Anthony; Tweedy, Sean Michael

2015-01-01

Paralympic throwing events for athletes with physical impairments comprise seated and standing javelin, shot put, discus and seated club throwing. Identification of talented throwers would enable prediction of future success and promote participation; however, a valid and reliable talent identification battery for Paralympic throwing has not been reported. This study evaluates the reliability and validity of a talent identification battery for Paralympic throws. Participants were non-disabled so that impairment would not confound analyses, and results would provide an indication of normative performance. Twenty-eight non-disabled participants (13 M; 15 F) aged 23.6 years (±5.44) performed five kinematically distinct criterion throws (three seated, two standing) and nine talent identification tests (three anthropometric, six motor); 23 were tested a second time to evaluate test-retest reliability. Talent identification test-retest reliability was evaluated using Intra-class Correlation Coefficient (ICC) and Bland-Altman plots (Limits of Agreement). Spearman's correlation assessed strength of association between criterion throws and talent identification tests. Reliability was generally acceptable (mean ICC = 0.89), but two seated talent identification tests require more extensive familiarisation. Correlation strength (mean rs = 0.76) indicated that the talent identification tests can be used to validly identify individuals with competitively advantageous attributes for each of the five kinematically distinct throwing activities. Results facilitate further research in this understudied area.

Patterns of Cognitive Strengths and Weaknesses: Identification Rates, Agreement, and Validity for Learning Disabilities Identification

ERIC Educational Resources Information Center

Miciak, Jeremy; Fletcher, Jack M.; Stuebing, Karla K.; Vaughn, Sharon; Tolar, Tammy D.

2014-01-01

Few empirical investigations have evaluated learning disabilities (LD) identification methods based on a pattern of cognitive strengths and weaknesses (PSW). This study investigated the reliability and validity of two proposed PSW methods: the concordance/discordance method (C/DM) and cross battery assessment (XBA) method. Cognitive assessment…

Identification of a novel MYO7A mutation in Usher syndrome type 1.

PubMed

Cheng, Ling; Yu, Hongsong; Jiang, Yan; He, Juan; Pu, Sisi; Li, Xin; Zhang, Li

2018-01-05

Usher syndrome (USH) is an autosomal recessive disease characterized by deafness and retinitis pigmentosa. In view of the high phenotypic and genetic heterogeneity in USH, performing genetic screening with traditional methods is impractical. In the present study, we carried out targeted next-generation sequencing (NGS) to uncover the underlying gene in an USH family (2 USH patients and 15 unaffected relatives). One hundred and thirty-five genes associated with inherited retinal degeneration were selected for deep exome sequencing. Subsequently, variant analysis, Sanger validation and segregation tests were utilized to identify the disease-causing mutations in this family. All affected individuals had a classic USH type I (USH1) phenotype which included deafness, vestibular dysfunction and retinitis pigmentosa. Targeted NGS and Sanger sequencing validation suggested that USH1 patients carried an unreported splice site mutation, c.5168+1G>A, as a compound heterozygous mutation with c.6070C>T (p.R2024X) in the MYO7A gene. A functional study revealed decreased expression of the MYO7A gene in the individuals carrying heterozygous mutations. In conclusion, targeted next-generation sequencing provided a comprehensive and efficient diagnosis for USH1. This study revealed the genetic defects in the MYO7A gene and expanded the spectrum of clinical phenotypes associated with USH1 mutations.

Systems-level modeling of mycobacterial metabolism for the identification of new (multi-)drug targets.

PubMed

Rienksma, Rienk A; Suarez-Diez, Maria; Spina, Lucie; Schaap, Peter J; Martins dos Santos, Vitor A P

2014-12-01

Systems-level metabolic network reconstructions and the derived constraint-based (CB) mathematical models are efficient tools to explore bacterial metabolism. Approximately one-fourth of the Mycobacterium tuberculosis (Mtb) genome contains genes that encode proteins directly involved in its metabolism. These represent potential drug targets that can be systematically probed with CB models through the prediction of genes essential (or the combination thereof) for the pathogen to grow. However, gene essentiality depends on the growth conditions and, so far, no in vitro model precisely mimics the host at the different stages of mycobacterial infection, limiting model predictions. These limitations can be circumvented by combining expression data from in vivo samples with a validated CB model, creating an accurate description of pathogen metabolism in the host. To this end, we present here a thoroughly curated and extended genome-scale CB metabolic model of Mtb quantitatively validated using 13C measurements. We describe some of the efforts made in integrating CB models and high-throughput data to generate condition specific models, and we will discuss challenges ahead. This knowledge and the framework herein presented will enable to identify potential new drug targets, and will foster the development of optimal therapeutic strategies. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygenmore » and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high-content screen for the identification of compounds that target cells in dormant tumor spheroid regions. • Metabolite profiling by NMR spectroscopy on 3D tumor spheroids. • Identification of respiratory chain inhibitors to specifically induce cell death in inner tumor spheroid regions. • Respiratory chain inhibitors enhance cytostatic based therapy in vitro.« less

CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool

PubMed Central

del Sol Keyer, Maria; Wittbrodt, Joachim; Mateo, Juan L.

2015-01-01

Engineering of the CRISPR/Cas9 system has opened a plethora of new opportunities for site-directed mutagenesis and targeted genome modification. Fundamental to this is a stretch of twenty nucleotides at the 5’ end of a guide RNA that provides specificity to the bound Cas9 endonuclease. Since a sequence of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas9 complex, an efficient and reliable in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, http://crispr.cos.uni-heidelberg.de) to overcome limitations of already available tools. CCTop provides an intuitive user interface with reasonable default parameters that can easily be tuned by the user. From a given query sequence, CCTop identifies and ranks all candidate sgRNA target sites according to their off-target quality and displays full documentation. CCTop was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair. Thus, CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites. PMID:25909470

from the Adolescents’ Perspective in Malaysia

PubMed

Mohd Zin, Faridah; Hillaluddin, Azlin Hilma; Mustaffa, Jamaludin

2017-05-01

Objective: This study aims to develop, validate and determine the reliability of an interactive multimedia strategy to prevent tobacco use among the young (TUPY-S) from an adolescents’ perspective. Methods: A descriptive study design was utilized. A modular instruction guideline by Russel (1974) was followed in the entire process, comprising a feasibility study, a review of existing modules, specification of the objectives, identification of the construct criterion items, learner analysis and entry behavior specification, establishment of the sequence instruction and media selection, a tryout with students and a field test. Result: Feasibility was agreed among the researchers and the school authorities. Culturally suitable rigorously developed tobacco use preventive strategies delivered using information technology (IT) are lacking in the literature. The objective of TUPY-S is to prevent tobacco use among adolescents living in Malaysia. Identified construct criterion items include knowledge, attitude, intention to use, self-efficacy, and refusal skill. The target population was early adolescents belonging to generation-Z. Content was developed from the adolescents’ perspective and delivered using IT in Malay language. Content validity, assessed by six experts in the field and module development, was good at 86%. The students’ tryout showed satisfactory face validity subjectively and objectively (85.5%) and high alpha Cronbach reliability (0.91). Conclusion: TUPY-S was confirmed to suit early adolescents of the current generation living in Malaysia. It demonstrated good content validity among the experts, satisfactory face validity and reliability among the target population. TUPY-S is ready to be evaluated for its effectiveness among early adolescents. Creative Commons Attribution License

Fragment-based approaches to the discovery of kinase inhibitors.

PubMed

Mortenson, Paul N; Berdini, Valerio; O'Reilly, Marc

2014-01-01

Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

PubMed

Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W; Ben Mamoun, Choukri; Altman, Sidney

2015-09-22

Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.

Global Identification of MicroRNAs and Their Targets in Barley under Salinity Stress

PubMed Central

Cui, Licao; Feng, Kewei; Liu, Fuyan; Du, Xianghong; Tong, Wei; Nie, Xiaojun; Ji, Wanquan; Weining, Song

2015-01-01

Salinity is a major limiting factor for agricultural production worldwide. A better understanding of the mechanisms of salinity stress response will aid efforts to improve plant salt tolerance. In this study, a combination of small RNA and mRNA degradome sequencing was used to identify salinity responsive-miRNAs and their targets in barley. A total of 152 miRNAs belonging to 126 families were identified, of which 44 were found to be salinity responsive with 30 up-regulated and 25 down-regulated respectively. The majority of the salinity-responsive miRNAs were up-regulated at the 8h time point, while down-regulated at the 3h and 27h time points. The targets of these miRNAs were further detected by degradome sequencing coupled with bioinformatics prediction. Finally, qRT-PCR was used to validate the identified miRNA and their targets. Our study systematically investigated the expression profile of miRNA and their targets in barley during salinity stress phase, which can contribute to understanding how miRNAs respond to salinity stress in barley and other cereal crops. PMID:26372557

Species and hybrid identification of sturgeon caviar: a new molecular approach to detect illegal trade.

PubMed

Boscari, E; Barmintseva, A; Pujolar, J M; Doukakis, P; Mugue, N; Congiu, L

2014-05-01

Overexploitation of wild populations due to the high economic value of caviar has driven sturgeons to near extinction. The high prices commanded by caviar on world markets have made it a magnet for illegal and fraudulent caviar trade, often involving low-value farmed caviar being sold as top-quality caviar. We present a new molecular approach for the identification of pure sturgeon species and hybrids that are among the most commercialized species in Europe and North America. Our test is based on the discovery of species-specific single nucleotide polymorphisms (SNPs) in the ribosomal protein S7, supplemented with the Vimentin gene and the mitochondrial D-loop. Test validations performed in 702 specimens of target and nontarget sturgeon species demonstrated a 100% identification success for Acipenser naccarii, A. fulvescens, A. stellatus, A. sinensis and A. transmontanus. In addition to species identification, our approach allows the identification of Bester and AL hybrids, two of the most economically important hybrids in the world, with 80% and 100% success, respectively. Moreover, the approach has the potential to identify many other existing sturgeon hybrids. The development of a standardized sturgeon identification tool will directly benefit trade law enforcement, providing the tools to monitor and regulate the legal trade of caviar and protect sturgeon stocks from illicit producers and traders, hence contributing to safeguarding this group of heavily threatened species. © 2013 John Wiley & Sons Ltd.

The use of alcohol use disorders identification test (AUDIT) in detecting alcohol use disorder and risk drinking in the general population: validation of AUDIT using schedules for clinical assessment in neuropsychiatry.

PubMed

Lundin, Andreas; Hallgren, Mats; Balliu, Natalja; Forsell, Yvonne

2015-01-01

The alcohol use disorders identification test (AUDIT) and AUDIT-Consumption (AUDIT-C) are commonly used in population surveys but there are few validations studies in the general population. Validity should be estimated in samples close to the targeted population and setting. This study aims to validate AUDIT and AUDIT-C in a general population sample (PART) in Stockholm, Sweden. We used a general population subsample age 20 to 64 that answered a postal questionnaire including AUDIT who later participated in a psychiatric interview (n = 1,093). Interviews using Schedules for Clinical Assessment in Neuropsychiatry was used as criterion standard. Diagnoses were set according to the fourth version of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). Agreement between the diagnostic test and criterion standard was measured with area under the receiver operator characteristics curve (AUC). A total of 1,086 (450 men and 636 women) of the interview participants completed AUDIT. There were 96 individuals with DSM-IV-alcohol dependence, 36 DSM-IV-Alcohol Abuse, and 153 Risk drinkers. AUCs were for DSM-IV-alcohol use disorder 0.90 (AUDIT-C 0.85); DSM-IV-dependence 0.94 (AUDIT-C 0.89); risk drinking 0.80 (AUDIT-C 0.80); and any criterion 0.87 (AUDIT-C 0.84). In this general population sample, AUDIT and AUDIT-C performed outstanding or excellent in identifying dependency, risk drinking, alcohol use disorder, any disorder, or risk drinking. Copyright © 2015 by the Research Society on Alcoholism.

Standardization of a two-step real-time polymerase chain reaction based method for species-specific detection of medically important Aspergillus species.

PubMed

Das, P; Pandey, P; Harishankar, A; Chandy, M; Bhattacharya, S; Chakrabarti, A

2017-01-01

Standardization of Aspergillus polymerase chain reaction (PCR) poses two technical challenges (a) standardization of DNA extraction, (b) optimization of PCR against various medically important Aspergillus species. Many cases of aspergillosis go undiagnosed because of relative insensitivity of conventional diagnostic methods such as microscopy, culture or antigen detection. The present study is an attempt to standardize real-time PCR assay for rapid sensitive and specific detection of Aspergillus DNA in EDTA whole blood. Three nucleic acid extraction protocols were compared and a two-step real-time PCR assay was developed and validated following the recommendations of the European Aspergillus PCR Initiative in our setup. In the first PCR step (pan-Aspergillus PCR), the target was 28S rDNA gene, whereas in the second step, species specific PCR the targets were beta-tubulin (for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus), gene and calmodulin gene (for Aspergillus niger). Species specific identification of four medically important Aspergillus species, namely, A. fumigatus, A. flavus, A. niger and A. terreus were achieved by this PCR. Specificity of the PCR was tested against 34 different DNA source including bacteria, virus, yeast, other Aspergillus sp., other fungal species and for human DNA and had no false-positive reactions. The analytical sensitivity of the PCR was found to be 102 CFU/ml. The present protocol of two-step real-time PCR assays for genus- and species-specific identification for commonly isolated species in whole blood for diagnosis of invasive Aspergillus infections offers a rapid, sensitive and specific assay option and requires clinical validation at multiple centers.

Diverse correlation patterns between microRNAs and their targets during tomato fruit development indicates different modes of microRNA actions.

PubMed

Lopez-Gomollon, Sara; Mohorianu, Irina; Szittya, Gyorgy; Moulton, Vincent; Dalmay, Tamas

2012-12-01

MicroRNAs negatively regulate the accumulation of mRNAs therefore when they are expressed in the same cells their expression profiles show an inverse correlation. We previously described one positively correlated miRNA/target pair, but it is not known how widespread this phenomenon is. Here, we investigated the correlation between the expression profiles of differentially expressed miRNAs and their targets during tomato fruit development using deep sequencing, Northern blot and RT-qPCR. We found an equal number of positively and negatively correlated miRNA/target pairs indicating that positive correlation is more frequent than previously thought. We also found that the correlation between microRNA and target expression profiles can vary between mRNAs belonging to the same gene family and even for the same target mRNA at different developmental stages. Since microRNAs always negatively regulate their targets, the high number of positively correlated microRNA/target pairs suggests that mutual exclusion could be as widespread as temporal regulation. The change of correlation during development suggests that the type of regulatory circuit directed by a microRNA can change over time and can be different for individual gene family members. Our results also highlight potential problems for expression profiling-based microRNA target identification/validation.

Real-time ultrasound image classification for spine anesthesia using local directional Hadamard features.

PubMed

Pesteie, Mehran; Abolmaesumi, Purang; Ashab, Hussam Al-Deen; Lessoway, Victoria A; Massey, Simon; Gunka, Vit; Rohling, Robert N

2015-06-01

Injection therapy is a commonly used solution for back pain management. This procedure typically involves percutaneous insertion of a needle between or around the vertebrae, to deliver anesthetics near nerve bundles. Most frequently, spinal injections are performed either blindly using palpation or under the guidance of fluoroscopy or computed tomography. Recently, due to the drawbacks of the ionizing radiation of such imaging modalities, there has been a growing interest in using ultrasound imaging as an alternative. However, the complex spinal anatomy with different wave-like structures, affected by speckle noise, makes the accurate identification of the appropriate injection plane difficult. The aim of this study was to propose an automated system that can identify the optimal plane for epidural steroid injections and facet joint injections. A multi-scale and multi-directional feature extraction system to provide automated identification of the appropriate plane is proposed. Local Hadamard coefficients are obtained using the sequency-ordered Hadamard transform at multiple scales. Directional features are extracted from local coefficients which correspond to different regions in the ultrasound images. An artificial neural network is trained based on the local directional Hadamard features for classification. The proposed method yields distinctive features for classification which successfully classified 1032 images out of 1090 for epidural steroid injection and 990 images out of 1052 for facet joint injection. In order to validate the proposed method, a leave-one-out cross-validation was performed. The average classification accuracy for leave-one-out validation was 94 % for epidural and 90 % for facet joint targets. Also, the feature extraction time for the proposed method was 20 ms for a native 2D ultrasound image. A real-time machine learning system based on the local directional Hadamard features extracted by the sequency-ordered Hadamard transform for detecting the laminae and facet joints in ultrasound images has been proposed. The system has the potential to assist the anesthesiologists in quickly finding the target plane for epidural steroid injections and facet joint injections.

Fast and Confident: Postdicting Eyewitness Identification Accuracy in a Field Study

ERIC Educational Resources Information Center

Sauerland, Melanie; Sporer, Siegfried L.

2009-01-01

The combined postdictive value of postdecision confidence, decision time, and Remember-Know-Familiar (RKF) judgments as markers of identification accuracy was evaluated with 10 targets and 720 participants. In a pedestrian area, passers-by were asked for directions. Identifications were made from target-absent or target-present lineups. Fast…

Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress.

PubMed

Zhu, Jianfeng; Li, Wanfeng; Yang, Wenhua; Qi, Liwang; Han, Suying

2013-09-01

142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, in C. intermedia . The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR. MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs and analysis of transcriptome data were performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. Furthermore, 36 potential target genes of 17 known miRNA families and 2 potential target genes of 1 novel miRNA were predicted; 4 of these were validated by 5' RACE. The expression of 12 miRNAs was validated in different tissues, and these and five target mRNAs were assessed by qRT-PCR after salt treatment. The expression levels of seven miRNAs (cin-miR157a, cin-miR159a, cin-miR165a, cin-miR167b, cin-miR172b, cin-miR390a and cin-miR396a) were upregulated, while cin-miR398a expression was downregulated after salt treatment. The targets of cin-miR157a, cin-miR165a, cin-miR172b and cin-miR396a were downregulated and showed an approximately negative correlation with their corresponding miRNAs under salt treatment. These results would help further understanding of miRNA regulation in response to abiotic stress in C. intermedia.

Target identification for small bioactive molecules: finding the needle in the haystack.

PubMed

Ziegler, Slava; Pries, Verena; Hedberg, Christian; Waldmann, Herbert

2013-03-04

Identification and confirmation of bioactive small-molecule targets is a crucial, often decisive step both in academic and pharmaceutical research. Through the development and availability of several new experimental techniques, target identification is, in principle, feasible, and the number of successful examples steadily grows. However, a generic methodology that can successfully be applied in the majority of the cases has not yet been established. Herein we summarize current methods for target identification of small molecules, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound. We describe the most frequently employed experimental approaches for target identification and provide several representative examples illustrating the state-of-the-art. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be most powerful and most frequently applied. To provide guidance for rapid entry into the field and based on our own experience we propose a typical workflow for target identification, which centers on the application of chemical proteomics as the key step to generate hypotheses for potential target proteins. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of Reduced-Order Thermal Therapy Models Using Thermal MR Images: Theory and Validation

PubMed Central

2013-01-01

In this paper, we develop and validate a method to identify computationally efficient site- and patient-specific models of ultrasound thermal therapies from MR thermal images. The models of the specific absorption rate of the transduced energy and the temperature response of the therapy target are identified in the reduced basis of proper orthogonal decomposition of thermal images, acquired in response to a mild thermal test excitation. The method permits dynamic reidentification of the treatment models during the therapy by recursively utilizing newly acquired images. Such adaptation is particularly important during high-temperature therapies, which are known to substantially and rapidly change tissue properties and blood perfusion. The developed theory was validated for the case of focused ultrasound heating of a tissue phantom. The experimental and computational results indicate that the developed approach produces accurate low-dimensional treatment models despite temporal and spatial noises in MR images and slow image acquisition rate. PMID:22531754

Detection of regulated herbs and plants in plant food supplements and traditional medicines using infrared spectroscopy.

PubMed

Deconinck, E; Djiogo, C A Sokeng; Bothy, J L; Courselle, P

2017-08-05

The identification of a specific toxic or regulated plant in herbal preparations or plant food supplements is a real challenge, since they are often powdered, mixed with other herbal or synthetic powders and compressed into tablets or capsules. The classical identification approaches based on micro- and macroscopy are therefore not possible anymore. In this paper infrared spectroscopy, combined with attenuated total reflectance was evaluated for the screening of plant based preparations for nine specific plants (five regulated and four common plants for herbal supplements). IR and NIR spectra were recorded for a series of self-made triturations of the targeted plants. After pretreatment of the spectral data chemometric classification techniques were applied to both data sets (IR and NIR) separately and the combination of both. The results show that the screening of herbal preparations or plant food supplements for specific plants, using infrared spectroscopy, is feasible. The best model was obtained with the Mid-IR data, using SIMCA as modelling technique. During validation of the model, using an external test set, 21 of 25 were correctly classified and six of the nine targeted plants showed no misclassifications for the selected test set. For the other three a success rate of 50% was obtained. Mid-IR combined with SIMCA can therefore be applied as a first step in the screening of unknown samples, before applying more sophisticated fingerprint approaches or identification tests described in several national and international pharmacopoeia. As a proof of concept five real suspicious samples were successfully screened for the targeted regulated plants. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

PubMed Central

Ge, Xie; Zhang, Yong; Jiang, Jianhao; Zhong, Yi; Yang, Xiaonan; Li, Zhiqian; Huang, Yongping; Tan, Anjiang

2013-01-01

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs. PMID:23289012

A quick responding quartz crystal microbalance sensor array based on molecular imprinted polyacrylic acids coating for selective identification of aldehydes in body odor.

PubMed

Jha, Sunil K; Hayashi, Kenshi

2015-03-01

In present work, a novel quartz crystal microbalance (QCM) sensor array has been developed for prompt identification of primary aldehydes in human body odor. Molecularly imprinted polymers (MIP) are prepared using the polyacrylic acid (PAA) polymer matrix and three organic acids (propenoic acid, hexanoic acid and octanoic acid) as template molecules, and utilized as QCM surface coating layer. The performance of MIP films is characterized by 4-element QCM sensor array (three coated with MIP layers and one with pure PAA for reference) dynamic and static responses to target aldehydes: hexanal, heptanal, and nonanal in single, binary, and tertiary mixtures at distinct concentrations. The target aldehydes were selected subsequent to characterization of body odor samples with solid phase-micro extraction gas chromatography mass spectrometer (SPME-GC-MS). The hexanoic acid and octanoic acid imprinted PAA exhibit fast response, and better sensitivity, selectivity and reproducibility than the propenoic acid, and non-imprinted PAA in array. The response time and recovery time for hexanoic acid imprinted PAA are obtained as 5 s and 12 s respectively to typical concentrations of binary and tertiary mixtures of aldehydes using the static response. Dynamic sensor array response matrix has been processed with principal component analysis (PCA) for visual, and support vector machine (SVM) classifier for quantitative identification of target odors. Aldehyde odors were identified successfully in principal component (PC) space. SVM classifier results maximum recognition rate 79% for three classes of binary odors and 83% including single, binary, and tertiary odor classes in 3-fold cross validation. Copyright © 2014 Elsevier B.V. All rights reserved.

Assessment of Dengue virus helicase and methyltransferase as targets for fragment-based drug discovery.

PubMed

Coutard, Bruno; Decroly, Etienne; Li, Changqing; Sharff, Andrew; Lescar, Julien; Bricogne, Gérard; Barral, Karine

2014-06-01

Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2'-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180μM to 9mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of the anti-tumor activity and mechanisms of nuciferine through a network pharmacology approach

PubMed Central

Qi, Quan; Li, Rui; Li, Hui-ying; Cao, Yu-bing; Bai, Ming; Fan, Xiao-jing; Wang, Shu-yan; Zhang, Bo; Li, Shao

2016-01-01

Aim: Nuciferine is an aporphine alkaloid extracted from lotus leaves, which is a raw material in Chinese medicinal herb for weight loss. In this study we used a network pharmacology approach to identify the anti-tumor activity of nuciferine and the underlying mechanisms. Methods: The pharmacological activities and mechanisms of nuciferine were identified through target profile prediction, clustering analysis and functional enrichment analysis using our traditional Chinese medicine (TCM) network pharmacology platform. The anti-tumor activity of nuciferine was validated by in vitro and in vivo experiments. The anti-tumor mechanisms of nuciferine were predicted through network target analysis and verified by in vitro experiments. Results: The nuciferine target profile was enriched with signaling pathways and biological functions, including “regulation of lipase activity”, “response to nicotine” and “regulation of cell proliferation”. Target profile clustering results suggested that nuciferine to exert anti-tumor effect. In experimental validation, nuciferine (0.8 mg/mL) markedly inhibited the viability of human neuroblastoma SY5Y cells and mouse colorectal cancer CT26 cells in vitro, and nuciferine (0.05 mg/mL) significantly suppressed the invasion of 6 cancer cell lines in vitro. Intraperitoneal injection of nuciferine (9.5 mg/mL, ip, 3 times a week for 3 weeks) significantly decreased the weight of SY5Y and CT26 tumor xenografts in nude mice. Network target analysis and experimental validation in SY5Y and CT26 cells showed that the anti-tumor effect of nuciferine was mediated through inhibiting the PI3K-AKT signaling pathway and IL-1 levels in SY5Y and CT26 cells. Conclusion: By using a TCM network pharmacology method, nuciferine is identified as an anti-tumor agent against human neuroblastoma and mouse colorectal cancer in vitro and in vivo, through inhibiting the PI3K-AKT signaling pathways and IL-1 levels. PMID:27180984

A convex optimization approach for identification of human tissue-specific interactomes.

PubMed

Mohammadi, Shahin; Grama, Ananth

2016-06-15

Analysis of organism-specific interactomes has yielded novel insights into cellular function and coordination, understanding of pathology, and identification of markers and drug targets. Genes, however, can exhibit varying levels of cell type specificity in their expression, and their coordinated expression manifests in tissue-specific function and pathology. Tissue-specific/tissue-selective interaction mechanisms have significant applications in drug discovery, as they are more likely to reveal drug targets. Furthermore, tissue-specific transcription factors (tsTFs) are significantly implicated in human disease, including cancers. Finally, disease genes and protein complexes have the tendency to be differentially expressed in tissues in which defects cause pathology. These observations motivate the construction of refined tissue-specific interactomes from organism-specific interactomes. We present a novel technique for constructing human tissue-specific interactomes. Using a variety of validation tests (Edge Set Enrichment Analysis, Gene Ontology Enrichment, Disease-Gene Subnetwork Compactness), we show that our proposed approach significantly outperforms state-of-the-art techniques. Finally, using case studies of Alzheimer's and Parkinson's diseases, we show that tissue-specific interactomes derived from our study can be used to construct pathways implicated in pathology and demonstrate the use of these pathways in identifying novel targets. http://www.cs.purdue.edu/homes/mohammas/projects/ActPro.html mohammadi@purdue.edu. © The Author 2016. Published by Oxford University Press.

Identification of Five Novel Variants in Chinese Oculocutaneous Albinism by Targeted Next-Generation Sequencing.

PubMed

Qiu, Biyuan; Ma, Tao; Peng, Chunyan; Zheng, Xiaoqin; Yang, Jiyun

2018-04-01

The diagnosis of oculocutaneous albinism (OCA) is established using clinical signs and symptoms. OCA is, however, a highly genetically heterogeneous disease with mutations identified in at least nineteen unique genes, many of which produce overlapping phenotypic traits. Thus, differentiating genetic OCA subtypes for diagnoses and genetic counseling is challenging, based on clinical presentation alone, and would benefit from a comprehensive molecular diagnostic. To develop and validate a more comprehensive, targeted, next-generation-sequencing-based diagnostic for the identification of OCA-causing variants. The genomic DNA samples from 28 OCA probands were analyzed by targeted next-generation sequencing (NGS), and the candidate variants were confirmed through Sanger sequencing. We observed mutations in the TYR, OCA2, and SLC45A2 genes in 25/28 (89%) patients with OCA. We identified 38 pathogenic variants among these three genes, including 5 novel variants: c.1970G>T (p.Gly657Val), c.1669A>C (p.Thr557Pro), c.2339-2A>C, and c.1349C>G (p.Thr450Arg) in OCA2; c.459_470delTTTTGCTGCCGA (p.Ala155_Phe158del) in SLC45A2. Our findings expand the mutational spectrum of OCA in the Chinese population, and the assay we developed should be broadly useful as a molecular diagnostic, and as an aid for genetic counseling for OCA patients.

Target specific compound identification using a support vector machine.

PubMed

Plewczynski, Dariusz; von Grotthuss, Marcin; Spieser, Stephane A H; Rychlewski, Leszek; Wyrwicz, Lucjan S; Ginalski, Krzysztof; Koch, Uwe

2007-03-01

In many cases at the beginning of an HTS-campaign, some information about active molecules is already available. Often known active compounds (such as substrate analogues, natural products, inhibitors of a related protein or ligands published by a pharmaceutical company) are identified in low-throughput validation studies of the biochemical target. In this study we evaluate the effectiveness of a support vector machine applied for those compounds and used to classify a collection with unknown activity. This approach was aimed at reducing the number of compounds to be tested against the given target. Our method predicts the biological activity of chemical compounds based on only the atom pairs (AP) two dimensional topological descriptors. The supervised support vector machine (SVM) method herein is trained on compounds from the MDL drug data report (MDDR) known to be active for specific protein target. For detailed analysis, five different biological targets were selected including cyclooxygenase-2, dihydrofolate reductase, thrombin, HIV-reverse transcriptase and antagonists of the estrogen receptor. The accuracy of compound identification was estimated using the recall and precision values. The sensitivities for all protein targets exceeded 80% and the classification performance reached 100% for selected targets. In another application of the method, we addressed the absence of an initial set of active compounds for a selected protein target at the beginning of an HTS-campaign. In such a case, virtual high-throughput screening (vHTS) is usually applied by using a flexible docking procedure. However, the vHTS experiment typically contains a large percentage of false positives that should be verified by costly and time-consuming experimental follow-up assays. The subsequent use of our machine learning method was found to improve the speed (since the docking procedure was not required for all compounds from the database) and also the accuracy of the HTS hit lists (the enrichment factor).

Extended version of the "Sniffin' Sticks" identification test: test-retest reliability and validity.

PubMed

Sorokowska, A; Albrecht, E; Haehner, A; Hummel, T

2015-03-30

The extended, 32-item version of the Sniffin' Sticks identification test was developed in order to create a precise tool enabling repeated, longitudinal testing of individual olfactory subfunctions. Odors of the previous test version had to be changed for technical reasons, and the odor identification test needed re-investigation in terms of reliability, validity, and normative values. In our study we investigated olfactory abilities of a group of 100 patients with olfactory dysfunction and 100 controls. We reconfirmed the high test-retest reliability of the extended version of the Sniffin' Sticks identification test and high correlations between the new and the original part of this tool. In addition, we confirmed the validity of the test as it discriminated clearly between controls and patients with olfactory loss. The additional set of 16 odor identification sticks can be either included in the current olfactory test, thus creating a more detailed diagnosis tool, or it can be used separately, enabling to follow olfactory function over time. Additionally, the normative values presented in our paper might provide useful guidelines for interpretation of the extended identification test results. The revised version of the Sniffin' Sticks 32-item odor identification test is a reliable and valid tool for the assessment of olfactory function. Copyright © 2015 Elsevier B.V. All rights reserved.

Public-Private Partnerships in Lead Discovery: Overview and Case Studies.

PubMed

Gottwald, Matthias; Becker, Andreas; Bahr, Inke; Mueller-Fahrnow, Anke

2016-09-01

The pharmaceutical industry is faced with significant challenges in its efforts to discover new drugs that address unmet medical needs. Safety concerns and lack of efficacy are the two main technical reasons for attrition. Improved early research tools including predictive in silico, in vitro, and in vivo models, as well as a deeper understanding of the disease biology, therefore have the potential to improve success rates. The combination of internal activities with external collaborations in line with the interests and needs of all partners is a successful approach to foster innovation and to meet the challenges. Collaboration can take place in different ways, depending on the requirements of the participants. In this review, the value of public-private partnership approaches will be discussed, using examples from the Innovative Medicines Initiative (IMI). These examples describe consortia approaches to develop tools and processes for improving target identification and validation, as well as lead identification and optimization. The project "Kinetics for Drug Discovery" (K4DD), focusing on the adoption of drug-target binding kinetics analysis in the drug discovery decision-making process, is described in more detail. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Clinical implications of omics and systems medicine: focus on predictive and individualized treatment.

PubMed

Benson, M

2016-03-01

Many patients with common diseases do not respond to treatment. This is a key challenge to modern health care, which causes both suffering and enormous costs. One important reason for the lack of treatment response is that common diseases are associated with altered interactions between thousands of genes, in combinations that differ between subgroups of patients who do or do not respond to a given treatment. Such subgroups, or even distinct disease entities, have been described recently in asthma, diabetes, autoimmune diseases and cancer. High-throughput techniques (omics) allow identification and characterization of such subgroups or entities. This may have important clinical implications, such as identification of diagnostic markers for individualized medicine, as well as new therapeutic targets for patients who do not respond to existing drugs. For example, whole-genome sequencing may be applied to more accurately guide treatment of neurodevelopmental diseases, or to identify drugs specifically targeting mutated genes in cancer. A study published in 2015 showed that 28% of hepatocellular carcinomas contained mutated genes that potentially could be targeted by drugs already approved by the US Food and Drug Administration. A translational study, which is described in detail, showed how combined omics, computational, functional and clinical studies could identify and validate a novel diagnostic and therapeutic candidate gene in allergy. Another important clinical implication is the identification of potential diagnostic markers and therapeutic targets for predictive and preventative medicine. By combining computational and experimental methods, early disease regulators may be identified and potentially used to predict and treat disease before it becomes symptomatic. Systems medicine is an emerging discipline, which may contribute to such developments through combining omics with computational, functional and clinical studies. The aims of this review are to provide a brief introduction to systems medicine and discuss how it may contribute to the clinical implementation of individualized treatment, using clinically relevant examples. © 2015 The Association for the Publication of the Journal of Internal Medicine.

Prediction of microRNA target genes using an efficient genetic algorithm-based decision tree.

PubMed

Rabiee-Ghahfarrokhi, Behzad; Rafiei, Fariba; Niknafs, Ali Akbar; Zamani, Behzad

2015-01-01

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression in almost all plants and animals. They play an important role in key processes, such as proliferation, apoptosis, and pathogen-host interactions. Nevertheless, the mechanisms by which miRNAs act are not fully understood. The first step toward unraveling the function of a particular miRNA is the identification of its direct targets. This step has shown to be quite challenging in animals primarily because of incomplete complementarities between miRNA and target mRNAs. In recent years, the use of machine-learning techniques has greatly increased the prediction of miRNA targets, avoiding the need for costly and time-consuming experiments to achieve miRNA targets experimentally. Among the most important machine-learning algorithms are decision trees, which classify data based on extracted rules. In the present work, we used a genetic algorithm in combination with C4.5 decision tree for prediction of miRNA targets. We applied our proposed method to a validated human datasets. We nearly achieved 93.9% accuracy of classification, which could be related to the selection of best rules.

Prediction of microRNA target genes using an efficient genetic algorithm-based decision tree

PubMed Central

Rabiee-Ghahfarrokhi, Behzad; Rafiei, Fariba; Niknafs, Ali Akbar; Zamani, Behzad

2015-01-01

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression in almost all plants and animals. They play an important role in key processes, such as proliferation, apoptosis, and pathogen–host interactions. Nevertheless, the mechanisms by which miRNAs act are not fully understood. The first step toward unraveling the function of a particular miRNA is the identification of its direct targets. This step has shown to be quite challenging in animals primarily because of incomplete complementarities between miRNA and target mRNAs. In recent years, the use of machine-learning techniques has greatly increased the prediction of miRNA targets, avoiding the need for costly and time-consuming experiments to achieve miRNA targets experimentally. Among the most important machine-learning algorithms are decision trees, which classify data based on extracted rules. In the present work, we used a genetic algorithm in combination with C4.5 decision tree for prediction of miRNA targets. We applied our proposed method to a validated human datasets. We nearly achieved 93.9% accuracy of classification, which could be related to the selection of best rules. PMID:26649272

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis.

PubMed

Puttini, Stefania; Ouvrard-Pascaud, Antoine; Palais, Gael; Beggah, Ahmed T; Gascard, Philippe; Cohen-Tannoudji, Michel; Babinet, Charles; Blot-Chabaud, Marcel; Jaisser, Frederic

2005-03-16

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.

Regulation of Isoflavone Biosynthesis by miRNAs in Two Contrasting Soybean Genotypes at Different Seed Developmental Stages.

PubMed

Gupta, Om P; Nigam, Deepti; Dahuja, Anil; Kumar, Sanjeev; Vinutha, T; Sachdev, Archana; Praveen, Shelly

2017-01-01

Owing to the presence of nutritionally important, health-promoting bioactive compounds, especially isoflavones, soybean has acquired the status of a functional food. miRNAs are tiny riboregulator of gene expression by either decreasing and/or increasing the expression of their corresponding target genes. Despite several works on identification and functional characterization of plant miRNAs, the role of miRNAs in the regulation of isoflavones metabolism is still a virgin field. In the present study, we identified a total of 31 new miRNAs along with their 245 putative target genes from soybean seed-specific ESTs using computational approach. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates metabolism and genetic information processing. Out of that, a total of 5 miRNAs ( Gma -miRNA12, Gma -miRNA24, Gma -miRNA26, Gma -miRNA28, and Gma -miRNA29) were predicted and validated for their probable role during isoflavone biosynthesis. We also validated their five target genes using RA-PCR, which is as good as 5'RLM-RACE. Temporal regulation [35 days after flowering, 45, 55, and 65 DAF] of miRNAs and their targets showed differential expression schema. Differential expression of Gma -miR26 and Gma -miRNA28 along with their corresponding target genes ( Glyma.10G197900 and Glyma.09G127200 ) showed a direct relationship with the total isoflavone content. Therefore, understanding the miRNA-based genetic regulation of isoflavone pathway would assist in selection and manipulation to get high-performing soybean genotypes with better isoflavone yield.

Prioritization of Biomarker Targets in Human Umbilical Cord Blood: Identification of Proteins in Infant Blood Serving as Validated Biomarkers in Adults

PubMed Central

Hansmeier, Nicole; Chao, Tzu-Chiao; Goldman, Lynn R.; Witter, Frank R.

2012-01-01

Background: Early diagnosis represents one of the best lines of defense in the fight against a wide array of human diseases. Umbilical cord blood (UCB) is one of the first easily available diagnostic biofluids and can inform about the health status of newborns. However, compared with adult blood, its diagnostic potential remains largely untapped. Objectives: Our goal was to accelerate biomarker research on UCB by exploring its detectable protein content and providing a priority list of potential biomarkers based on known proteins involved in disease pathways. Methods: We explored cord blood serum proteins by profiling a UCB pool of 12 neonates with different backgrounds using a combination of isoelectric focusing and liquid chromatography coupled with matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) and by comparing results with information contained in metabolic and disease databases available for adult blood. Results: A total of 1,210 UCB proteins were identified with a protein-level false discovery rate of ~ 5% as estimated by naïve target-decoy and MAYU approaches, signifying a 6-fold increase in the number of UCB proteins described to date. Identified proteins correspond to 138 different metabolic and disease pathways and provide a platform of mechanistically linked biomarker candidates for tracking disruptions in cellular processes. Moreover, among the identified proteins, 38 were found to be approved biomarkers for adult blood. Conclusions: The results of this study advance current knowledge of the human cord blood serum proteome. They showcase the potential of UCB as a diagnostic medium for assessing infant health by detection and identification of candidate biomarkers for known disease pathways using a global, nontargeted approach. These biomarkers may inform about mechanisms of exposure–disease relationships. Furthermore, biomarkers approved by the U.S. Food and Drug Administration for screening in adult blood were detected in UCB and represent high-priority targets for immediate validation. PMID:22538116

Prioritization of biomarker targets in human umbilical cord blood: identification of proteins in infant blood serving as validated biomarkers in adults.

PubMed

Hansmeier, Nicole; Chao, Tzu-Chiao; Goldman, Lynn R; Witter, Frank R; Halden, Rolf U

2012-05-01

Early diagnosis represents one of the best lines of defense in the fight against a wide array of human diseases. Umbilical cord blood (UCB) is one of the first easily available diagnostic biofluids and can inform about the health status of newborns. However, compared with adult blood, its diagnostic potential remains largely untapped. Our goal was to accelerate biomarker research on UCB by exploring its detectable protein content and providing a priority list of potential biomarkers based on known proteins involved in disease pathways. We explored cord blood serum proteins by profiling a UCB pool of 12 neonates with different backgrounds using a combination of isoelectric focusing and liquid chromatography coupled with matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) and by comparing results with information contained in metabolic and disease databases available for adult blood. A total of 1,210 UCB proteins were identified with a protein-level false discovery rate of ~ 5% as estimated by naïve target-decoy and MAYU approaches, signifying a 6-fold increase in the number of UCB proteins described to date. Identified proteins correspond to 138 different metabolic and disease pathways and provide a platform of mechanistically linked biomarker candidates for tracking disruptions in cellular processes. Moreover, among the identified proteins, 38 were found to be approved biomarkers for adult blood. The results of this study advance current knowledge of the human cord blood serum proteome. They showcase the potential of UCB as a diagnostic medium for assessing infant health by detection and identification of candidate biomarkers for known disease pathways using a global, nontargeted approach. These biomarkers may inform about mechanisms of exposure-disease relationships. Furthermore, biomarkers approved by the U.S. Food and Drug Administration for screening in adult blood were detected in UCB and represent high-priority targets for immediate validation.

Effects of postidentification feedback on eyewitness identification and nonidentification confidence.

PubMed

Semmler, Carolyn; Brewer, Neil; Wells, Gary L

2004-04-01

Two experiments investigated new dimensions of the effect of confirming feedback on eyewitness identification confidence using target-absent and target-present lineups and (previously unused) unbiased witness instructions (i.e., "offender not present" option highlighted). In Experiment 1, participants viewed a crime video and were later asked to try to identify the thief from an 8-person target-absent photo array. Feedback inflated witness confidence for both mistaken identifications and correct lineup rejections. With target-present lineups in Experiment 2, feedback inflated confidence for correct and mistaken identifications and lineup rejections. Although feedback had no influence on the confidence-accuracy correlation, it produced clear overconfidence. Confidence inflation varied with the confidence measure reference point (i.e., retrospective vs. current confidence) and identification response latency.

A database de-identification framework to enable direct queries on medical data for secondary use.

PubMed

Erdal, B S; Liu, J; Ding, J; Chen, J; Marsh, C B; Kamal, J; Clymer, B D

2012-01-01

To qualify the use of patient clinical records as non-human-subject for research purpose, electronic medical record data must be de-identified so there is minimum risk to protected health information exposure. This study demonstrated a robust framework for structured data de-identification that can be applied to any relational data source that needs to be de-identified. Using a real world clinical data warehouse, a pilot implementation of limited subject areas were used to demonstrate and evaluate this new de-identification process. Query results and performances are compared between source and target system to validate data accuracy and usability. The combination of hashing, pseudonyms, and session dependent randomizer provides a rigorous de-identification framework to guard against 1) source identifier exposure; 2) internal data analyst manually linking to source identifiers; and 3) identifier cross-link among different researchers or multiple query sessions by the same researcher. In addition, a query rejection option is provided to refuse queries resulting in less than preset numbers of subjects and total records to prevent users from accidental subject identification due to low volume of data. This framework does not prevent subject re-identification based on prior knowledge and sequence of events. Also, it does not deal with medical free text de-identification, although text de-identification using natural language processing can be included due its modular design. We demonstrated a framework resulting in HIPAA Compliant databases that can be directly queried by researchers. This technique can be augmented to facilitate inter-institutional research data sharing through existing middleware such as caGrid.

The Development of Target-Specific Pose Filter Ensembles To Boost Ligand Enrichment for Structure-Based Virtual Screening.

PubMed

Xia, Jie; Hsieh, Jui-Hua; Hu, Huabin; Wu, Song; Wang, Xiang Simon

2017-06-26

Structure-based virtual screening (SBVS) has become an indispensable technique for hit identification at the early stage of drug discovery. However, the accuracy of current scoring functions is not high enough to confer success to every target and thus remains to be improved. Previously, we had developed binary pose filters (PFs) using knowledge derived from the protein-ligand interface of a single X-ray structure of a specific target. This novel approach had been validated as an effective way to improve ligand enrichment. Continuing from it, in the present work we attempted to incorporate knowledge collected from diverse protein-ligand interfaces of multiple crystal structures of the same target to build PF ensembles (PFEs). Toward this end, we first constructed a comprehensive data set to meet the requirements of ensemble modeling and validation. This set contains 10 diverse targets, 118 well-prepared X-ray structures of protein-ligand complexes, and large benchmarking actives/decoys sets. Notably, we designed a unique workflow of two-layer classifiers based on the concept of ensemble learning and applied it to the construction of PFEs for all of the targets. Through extensive benchmarking studies, we demonstrated that (1) coupling PFE with Chemgauss4 significantly improves the early enrichment of Chemgauss4 itself and (2) PFEs show greater consistency in boosting early enrichment and larger overall enrichment than our prior PFs. In addition, we analyzed the pairwise topological similarities among cognate ligands used to construct PFEs and found that it is the higher chemical diversity of the cognate ligands that leads to the improved performance of PFEs. Taken together, the results so far prove that the incorporation of knowledge from diverse protein-ligand interfaces by ensemble modeling is able to enhance the screening competence of SBVS scoring functions.

A novel approach for predicting microRNA-disease associations by unbalanced bi-random walk on heterogeneous network.

PubMed

Luo, Jiawei; Xiao, Qiu

2017-02-01

MicroRNAs (miRNAs) play a critical role by regulating their targets in post-transcriptional level. Identification of potential miRNA-disease associations will aid in deciphering the pathogenesis of human polygenic diseases. Several computational models have been developed to uncover novel miRNA-disease associations based on the predicted target genes. However, due to the insufficient number of experimentally validated miRNA-target interactions as well as the relatively high false-positive and false-negative rates of predicted target genes, it is still challenging for these prediction models to obtain remarkable performances. The purpose of this study is to prioritize miRNA candidates for diseases. We first construct a heterogeneous network, which consists of a disease similarity network, a miRNA functional similarity network and a known miRNA-disease association network. Then, an unbalanced bi-random walk-based algorithm on the heterogeneous network (BRWH) is adopted to discover potential associations by exploiting bipartite subgraphs. Based on 5-fold cross validation, the proposed network-based method achieves AUC values ranging from 0.782 to 0.907 for the 22 human diseases and an average AUC of almost 0.846. The experiments indicated that BRWH can achieve better performances compared with several popular methods. In addition, case studies of some common diseases further demonstrated the superior performance of our proposed method on prioritizing disease-related miRNA candidates. Copyright © 2017 Elsevier Inc. All rights reserved.

Social judgment of abortion: a black-sheep effect in a Catholic sheepfold.

PubMed

Bègue, L

2001-10-01

French Catholic participants (N = 340) with high or low religious identification read 1 of 8 scenarios presented as an interview with a female target 2 months after she had had an abortion. The experimental device varied situational pressure (pressure vs. no pressure), the target's religious social identity (Catholic vs. neutral), and the consequences of abortion for the target (positive vs. negative). The participants then rated the acceptability of the target's decision. The participants judged abortion more negatively in the no-pressure condition. Moreover, the participants with high religious identification judged abortion more negatively than did those with low religious identification. In partial support of a black-sheep effect, the participants with high religious identification judged the Catholic target more negatively than they judged the neutral one in some conditions (pressure, negative consequences). In other conditions (no pressure, both positive and negative consequences), the participants with low religious identification judged the Catholic target more positively than they judged the neutral one.

Beyond decoding: phonological processing during silent reading in beginning readers.

PubMed

Blythe, Hazel I; Pagán, Ascensión; Dodd, Megan

2015-07-01

In this experiment, the extent to which beginning readers process phonology during lexical identification in silent sentence reading was investigated. The eye movements of children aged seven to nine years and adults were recorded as they read sentences containing either a correctly spelled target word (e.g., girl), a pseudohomophone (e.g., gerl), or a spelling control (e.g., garl). Both children and adults showed a benefit from the valid phonology of the pseudohomophone, compared to the spelling control during reading. This indicates that children as young as seven years old exhibit relatively skilled phonological processing during reading, despite having moved past the use of overt phonological decoding strategies. In addition, in comparison to adults, children's lexical processing was more disrupted by the presence of spelling errors, suggesting a developmental change in the relative dependence upon phonological and orthographic processing in lexical identification during silent sentence reading. (c) 2015 APA, all rights reserved.

ATAQS: A computational software tool for high throughput transition optimization and validation for selected reaction monitoring mass spectrometry

PubMed Central

2011-01-01

Background Since its inception, proteomics has essentially operated in a discovery mode with the goal of identifying and quantifying the maximal number of proteins in a sample. Increasingly, proteomic measurements are also supporting hypothesis-driven studies, in which a predetermined set of proteins is consistently detected and quantified in multiple samples. Selected reaction monitoring (SRM) is a targeted mass spectrometric technique that supports the detection and quantification of specific proteins in complex samples at high sensitivity and reproducibility. Here, we describe ATAQS, an integrated software platform that supports all stages of targeted, SRM-based proteomics experiments including target selection, transition optimization and post acquisition data analysis. This software will significantly facilitate the use of targeted proteomic techniques and contribute to the generation of highly sensitive, reproducible and complete datasets that are particularly critical for the discovery and validation of targets in hypothesis-driven studies in systems biology. Result We introduce a new open source software pipeline, ATAQS (Automated and Targeted Analysis with Quantitative SRM), which consists of a number of modules that collectively support the SRM assay development workflow for targeted proteomic experiments (project management and generation of protein, peptide and transitions and the validation of peptide detection by SRM). ATAQS provides a flexible pipeline for end-users by allowing the workflow to start or end at any point of the pipeline, and for computational biologists, by enabling the easy extension of java algorithm classes for their own algorithm plug-in or connection via an external web site. This integrated system supports all steps in a SRM-based experiment and provides a user-friendly GUI that can be run by any operating system that allows the installation of the Mozilla Firefox web browser. Conclusions Targeted proteomics via SRM is a powerful new technique that enables the reproducible and accurate identification and quantification of sets of proteins of interest. ATAQS is the first open-source software that supports all steps of the targeted proteomics workflow. ATAQS also provides software API (Application Program Interface) documentation that enables the addition of new algorithms to each of the workflow steps. The software, installation guide and sample dataset can be found in http://tools.proteomecenter.org/ATAQS/ATAQS.html PMID:21414234

Natural Products as a Source for Novel Antibiotics.

PubMed

Moloney, Mark G

2016-08-01

Natural products have historically been of crucial importance in the identification and development of antibacterial agents. Interest in these systems has waned in recent years, but the rapid emergence of resistant bacterial strains has forced their re-evaluation as a route to identify novel chemical skeletons with antibacterial activity for elaboration in drug development. This overview examines the current situation, highlights new natural product systems which have been found, together with re-examination of some old ones, and new technologies for their identification. While natural products certainly have the potential to re-emerge as a key start-point in antibacterial drug discovery, reports of new or reinvestigated structures need to be supported with sufficient quality chemical (solubility, stability), biochemical (including toxicity in particular, along with target information) and microbiological [minimum inhibitory concentration (MIC) and resistance frequency] validation data to assist in the identification of promising hit structures and to avoid wasted effort from trawling over already cultivated territory. This is particularly important in a resource-limited research environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis.

PubMed

Tsuchiya, Megumi; Karim, M Rezaul; Matsumoto, Taro; Ogawa, Hidesato; Taniguchi, Hiroaki

2017-01-24

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

LIQUID: an-open source software for identifying lipids in LC-MS/MS-based lipidomics data.

PubMed

Kyle, Jennifer E; Crowell, Kevin L; Casey, Cameron P; Fujimoto, Grant M; Kim, Sangtae; Dautel, Sydney E; Smith, Richard D; Payne, Samuel H; Metz, Thomas O

2017-06-01

We introduce an open-source software, LIQUID, for semi-automated processing and visualization of LC-MS/MS-based lipidomics data. LIQUID provides users with the capability to process high throughput data and contains a customizable target library and scoring model per project needs. The graphical user interface provides visualization of multiple lines of spectral evidence for each lipid identification, allowing rapid examination of data for making confident identifications of lipid molecular species. LIQUID was compared to other freely available software commonly used to identify lipids and other small molecules (e.g. CFM-ID, MetFrag, GNPS, LipidBlast and MS-DIAL), and was found to have a faster processing time to arrive at a higher number of validated lipid identifications. LIQUID is available at http://github.com/PNNL-Comp-Mass-Spec/LIQUID . jennifer.kyle@pnnl.gov or thomas.metz@pnnl.gov. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae)

PubMed Central

Li, Dongmei; Fan, Qing-Hai; Waite, David W.; Gunawardana, Disna; George, Sherly; Kumarasinghe, Lalith

2015-01-01

Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand’s borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide. PMID:26147599

Pharmacophore modeling using Site-Identification by Ligand Competitive Saturation (SILCS) with multiple probe molecules

PubMed Central

Yu, Wenbo; Lakkaraju, Sirish Kaushik; Raman, E. Prabhu; Fang, Lei; MacKerell, Alexander D.

2015-01-01

Receptor-based pharmacophore modeling is an efficient computer-aided drug design technique that uses the structure of the target protein to identify novel leads. However, most methods consider protein flexibility and desolvation effects in a very approximate way, which may limit their use in practice. The Site-Identification by Ligand Competitive Saturation (SILCS) assisted pharmacophore modeling protocol (SILCS-Pharm) was introduced recently to address these issues as SILCS naturally takes both protein flexibility and desolvation effects into account by using full MD simulations to determine 3D maps of the functional group-affinity patterns on a target receptor. In the present work, the SILCS-Pharm protocol is extended to use a wider range of probe molecules including benzene, propane, methanol, formamide, acetaldehyde, methylammonium, acetate and water. This approach removes the previous ambiguity brought by using water as both the hydrogen-bond donor and acceptor probe molecule. The new SILCS-Pharm protocol is shown to yield improved screening results as compared to the previous approach based on three target proteins. Further validation of the new protocol using five additional protein targets showed improved screening compared to those using common docking methods, further indicating improvements brought by the explicit inclusion of additional feature types associated with the wider collection of probe molecules in the SILCS simulations. The advantage of using complementary features and volume constraints, based on exclusion maps of the protein defined from the SILCS simulations, is presented. In addition, re-ranking using SILCS-based ligand grid free energies is shown to enhance the diversity of identified ligands for the majority of targets. These results suggest that the SILCS-Pharm protocol will be of utility in rational drug design. PMID:25622696

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics

PubMed Central

Radonić, Aleksandar; Kocak Tufan, Zeliha; Domingo, Cristina

2017-01-01

Background We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. Methodology An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. Principal findings The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1–10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. Conclusions Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring. PMID:29155823

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

PubMed

Brinkmann, Annika; Ergünay, Koray; Radonić, Aleksandar; Kocak Tufan, Zeliha; Domingo, Cristina; Nitsche, Andreas

2017-11-01

We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

A new PCR-CGE (size and color) method for simultaneous detection of genetically modified maize events.

PubMed

Nadal, Anna; Coll, Anna; La Paz, Jose-Luis; Esteve, Teresa; Pla, Maria

2006-10-01

We present a novel multiplex PCR assay for simultaneous detection of multiple transgenic events in maize. Initially, five PCR primers pairs specific to events Bt11, GA21, MON810, and NK603, and Zea mays L. (alcohol dehydrogenase) were included. The event specificity was based on amplification of transgene/plant genome flanking regions, i.e., the same targets as for validated real-time PCR assays. These short and similarly sized amplicons were selected to achieve high and similar amplification efficiency for all targets; however, its unambiguous identification was a technical challenge. We achieved a clear distinction by a novel CGE approach that combined the identification by size and color (CGE-SC). In one single step, all five targets were amplified and specifically labeled with three different fluorescent dyes. The assay was specific and displayed an LOD of 0.1% of each genetically modified organism (GMO). Therefore, it was adequate to fulfill legal thresholds established, e.g., in the European Union. Our CGE-SC based strategy in combination with an adequate labeling design has the potential to simultaneously detect higher numbers of targets. As an example, we present the detection of up to eight targets in a single run. Multiplex PCR-CGE-SC only requires a conventional sequencer device and enables automation and high throughput. In addition, it proved to be transferable to a different laboratory. The number of authorized GMO events is rapidly growing; and the acreage of genetically modified (GM) varieties cultivated and commercialized worldwide is rapidly increasing. In this context, our multiplex PCR-CGE-SC can be suitable for screening GM contents in food.

Use of existing patient-reported outcome (PRO) instruments and their modification: the ISPOR Good Research Practices for Evaluating and Documenting Content Validity for the Use of Existing Instruments and Their Modification PRO Task Force Report.

PubMed

Rothman, Margaret; Burke, Laurie; Erickson, Pennifer; Leidy, Nancy Kline; Patrick, Donald L; Petrie, Charles D

2009-01-01

Patient-reported outcome (PRO) instruments are used to evaluate the effect of medical products on how patients feel or function. This article presents the results of an ISPOR task force convened to address good clinical research practices for the use of existing or modified PRO instruments to support medical product labeling claims. The focus of the article is on content validity, with specific reference to existing or modified PRO instruments, because of the importance of content validity in selecting or modifying an existing PRO instrument and the lack of consensus in the research community regarding best practices for establishing and documenting this measurement property. Topics addressed in the article include: definition and general description of content validity; PRO concept identification as the important first step in establishing content validity; instrument identification and the initial review process; key issues in qualitative methodology; and potential threats to content validity, with three case examples used to illustrate types of threats and how they might be resolved. A table of steps used to identify and evaluate an existing PRO instrument is provided, and figures are used to illustrate the meaning of content validity in relationship to instrument development and evaluation. RESULTS & RECOMMENDATIONS: Four important threats to content validity are identified: unclear conceptual match between the PRO instrument and the intended claim, lack of direct patient input into PRO item content from the target population in which the claim is desired, no evidence that the most relevant and important item content is contained in the instrument, and lack of documentation to support modifications to the PRO instrument. In some cases, careful review of the threats to content validity in a specific application may be reduced through additional well documented qualitative studies that specifically address the issue of concern. Published evidence of the content validity of a PRO instrument for an intended application is often limited. Such evidence is, however, important to evaluating the adequacy of a PRO instrument for the intended application. This article provides an overview of key issues involved in assessing and documenting content validity as it relates to using existing instruments in the drug approval process.

Group-level self-definition and self-investment: a hierarchical (multicomponent) model of in-group identification.

PubMed

Leach, Colin Wayne; van Zomeren, Martijn; Zebel, Sven; Vliek, Michael L W; Pennekamp, Sjoerd F; Doosje, Bertjan; Ouwerkerk, Jaap W; Spears, Russell

2008-07-01

Recent research shows individuals' identification with in-groups to be psychologically important and socially consequential. However, there is little agreement about how identification should be conceptualized or measured. On the basis of previous work, the authors identified 5 specific components of in-group identification and offered a hierarchical 2-dimensional model within which these components are organized. Studies 1 and 2 used confirmatory factor analysis to validate the proposed model of self-definition (individual self-stereotyping, in-group homogeneity) and self-investment (solidarity, satisfaction, and centrality) dimensions, across 3 different group identities. Studies 3 and 4 demonstrated the construct validity of the 5 components by examining their (concurrent) correlations with established measures of in-group identification. Studies 5-7 demonstrated the predictive and discriminant validity of the 5 components by examining their (prospective) prediction of individuals' orientation to, and emotions about, real intergroup relations. Together, these studies illustrate the conceptual and empirical value of a hierarchical multicomponent model of in-group identification.

DNA barcoding and real-time PCR detection of Bactrocera xanthodes (Tephritidae: Diptera) complex.

PubMed

Li, D; Waite, D W; Gunawardana, D N; McCarthy, B; Anderson, D; Flynn, A; George, S

2018-05-06

Immature fruit fly stages of the family Tephritidae are commonly intercepted on breadfruit from Pacific countries at the New Zealand border but are unable to be identified to the species level using morphological characters. Subsequent molecular identification showed that they belong to Bactrocera xanthodes, which is part of a species complex that includes Bactrocera paraxanthodes, Bactrocera neoxanthodes and an undescribed species. To establish a more reliable molecular identification system for B. xanthodes, a reference database of DNA barcode sequences for the 5'-fragment of COI gene region was constructed for B. xanthodes from Fiji, Samoa and Tonga. To better understand the species complex, B. neoxanthodes from Vanuatu and B. paraxanthodes from New Caledonia were also barcoded. Using the results of this analysis, real-time TaqMan polymerase chain reaction (PCR) assays for the detection of B. xanthodes complex and for the three individual species of the complex were developed and validated. The assay showed high specificity for the target species, with no cross-reaction observed for closely related organisms. Each of the real-time PCR assays is sensitive, detecting the target sequences at concentrations as low as ten copies µl-1 and can be used as either singleplex or multiplex formats. This real-time PCR assay for B. xanthodes has been successfully applied at the borders in New Zealand, leading to the rapid identification of intercepted Tephritidae eggs and larvae. The developed assays will be useful biosecurity tools for rapid detection of species in the B. xanthodes complex worldwide.

Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subculture.

PubMed

Verroken, A; Defourny, L; Lechgar, L; Magnette, A; Delmée, M; Glupczynski, Y

2015-02-01

Speeding up the turn-around time of positive blood culture identifications is essential in order to optimize the treatment of septic patients. Several sample preparation techniques have been developed allowing direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of positive blood cultures. Yet, the hands-on time restrains their routine workflow. In this study, we evaluated an approach whereby MALDI-TOF MS identification without any additional steps was carried out on short subcultured colonies from positive blood bottles with the objective of allowing results reporting on the day of positivity detection. Over a 7-month period in 2012, positive blood cultures detected by 9 am with an automated system were inoculated onto a Columbia blood agar and processed after a 5-h incubation on a MALDI-TOF MicroFlex platform (Bruker Daltonik GmbH). Single-spotted colonies were covered with 1 μl formic acid and 1 μl matrix solution. The results were compared to the validated identification techniques. A total of 925 positive blood culture bottles (representing 470 bacteremic episodes) were included. Concordant identification was obtained in 727 (81.1 %) of the 896 monomicrobial blood cultures, with failure being mostly observed with anaerobes and yeasts. In 17 episodes of polymicrobic bacteremia, the identification of one of the two isolates was achieved in 24/29 (82.7 %) positive cultures. Routine implementation of MALDI-TOF MS identification on young positive blood subcultures provides correct results to the clinician in more than 80 % of the bacteremic episodes and allows access to identification results on the day of blood culture positivity detection, potentially accelerating the implementation of targeted clinical treatments.

Open-source chemogenomic data-driven algorithms for predicting drug-target interactions.

PubMed

Hao, Ming; Bryant, Stephen H; Wang, Yanli

2018-02-06

While novel technologies such as high-throughput screening have advanced together with significant investment by pharmaceutical companies during the past decades, the success rate for drug development has not yet been improved prompting researchers looking for new strategies of drug discovery. Drug repositioning is a potential approach to solve this dilemma. However, experimental identification and validation of potential drug targets encoded by the human genome is both costly and time-consuming. Therefore, effective computational approaches have been proposed to facilitate drug repositioning, which have proved to be successful in drug discovery. Doubtlessly, the availability of open-accessible data from basic chemical biology research and the success of human genome sequencing are crucial to develop effective in silico drug repositioning methods allowing the identification of potential targets for existing drugs. In this work, we review several chemogenomic data-driven computational algorithms with source codes publicly accessible for predicting drug-target interactions (DTIs). We organize these algorithms by model properties and model evolutionary relationships. We re-implemented five representative algorithms in R programming language, and compared these algorithms by means of mean percentile ranking, a new recall-based evaluation metric in the DTI prediction research field. We anticipate that this review will be objective and helpful to researchers who would like to further improve existing algorithms or need to choose appropriate algorithms to infer potential DTIs in the projects. The source codes for DTI predictions are available at: https://github.com/minghao2016/chemogenomicAlg4DTIpred. Published by Oxford University Press 2018. This work is written by US Government employees and is in the public domain in the US.

Genome-wide identification of Hami melon miRNAs with putative roles during fruit development

PubMed Central

Wang, Guangzhi; Ma, Xinli; Li, Meihua; Wu, Haibo; Fu, Qiushi; Zhang, Yi; Yi, Hongping

2017-01-01

MicroRNAs represent a family of small endogenous, non-coding RNAs that play critical regulatory roles in plant growth, development, and environmental stress responses. Hami melon is famous for its attractive flavor and excellent nutritional value, however, the mechanisms underlying the fruit development and ripening remains largely unknown. Here, we performed small RNA sequencing to investigate the roles of miRNAs during Hami melon fruit development. Two batches of flesh samples were collected at four fruit development stages. Small RNA sequencing yielded a total of 54,553,424 raw reads from eight libraries. 113 conserved miRNAs belonging to 30 miRNA families and nine novel miRNAs comprising nine miRNA families were identified. The expression of 42 conserved miRNAs and three Hami melon-specific miRNAs significantly changed during fruit development. Furthermore, 484 and 124 melon genes were predicted as putative targets of 29 conserved and nine Hami melon-specific miRNA families, respectively. GO enrichment analysis were performed on target genes, “transcription, DNA-dependent”, “rRNA processing”, “oxidation reduction”, “signal transduction”, “regulation of transcription, DNA-dependent”, and “metabolic process” were the over-represented biological process terms. Cleavage sites of six target genes were validated using 5’ RACE. Our results present a comprehensive set of identification and characterization of Hami melon fruit miRNAs and their potential targets, which provide valuable basis towards understanding the regulatory mechanisms in programmed process of normal Hami fruit development and ripening. Specific miRNAs could be selected for further research and applications in breeding practices. PMID:28742088

Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

PubMed

Byrum, Stephanie D; Larson, Signe K; Avaritt, Nathan L; Moreland, Linley E; Mackintosh, Samuel G; Cheung, Wang L; Tackett, Alan J

2013-03-01

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

Micro-Doppler Signal Time-Frequency Algorithm Based on STFRFT.

PubMed

Pang, Cunsuo; Han, Yan; Hou, Huiling; Liu, Shengheng; Zhang, Nan

2016-09-24

This paper proposes a time-frequency algorithm based on short-time fractional order Fourier transformation (STFRFT) for identification of a complicated movement targets. This algorithm, consisting of a STFRFT order-changing and quick selection method, is effective in reducing the computation load. A multi-order STFRFT time-frequency algorithm is also developed that makes use of the time-frequency feature of each micro-Doppler component signal. This algorithm improves the estimation accuracy of time-frequency curve fitting through multi-order matching. Finally, experiment data were used to demonstrate STFRFT's performance in micro-Doppler time-frequency analysis. The results validated the higher estimate accuracy of the proposed algorithm. It may be applied to an LFM (Linear frequency modulated) pulse radar, SAR (Synthetic aperture radar), or ISAR (Inverse synthetic aperture radar), for improving the probability of target recognition.

Mapping the pathways of resistance to targeted therapies

PubMed Central

Wood, Kris C.

2015-01-01

Resistance substantially limits the depth and duration of clinical responses to targeted anticancer therapies. Through the use of complementary experimental approaches, investigators have revealed that cancer cells can achieve resistance through adaptation or selection driven by specific genetic, epigenetic, or microenvironmental alterations. Ultimately, these diverse alterations often lead to the activation of signaling pathways that, when co-opted, enable cancer cells to survive drug treatments. Recently developed methods enable the direct and scalable identification of the signaling pathways capable of driving resistance in specific contexts. Using these methods, novel pathways of resistance to clinically approved drugs have been identified and validated. By combining systematic resistance pathway mapping methods with studies revealing biomarkers of specific resistance pathways and pharmacological approaches to block these pathways, it may be possible to rationally construct drug combinations that yield more penetrant and lasting responses in patients. PMID:26392071

Therapeutic Targeting of Eosinophil Adhesion and Accumulation in Allergic Conjunctivitis

PubMed Central

Baiula, Monica; Bedini, Andrea; Carbonari, Gioia; Dattoli, Samantha Deianira; Spampinato, Santi

2012-01-01

Considerable evidence indicates that eosinophils are important effectors of ocular allergy. Increased worldwide prevalence of allergic eye pathologies has stimulated the identification of novel drug targets, including eosinophils and adhesion molecules. Accumulation of eosinophils in the eye is a key event in the onset and maintenance of allergic inflammation and is mediated by different adhesion molecules. Antihistamines with multiple mechanisms of action can be effective during the early and late phases of allergic conjunctivitis by blocking the interaction between β1 integrins and vascular cell adhesion molecule (VCAM)-1. Small molecule antagonists that target key elements in the process of eosinophil recruitment have been identified and reinforce the validity of α4β1 integrin as a therapeutic target. Glucocorticoids are among the most effective drugs for ocular allergy, but their use is limited by adverse effects. Novel dissociated glucocorticoids can prevent eosinophil accumulation and induce apoptosis of eosinophils, making them promising candidates for ophthalmic drugs. This article reviews recent understanding of the role of adhesion molecules in eosinophil recruitment in the inflamed conjunctiva along with effective treatments for allergic conjunctivitis. PMID:23271999

Does methodology matter in eyewitness identification research? The effect of live versus video exposure on eyewitness identification accuracy.

PubMed

Pozzulo, Joanna D; Crescini, Charmagne; Panton, Tasha

2008-01-01

The present study examined the effect of mode of target exposure (live versus video) on eyewitness identification accuracy. Adult participants (N=104) were exposed to a staged crime that they witnessed either live or on videotape. Participants were then asked to rate their stress and arousal levels prior to being presented with either a target-present or -absent simultaneous lineup. Across target-present and -absent lineups, mode of target exposure did not have a significant effect on identification accuracy. However, mode of target exposure was found to have a significant effect on stress and arousal levels. Participants who witnessed the crime live had higher levels of stress and arousal than those who were exposed to the videotaped crime. A higher level of arousal was significantly related to poorer identification accuracy for those in the video condition. For participants in the live condition however, stress and arousal had no effect on eyewitness identification accuracy. Implications of these findings in regards to the generalizability of laboratory-based research on eyewitness testimony to real-life crime are discussed.

Identification and characterization of microRNAs and their targets in high-altitude stress-adaptive plant maca (Lepidium meyenii Walp).

PubMed

Paul, Sujay

2017-06-01

MicroRNAs (miRNAs) are endogenous, short (~21-nucleotide), non-coding RNA molecules that play pivotal roles in plant growth, development, and stress response signaling. In this study using recently published draft genome sequence of a high-altitude plant maca (Lepidium meyenii Walp) and applying genome-wide computational-based approaches, a total of 62 potentially conserved miRNAs belonging to 28 families were identified and four (lme-miR160a, lme-miR164c, lme-miR 166a, and lme-miR 319a) of them further validated by RT-PCR. Deploying psRNATarget tool a total of 99 potential miRNA target transcripts were also identified in maca. Targets include a number of transcription factors like Squamosa promoter-binding, NAC, MYB, auxin response factor, APETALA, WRKY, and F-box protein. To the best of my knowledge, this is the first genome-based miRNA profiling of a high-altitude plant.

Searching for new pharmacological targets for the treatment of Alzheimer's disease in Down syndrome.

PubMed

Caraci, Filippo; Iulita, M Florencia; Pentz, Rowan; Flores Aguilar, Lisi; Orciani, Chiara; Barone, Concetta; Romano, Corrado; Drago, Filippo; Cuello, A Claudio

2017-12-15

Individuals with Down syndrome are at increased risk of developing Alzheimer's disease due to increase gene dosage resulting from chromosome 21 triplication. Although virtually all adults with Down syndrome will exhibit the major neuropathological hallmarks that define Alzheimer's disease, not all of them will develop the clinical symptoms associated with this disorder (i.e. dementia). Therefore, a good understanding of the pathophysiology of Alzheimer's disease in Down syndrome will be crucial for the identification of novel pharmacological targets to develop disease-modifying therapies for the benefit of Down syndrome individuals and for Alzheimer's sufferers alike. The study of biomarkers will also be essential for the development of better screening tools to identify dementia at its incipient stages. This review discusses the best-validated pharmacological targets for the treatment of cognitive impairment and Alzheimer's disease in Down syndrome. We further examine the relevance of newly discovered biological markers for earlier dementia diagnosis in this population. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Inhibition of CDC25B Phosphatase Through Disruption of Protein–Protein Interaction

DOE PAGES

Lund, George; Dudkin, Sergii; Borkin, Dmitry; ...

2014-11-25

CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here in this paper, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation ofmore » CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.« less

Bioinformatics in protein kinases regulatory network and drug discovery.

PubMed

Chen, Qingfeng; Luo, Haiqiong; Zhang, Chengqi; Chen, Yi-Ping Phoebe

2015-04-01

Protein kinases have been implicated in a number of diseases, where kinases participate many aspects that control cell growth, movement and death. The deregulated kinase activities and the knowledge of these disorders are of great clinical interest of drug discovery. The most critical issue is the development of safe and efficient disease diagnosis and treatment for less cost and in less time. It is critical to develop innovative approaches that aim at the root cause of a disease, not just its symptoms. Bioinformatics including genetic, genomic, mathematics and computational technologies, has become the most promising option for effective drug discovery, and has showed its potential in early stage of drug-target identification and target validation. It is essential that these aspects are understood and integrated into new methods used in drug discovery for diseases arisen from deregulated kinase activity. This article reviews bioinformatics techniques for protein kinase data management and analysis, kinase pathways and drug targets and describes their potential application in pharma ceutical industry. Copyright © 2015 Elsevier Inc. All rights reserved.

Targeting mammalian organelles with internalizing phage (iPhage) libraries

PubMed Central

Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

2015-01-01

Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

PubMed

Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

2015-12-15

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

PubMed Central

Yang, Jae-Seong; Kwon, Oh Sung; Kim, Sanguk; Jang, Sung Key

2013-01-01

Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets. PMID:23593195

Using PeptideAtlas, SRMAtlas and PASSEL – Comprehensive Resources for discovery and targeted proteomics

PubMed Central

Kusebauch, Ulrike; Deutsch, Eric W.; Campbell, David S.; Sun, Zhi; Farrah, Terry; Moritz, Robert L.

2014-01-01

PeptideAtlas, SRMAtlas and PASSEL are web-accessible resources to support discovery and targeted proteomics research. PeptideAtlas is a multi-species compendium of shotgun proteomic data provided by the scientific community, SRMAtlas is a resource of high-quality, complete proteome SRM assays generated in a consistent manner for the targeted identification and quantification of proteins, and PASSEL is a repository that compiles and represents selected reaction monitoring data, all in an easy to use interface. The databases are generated from native mass spectrometry data files that are analyzed in a standardized manner including statistical validation of the results. Each resource offers search functionalities and can be queried by user defined constraints; the query results are provided in tables or are graphically displayed. PeptideAtlas, SRMAtlas and PASSEL are publicly available freely via the website http://www.peptideatlas.org. In this protocol, we describe the use of these resources, we highlight how to submit, search, collate and download data. PMID:24939129

Pharmacophore modeling for identification of anti-IGF-1R drugs and in-vitro validation of fulvestrant as a potential inhibitor

PubMed Central

Hanif, Rumeza; Jabeen, Ishrat; Mansoor, Qaisar; Ismail, Muhammad

2018-01-01

Insulin-like growth factor 1 receptor (IGF-1R) is an important therapeutic target for breast cancer treatment. The alteration in the IGF-1R associated signaling network due to various genetic and environmental factors leads the system towards metastasis. The pharmacophore modeling and logical approaches have been applied to analyze the behaviour of complex regulatory network involved in breast cancer. A total of 23 inhibitors were selected to generate ligand based pharmacophore using the tool, Molecular Operating Environment (MOE). The best model consisted of three pharmacophore features: aromatic hydrophobic (HyD/Aro), hydrophobic (HyD) and hydrogen bond acceptor (HBA). This model was validated against World drug bank (WDB) database screening to identify 189 hits with the required pharmacophore features and was further screened by using Lipinski positive compounds. Finally, the most effective drug, fulvestrant, was selected. Fulvestrant is a selective estrogen receptor down regulator (SERD). This inhibitor was further studied by using both in-silico and in-vitro approaches that showed the targeted effect of fulvestrant in ER+ MCF-7 cells. Results suggested that fulvestrant has selective cytotoxic effect and a dose dependent response on IRS-1, IGF-1R, PDZK1 and ER-α in MCF-7 cells. PDZK1 can be an important inhibitory target using fulvestrant because it directly regulates IGF-1R. PMID:29787591

Pharmacophore modeling for identification of anti-IGF-1R drugs and in-vitro validation of fulvestrant as a potential inhibitor.

PubMed

Khalid, Samra; Hanif, Rumeza; Jabeen, Ishrat; Mansoor, Qaisar; Ismail, Muhammad

2018-01-01

Insulin-like growth factor 1 receptor (IGF-1R) is an important therapeutic target for breast cancer treatment. The alteration in the IGF-1R associated signaling network due to various genetic and environmental factors leads the system towards metastasis. The pharmacophore modeling and logical approaches have been applied to analyze the behaviour of complex regulatory network involved in breast cancer. A total of 23 inhibitors were selected to generate ligand based pharmacophore using the tool, Molecular Operating Environment (MOE). The best model consisted of three pharmacophore features: aromatic hydrophobic (HyD/Aro), hydrophobic (HyD) and hydrogen bond acceptor (HBA). This model was validated against World drug bank (WDB) database screening to identify 189 hits with the required pharmacophore features and was further screened by using Lipinski positive compounds. Finally, the most effective drug, fulvestrant, was selected. Fulvestrant is a selective estrogen receptor down regulator (SERD). This inhibitor was further studied by using both in-silico and in-vitro approaches that showed the targeted effect of fulvestrant in ER+ MCF-7 cells. Results suggested that fulvestrant has selective cytotoxic effect and a dose dependent response on IRS-1, IGF-1R, PDZK1 and ER-α in MCF-7 cells. PDZK1 can be an important inhibitory target using fulvestrant because it directly regulates IGF-1R.

Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

PubMed Central

Wu, Xiaoqing; Lan, Lan; Wilson, David Michael; Marquez, Rebecca T.; Tsao, Wei-chung; Gao, Philip; Roy, Anuradha; Turner, Benjamin Andrew; McDonald, Peter; Tunge, Jon A; Rogers, Steven A; Dixon, Dan A.; Aubé, Jeffrey; Xu, Liang

2015-01-01

HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3′-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR–ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ~6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR–ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers. PMID:25750985

Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition.

PubMed

Gallipoli, Paolo; Giotopoulos, George; Tzelepis, Konstantinos; Costa, Ana S H; Vohra, Shabana; Medina-Perez, Paula; Basheer, Faisal; Marando, Ludovica; Di Lisio, Lorena; Dias, Joao M L; Yun, Haiyang; Sasca, Daniel; Horton, Sarah J; Vassiliou, George; Frezza, Christian; Huntly, Brian J P

2018-04-12

FLT3 internal tandem duplication (FLT3 ITD ) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3 ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3 ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3 ITD and other TK activating mutation-driven leukemias. © 2018 by The American Society of Hematology.

Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setaria italica).

PubMed

Han, Jun; Xie, Hao; Sun, Qingpeng; Wang, Jun; Lu, Min; Wang, Weixiang; Guo, Erhu; Pan, Jinbao

2014-08-10

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5'-RLM-RACE) method. Copyright © 2014 Elsevier B.V. All rights reserved.

Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

PubMed Central

Pierce, M L; Ruffner, D E

1998-01-01

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

GEPSI: A Gene Expression Profile Similarity-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula.

PubMed

Zhang, Baixia; He, Shuaibing; Lv, Chenyang; Zhang, Yanling; Wang, Yun

2018-01-01

The identification of bioactive components in traditional Chinese medicine (TCM) is an important part of the TCM material foundation research. Recently, molecular docking technology has been extensively used for the identification of TCM bioactive components. However, target proteins that are used in molecular docking may not be the actual TCM target. For this reason, the bioactive components would likely be omitted or incorrect. To address this problem, this study proposed the GEPSI method that identified the target proteins of TCM based on the similarity of gene expression profiles. The similarity of the gene expression profiles affected by TCM and small molecular drugs was calculated. The pharmacological action of TCM may be similar to that of small molecule drugs that have a high similarity score. Indeed, the target proteins of the small molecule drugs could be considered TCM targets. Thus, we identified the bioactive components of a TCM by molecular docking and verified the reliability of this method by a literature investigation. Using the target proteins that TCM actually affected as targets, the identification of the bioactive components was more accurate. This study provides a fast and effective method for the identification of TCM bioactive components.

GEPSI: A Gene Expression Profile Similarity-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula

PubMed Central

Zhang, Baixia; He, Shuaibing; Lv, Chenyang; Zhang, Yanling

2018-01-01

The identification of bioactive components in traditional Chinese medicine (TCM) is an important part of the TCM material foundation research. Recently, molecular docking technology has been extensively used for the identification of TCM bioactive components. However, target proteins that are used in molecular docking may not be the actual TCM target. For this reason, the bioactive components would likely be omitted or incorrect. To address this problem, this study proposed the GEPSI method that identified the target proteins of TCM based on the similarity of gene expression profiles. The similarity of the gene expression profiles affected by TCM and small molecular drugs was calculated. The pharmacological action of TCM may be similar to that of small molecule drugs that have a high similarity score. Indeed, the target proteins of the small molecule drugs could be considered TCM targets. Thus, we identified the bioactive components of a TCM by molecular docking and verified the reliability of this method by a literature investigation. Using the target proteins that TCM actually affected as targets, the identification of the bioactive components was more accurate. This study provides a fast and effective method for the identification of TCM bioactive components. PMID:29692857

Panorama: A Targeted Proteomics Knowledge Base

PubMed Central

2015-01-01

Panorama is a web application for storing, sharing, analyzing, and reusing targeted assays created and refined with Skyline,1 an increasingly popular Windows client software tool for targeted proteomics experiments. Panorama allows laboratories to store and organize curated results contained in Skyline documents with fine-grained permissions, which facilitates distributed collaboration and secure sharing of published and unpublished data via a web-browser interface. It is fully integrated with the Skyline workflow and supports publishing a document directly to a Panorama server from the Skyline user interface. Panorama captures the complete Skyline document information content in a relational database schema. Curated results published to Panorama can be aggregated and exported as chromatogram libraries. These libraries can be used in Skyline to pick optimal targets in new experiments and to validate peak identification of target peptides. Panorama is open-source and freely available. It is distributed as part of LabKey Server,2 an open source biomedical research data management system. Laboratories and organizations can set up Panorama locally by downloading and installing the software on their own servers. They can also request freely hosted projects on https://panoramaweb.org, a Panorama server maintained by the Department of Genome Sciences at the University of Washington. PMID:25102069

Targeting Host Factors to Treat West Nile and Dengue Viral Infections

PubMed Central

Krishnan, Manoj N.; Garcia-Blanco, Mariano A.

2014-01-01

West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus Flavivirus. At the current time, there are no approved prophylactics (e.g., vaccines) or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans. PMID:24517970

Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiangbo; Zhang, Lu; Ding, Nianhua

2015-05-29

Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possessmore » efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy. - Highlights: • Identified DNMT1-targeted DNAzymes by multiplex selection system. • Biochemically characterized a lead DNAzyme with high kinetic efficiency. • Validated DNMT1-targeted DNAzyme in its enzymatic and cellular activities.« less

Targeting host factors to treat West Nile and dengue viral infections.

PubMed

Krishnan, Manoj N; Garcia-Blanco, Mariano A

2014-02-10

West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus Flavivirus. At the current time, there are no approved prophylactics (e.g., vaccines) or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans.

Abnormal expression of leiomyoma cytoskeletal proteins involved in cell migration.

PubMed

Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Athanasakis, Emmanouil; Aloisio, Michelangelo; Monasta, Lorenzo; Ricci, Giuseppe

2016-05-01

Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two‑dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches.

Molecular markers associated with development and progression of potentially premalignant oral epithelial lesions: Current knowledge and future implications.

PubMed

Nikitakis, Nikolaos G; Pentenero, Monica; Georgaki, Maria; Poh, Catherine F; Peterson, Douglas E; Edwards, Paul; Lingen, Mark; Sauk, John J

2018-06-01

Identification and management of potentially premalignant oral epithelial lesions (PPOELs) at highest risk of malignant transformation holds great promise for successful secondary prevention of oral squamous cell carcinoma, potentially reducing oral cancer morbidity and mortality. However, to date, neither clinical nor histopathologic validated risk predictors that can reliably predict which PPOELs will definitively progress to malignancy have been identified. In addition, the management of PPOELs remains a major challenge. Arguably, progress in the prevention and treatment of oral premalignancy and cancer will require improved understanding of the underlying molecular mechanisms, facilitating the discovery of diagnostic, prognostic, and predictive markers, as well as the identification of novel targeted therapeutics. This review provides a synopsis of the molecular biomarkers that have been studied in PPOELs and have been correlated with the presence and grade of dysplasia and/or their propensity to undergo malignant transformation to oral squamous cell carcinoma. The emphasis is on highlighting new emerging research fields, particularly epigenetic events, including methylation and micro-RNA regulation. Several promising biomarkers are highlighted. Current limitations and challenges are discussed. Recommendations for future focused research areas, to validate and promote clinically useful applications, are offered. Copyright © 2018 Elsevier Inc. All rights reserved.

A refined Frequency Domain Decomposition tool for structural modal monitoring in earthquake engineering

NASA Astrophysics Data System (ADS)

Pioldi, Fabio; Rizzi, Egidio

2017-07-01

Output-only structural identification is developed by a refined Frequency Domain Decomposition ( rFDD) approach, towards assessing current modal properties of heavy-damped buildings (in terms of identification challenge), under strong ground motions. Structural responses from earthquake excitations are taken as input signals for the identification algorithm. A new dedicated computational procedure, based on coupled Chebyshev Type II bandpass filters, is outlined for the effective estimation of natural frequencies, mode shapes and modal damping ratios. The identification technique is also coupled with a Gabor Wavelet Transform, resulting in an effective and self-contained time-frequency analysis framework. Simulated response signals generated by shear-type frames (with variable structural features) are used as a necessary validation condition. In this context use is made of a complete set of seismic records taken from the FEMA P695 database, i.e. all 44 "Far-Field" (22 NS, 22 WE) earthquake signals. The modal estimates are statistically compared to their target values, proving the accuracy of the developed algorithm in providing prompt and accurate estimates of all current strong ground motion modal parameters. At this stage, such analysis tool may be employed for convenient application in the realm of Earthquake Engineering, towards potential Structural Health Monitoring and damage detection purposes.

Identification of five highly priced tuna species by quantitative real-time polymerase chain reaction.

PubMed

Liu, Shasha; Xu, Kunhua; Wu, Zhigang; Xie, Xiao; Feng, Junli

2016-09-01

Tunas are economically important fishery worldwide, and are often used for commercial processed production. For effective fishery management and protection of consumers' rights, it is important to develop a molecular method to identify species in canned tuna products rapidly and reliably. Here, we have developed a duplex quantitative real-time PCR (qPCR) for identification of five highly priced tuna species (Thunnus maccoyii, Thunnus obesus, Thunnus albacares, Thunnus alalunga and Katsuwonus pelamis) from processed as well as fresh fish. After amplification and sequencing of seven genetic markers commonly used for species identification, 16S rDNA and control region (CR) of mitochondrial DNA were selected as the reference gene markers for genus Thunnus and tuna species identification, respectively. Subsequently, a 73 bp fragment of 16S rDNA and 85-99 bp fragment of CR were simultaneously amplified from each target species by qPCR. The qPCR efficiency of each reaction was calculated according to the standard curves, and the method was validated by amplification DNA extracted from single or mixed tuna specimen. The developed duplex qPCR system was applied to authenticate species of 14 commercial tuna products successfully, which demonstrated it was really a useful and academic technique to identify highly priced tuna species.

INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE

PubMed Central

Grzegorzewicz, Anna E.; Pham, Ha; Gundi, Vijay A. K. B.; Scherman, Michael S.; North, Elton J.; Hess, Tamara; Jones, Victoria; Gruppo, Veronica; Born, Sarah E. M.; Korduláková, Jana; Chavadi, Sivagami Sundaram; Morisseau, Christophe; Lenaerts, Anne J.; Lee, Richard E.; McNeil, Michael R.; Jackson, Mary

2011-01-01

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane. PMID:22344175

Galileo Attitude Determination: Experiences with a Rotating Star Scanner

NASA Technical Reports Server (NTRS)

Merken, L.; Singh, G.

1991-01-01

The Galileo experience with a rotating star scanner is discussed in terms of problems encountered in flight, solutions implemented, and lessons learned. An overview of the Galileo project and the attitude and articulation control subsystem is given and the star scanner hardware and relevant software algorithms are detailed. The star scanner is the sole source of inertial attitude reference for this spacecraft. Problem symptoms observed in flight are discussed in terms of effects on spacecraft performance and safety. Sources of thse problems include contributions from flight software idiosyncrasies and inadequate validation of the ground procedures used to identify target stars for use by the autonomous on-board star identification algorithm. Problem fixes (some already implemented and some only proposed) are discussed. A general conclusion is drawn regarding the inherent difficulty of performing simulation tests to validate algorithms which are highly sensitive to external inputs of statistically 'rare' events.

Early Numeracy Assessment: The Development of the Preschool Numeracy Scales

PubMed Central

Purpura, David J.; Lonigan, Christopher J.

2015-01-01

Research Findings The focus of this study was to construct and validate twelve brief early numeracy assessment tasks that measure the skills and concepts identified as key to early mathematics development by the National Council of Teachers of Mathematics (2006) and the National Mathematics Advisory Panel (2008)—as well as critical developmental precursors to later mathematics skill by the Common Core State Standards (CCSS; 2010). Participants were 393 preschool children ages 3 to 5 years old. Measure development and validation occurred through three analytic phases designed to ensure that the measures were brief, reliable, and valid. These measures included: one-to-one counting, cardinality, counting subsets, subitizing, number comparison, set comparison, number order, numeral identification, set-to-numerals, story problems, number combinations, and verbal counting. Practice or Policy Teachers have extensive demands on their time, yet, they are tasked with ensuring that all students’ academic needs are met. To identify individual instructional needs and measure progress, they need to be able to efficiently assess children’s numeracy skills. The measures developed in this study are not only reliable and valid, but also easy to use and can be utilized for measuring the effects of targeted instruction on individual numeracy skills. PMID:25709375

Atlas-based identification of targets for functional radiosurgery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stancanello, Joseph; Romanelli, Pantaleo; Modugno, Nicola

2006-06-15

Functional disorders of the brain, such as Parkinson's disease, dystonia, epilepsy, and neuropathic pain, may exhibit poor response to medical therapy. In such cases, surgical intervention may become necessary. Modern surgical approaches to such disorders include radio-frequency lesioning and deep brain stimulation (DBS). The subthalamic nucleus (STN) is one of the most useful stereotactic targets available: STN DBS is known to induce substantial improvement in patients with end-stage Parkinson's disease. Other targets include the Globus Pallidus pars interna (GPi) for dystonia and Parkinson's disease, and the centromedian nucleus of the thalamus (CMN) for neuropathic pain. Radiosurgery is an attractive noninvasivemore » alternative to treat some functional brain disorders. The main technical limitation to radiosurgery is that the target can be selected only on the basis of magnetic resonance anatomy without electrophysiological confirmation. The aim of this work is to provide a method for the correct atlas-based identification of the target to be used in functional neurosurgery treatment planning. The coordinates of STN, CMN, and GPi were identified in the Talairach and Tournoux atlas and transformed to the corresponding regions of the Montreal Neurological Institute (MNI) electronic atlas. Binary masks describing the target nuclei were created. The MNI electronic atlas was deformed onto the patient magnetic resonance imaging-T1 scan by applying an affine transformation followed by a local nonrigid registration. The first transformation was based on normalized cross correlation and the second on optimization of a two-part objective function consisting of similarity criteria and weighted regularization. The obtained deformation field was then applied to the target masks. The minimum distance between the surface of an implanted electrode and the surface of the deformed mask was calculated. The validation of the method consisted of comparing the electrode-mask distance to the clinical outcome of the treatments in ten cases of bilateral DBS implants. Electrode placement may have an effect within a radius of stimulation equal to 2 mm, therefore the registration process is considered successful if error is less than 2 mm. The registrations of the MNI atlas onto the patient space succeeded in all cases. The comparison of the distance to the clinical outcome revealed good agreement: where the distance was high (at least in one implant), the clinical outcome was poor; where there was a close correlation between the structures, clinical outcome revealed an improvement of the pathological condition. In conclusion, the proposed method seems to provide a useful tool for the identification of the target nuclei for functional radiosurgery. Also, the method is applicable to other types of functional treatment.« less

Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein-Protein Interaction.

PubMed

Sail, Vibhavari; Rizzo, Alessandro A; Chatterjee, Nimrat; Dash, Radha C; Ozen, Zuleyha; Walker, Graham C; Korzhnev, Dmitry M; Hadden, M Kyle

2017-07-21

Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

Regulation of Isoflavone Biosynthesis by miRNAs in Two Contrasting Soybean Genotypes at Different Seed Developmental Stages

PubMed Central

Gupta, Om P.; Nigam, Deepti; Dahuja, Anil; Kumar, Sanjeev; Vinutha, T.; Sachdev, Archana; Praveen, Shelly

2017-01-01

Owing to the presence of nutritionally important, health-promoting bioactive compounds, especially isoflavones, soybean has acquired the status of a functional food. miRNAs are tiny riboregulator of gene expression by either decreasing and/or increasing the expression of their corresponding target genes. Despite several works on identification and functional characterization of plant miRNAs, the role of miRNAs in the regulation of isoflavones metabolism is still a virgin field. In the present study, we identified a total of 31 new miRNAs along with their 245 putative target genes from soybean seed-specific ESTs using computational approach. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates metabolism and genetic information processing. Out of that, a total of 5 miRNAs (Gma-miRNA12, Gma-miRNA24, Gma-miRNA26, Gma-miRNA28, and Gma-miRNA29) were predicted and validated for their probable role during isoflavone biosynthesis. We also validated their five target genes using RA-PCR, which is as good as 5'RLM-RACE. Temporal regulation [35 days after flowering, 45, 55, and 65 DAF] of miRNAs and their targets showed differential expression schema. Differential expression of Gma-miR26 and Gma-miRNA28 along with their corresponding target genes (Glyma.10G197900 and Glyma.09G127200) showed a direct relationship with the total isoflavone content. Therefore, understanding the miRNA-based genetic regulation of isoflavone pathway would assist in selection and manipulation to get high-performing soybean genotypes with better isoflavone yield. PMID:28450878

Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

PubMed

Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

2017-03-21

This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon carcinogenesis.

Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic corynebacteria.

PubMed

De Zoysa, Aruni; Efstratiou, Androulla; Mann, Ginder; Harrison, Timothy G; Fry, Norman K

2016-12-01

Toxigenic corynebacteria are uncommon in the UK; however, laboratory confirmation by the national reference laboratory can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 to ≥48 h from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase β-subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. qPCR results were obtained within 3 to 4 h after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91 to 100 % and 100 %, respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR assay were the inability to distinguish between C. ulcerans and C. pseudotuberculosis and that the presence of the toxin gene as demonstrated by qPCR may not always predict toxin expression. Thus, confirmation of expression of diphtheria toxin is always sought using the phenotypic Elek test. The new qPCR assay was formally introduced as the front-line test for putative toxigenic corynebacteria to inform public health action in England and Wales on 1 April 2014.

Validation of automatic landmark identification for atlas-based segmentation for radiation treatment planning of the head-and-neck region

NASA Astrophysics Data System (ADS)

Leavens, Claudia; Vik, Torbjørn; Schulz, Heinrich; Allaire, Stéphane; Kim, John; Dawson, Laura; O'Sullivan, Brian; Breen, Stephen; Jaffray, David; Pekar, Vladimir

2008-03-01

Manual contouring of target volumes and organs at risk in radiation therapy is extremely time-consuming, in particular for treating the head-and-neck area, where a single patient treatment plan can take several hours to contour. As radiation treatment delivery moves towards adaptive treatment, the need for more efficient segmentation techniques will increase. We are developing a method for automatic model-based segmentation of the head and neck. This process can be broken down into three main steps: i) automatic landmark identification in the image dataset of interest, ii) automatic landmark-based initialization of deformable surface models to the patient image dataset, and iii) adaptation of the deformable models to the patient-specific anatomical boundaries of interest. In this paper, we focus on the validation of the first step of this method, quantifying the results of our automatic landmark identification method. We use an image atlas formed by applying thin-plate spline (TPS) interpolation to ten atlas datasets, using 27 manually identified landmarks in each atlas/training dataset. The principal variation modes returned by principal component analysis (PCA) of the landmark positions were used by an automatic registration algorithm, which sought the corresponding landmarks in the clinical dataset of interest using a controlled random search algorithm. Applying a run time of 60 seconds to the random search, a root mean square (rms) distance to the ground-truth landmark position of 9.5 +/- 0.6 mm was calculated for the identified landmarks. Automatic segmentation of the brain, mandible and brain stem, using the detected landmarks, is demonstrated.

49 CFR Appendix D to Part 236 - Independent Review of Verification and Validation

Code of Federal Regulations, 2010 CFR

2010-10-01

... standards. (f) The reviewer shall analyze all Fault Tree Analyses (FTA), Failure Mode and Effects... for each product vulnerability cited by the reviewer; (4) Identification of any documentation or... not properly followed; (6) Identification of the software verification and validation procedures, as...

Novel targets of sulforaphane in primary cardiomyocytes identified by proteomic analysis.

PubMed

Angeloni, Cristina; Turroni, Silvia; Bianchi, Laura; Fabbri, Daniele; Motori, Elisa; Malaguti, Marco; Leoncini, Emanuela; Maraldi, Tullia; Bini, Luca; Brigidi, Patrizia; Hrelia, Silvana

2013-01-01

Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.

Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains▿†

PubMed Central

Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

2011-01-01

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC. PMID:21123525

Fast Open-World Person Re-Identification.

PubMed

Zhu, Xiatian; Wu, Botong; Huang, Dongcheng; Zheng, Wei-Shi

2018-05-01

Existing person re-identification (re-id) methods typically assume that: 1) any probe person is guaranteed to appear in the gallery target population during deployment (i.e., closed-world) and 2) the probe set contains only a limited number of people (i.e., small search scale). Both assumptions are artificial and breached in real-world applications, since the probe population in target people search can be extremely vast in practice due to the ambiguity of probe search space boundary. Therefore, it is unrealistic that any probe person is assumed as one target people, and a large-scale search in person images is inherently demanded. In this paper, we introduce a new person re-id search setting, called large scale open-world (LSOW) re-id, characterized by huge size probe images and open person population in search thus more close to practical deployments. Under LSOW, the under-studied problem of person re-id efficiency is essential in addition to that of commonly studied re-id accuracy. We, therefore, develop a novel fast person re-id method, called Cross-view Identity Correlation and vErification (X-ICE) hashing, for joint learning of cross-view identity representation binarisation and discrimination in a unified manner. Extensive comparative experiments on three large-scale benchmarks have been conducted to validate the superiority and advantages of the proposed X-ICE method over a wide range of the state-of-the-art hashing models, person re-id methods, and their combinations.

Validation of photo-identification as a mark-recapture method in the spotted eagle ray Aetobatus narinari.

PubMed

González-Ramos, M S; Santos-Moreno, A; Rosas-Alquicira, E F; Fuentes-Mascorro, G

2017-03-01

The spotted eagle ray Aetobatus narinari is characterized by pigmentation patterns that are retained for up to 3·5 years. These pigmentations can be used to identify individuals through photo-identification. Only one study has validated this technique, but no study has estimated the percentage of correct identification of the rays using this technique. In order to carry out demographic research, a reliable photographic identification technique is needed. To achieve this validation for A. narinari, a double-mark system was established over 11 months and photographs of the dorsal surface of 191 rays were taken. Three body parts with distinctive natural patterns were analysed (dorsal surface of the cephalic region, dorsal surface of the pectoral fins and dorsal surface of the pelvic fins) in order to determine the body part that could be used to give the highest percentage of correct identification. The dorsal surface of the pectoral fins of A. narinari provides the most accurate photo-identification to distinguish individuals (88·2%). © 2016 The Fisheries Society of the British Isles.

Sleep and eyewitness memory: Fewer false identifications after sleep when the target is absent from the lineup.

PubMed

Stepan, Michelle E; Dehnke, Taylor M; Fenn, Kimberly M

2017-01-01

Inaccurate eyewitness identifications are the leading cause of known false convictions in the United States. Moreover, improving eyewitness memory is difficult and often unsuccessful. Sleep consistently strengthens and protects memory from interference, particularly when a recall test is used. However, the effect of sleep on recognition memory is more equivocal. Eyewitness identification tests are often recognition based, thus leaving open the question of how sleep affects recognition performance in an eyewitness context. In the current study, we investigated the effect of sleep on eyewitness memory. Participants watched a video of a mock-crime and attempted to identify the perpetrator from a simultaneous lineup after a 12-hour retention interval that either spanned a waking day or night of sleep. In Experiment 1, we used a target-present lineup and, in Experiment 2, we used a target-absent lineup in order to investigate correct and false identifications, respectively. Sleep reduced false identifications in the target-absent lineup (Experiment 2) but had no effect on correct identifications in the target-present lineup (Experiment 1). These results are discussed with respect to memory strength and decision making strategies.

Sleep and eyewitness memory: Fewer false identifications after sleep when the target is absent from the lineup

PubMed Central

Dehnke, Taylor M.; Fenn, Kimberly M.

2017-01-01

Inaccurate eyewitness identifications are the leading cause of known false convictions in the United States. Moreover, improving eyewitness memory is difficult and often unsuccessful. Sleep consistently strengthens and protects memory from interference, particularly when a recall test is used. However, the effect of sleep on recognition memory is more equivocal. Eyewitness identification tests are often recognition based, thus leaving open the question of how sleep affects recognition performance in an eyewitness context. In the current study, we investigated the effect of sleep on eyewitness memory. Participants watched a video of a mock-crime and attempted to identify the perpetrator from a simultaneous lineup after a 12-hour retention interval that either spanned a waking day or night of sleep. In Experiment 1, we used a target-present lineup and, in Experiment 2, we used a target-absent lineup in order to investigate correct and false identifications, respectively. Sleep reduced false identifications in the target-absent lineup (Experiment 2) but had no effect on correct identifications in the target-present lineup (Experiment 1). These results are discussed with respect to memory strength and decision making strategies. PMID:28877169

Aeroservoelastic Model Validation and Test Data Analysis of the F/A-18 Active Aeroelastic Wing

NASA Technical Reports Server (NTRS)

Brenner, Martin J.; Prazenica, Richard J.

2003-01-01

Model validation and flight test data analysis require careful consideration of the effects of uncertainty, noise, and nonlinearity. Uncertainty prevails in the data analysis techniques and results in a composite model uncertainty from unmodeled dynamics, assumptions and mechanics of the estimation procedures, noise, and nonlinearity. A fundamental requirement for reliable and robust model development is an attempt to account for each of these sources of error, in particular, for model validation, robust stability prediction, and flight control system development. This paper is concerned with data processing procedures for uncertainty reduction in model validation for stability estimation and nonlinear identification. F/A-18 Active Aeroelastic Wing (AAW) aircraft data is used to demonstrate signal representation effects on uncertain model development, stability estimation, and nonlinear identification. Data is decomposed using adaptive orthonormal best-basis and wavelet-basis signal decompositions for signal denoising into linear and nonlinear identification algorithms. Nonlinear identification from a wavelet-based Volterra kernel procedure is used to extract nonlinear dynamics from aeroelastic responses, and to assist model development and uncertainty reduction for model validation and stability prediction by removing a class of nonlinearity from the uncertainty.

Complex network theory for the identification and assessment of candidate protein targets.

PubMed

McGarry, Ken; McDonald, Sharon

2018-06-01

In this work we use complex network theory to provide a statistical model of the connectivity patterns of human proteins and their interaction partners. Our intention is to identify important proteins that may be predisposed to be potential candidates as drug targets for therapeutic interventions. Target proteins usually have more interaction partners than non-target proteins, but there are no hard-and-fast rules for defining the actual number of interactions. We devise a statistical measure for identifying hub proteins, we score our target proteins with gene ontology annotations. The important druggable protein targets are likely to have similar biological functions that can be assessed for their potential therapeutic value. Our system provides a statistical analysis of the local and distant neighborhood protein interactions of the potential targets using complex network measures. This approach builds a more accurate model of drug-to-target activity and therefore the likely impact on treating diseases. We integrate high quality protein interaction data from the HINT database and disease associated proteins from the DrugTarget database. Other sources include biological knowledge from Gene Ontology and drug information from DrugBank. The problem is a very challenging one since the data is highly imbalanced between target proteins and the more numerous nontargets. We use undersampling on the training data and build Random Forest classifier models which are used to identify previously unclassified target proteins. We validate and corroborate these findings from the available literature. Copyright © 2018 Elsevier Ltd. All rights reserved.

Identification of inhibitors for putative malaria drug targets amongst novel antimalarial compounds

PubMed Central

Crowther, Gregory J.; Napuli, Alberto J.; Gilligan, James H.; Gagaring, Kerstin; Borboa, Rachel; Francek, Carolyn; Chen, Zhong; Dagostino, Eleanor F.; Stockmyer, Justin B.; Wang, Yu; Rodenbough, Philip P.; Castaneda, Lisa J.; Leibly, David J.; Bhandari, Janhavi; Gelb, Michael H.; Brinker, Achim; Engels, Ingo; Taylor, Jennifer; Chatterjee, Arnab K.; Fantauzzi, Pascal; Glynne, Richard J.; Van Voorhis, Wesley C.; Kuhen, Kelli L.

2011-01-01

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for P. falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5,655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC50 values below 1.25 μM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5′-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology. PMID:20813141

Identification of inhibitors for putative malaria drug targets among novel antimalarial compounds.

PubMed

Crowther, Gregory J; Napuli, Alberto J; Gilligan, James H; Gagaring, Kerstin; Borboa, Rachel; Francek, Carolyn; Chen, Zhong; Dagostino, Eleanor F; Stockmyer, Justin B; Wang, Yu; Rodenbough, Philip P; Castaneda, Lisa J; Leibly, David J; Bhandari, Janhavi; Gelb, Michael H; Brinker, Achim; Engels, Ingo H; Taylor, Jennifer; Chatterjee, Arnab K; Fantauzzi, Pascal; Glynne, Richard J; Van Voorhis, Wesley C; Kuhen, Kelli L

2011-01-01

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC(50) values below 1.25μM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5'-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology. Copyright © 2010 Elsevier B.V. All rights reserved.

Bioinformatic identification and expression analysis of banana microRNAs and their targets.

PubMed

Chai, Juan; Feng, Renjun; Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

2015-01-01

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions.

Bioinformatic Identification and Expression Analysis of Banana MicroRNAs and Their Targets

PubMed Central

Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

2015-01-01

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions. PMID:25856313

Reference curve for the first-year growth response to growth hormone treatment in prepubertal children with idiopathic growth hormone deficiency: validation of the KIGS first-year growth response curve using the Belgian Register for the Study of Growth and Puberty Problems.

PubMed

Straetemans, Saartje; Roelants, Mathieu; Thomas, Muriel; Rooman, Raoul; De Schepper, Jean

2014-01-01

Comparing observed and expected growth after first-year growth hormone (GH) therapy is useful for identifying a poor growth response to GH. To generate a first-year, age-specific growth response reference curve for prepubertal Belgian children with idiopathic growth hormone deficiency (iGHD) treated with a standard weight-adjusted GH dose and to compare this national reference with the response references derived from KIGS. First-year height data of 357 prepubertal children (240 males) with iGHD were analyzed. Smooth reference curves of first-year height velocity (HV) in relation to age were created. Differences with the KIGS targets were evaluated after z-score transformation. The observed first-year HVs were log-normal distributed by age and decreased significantly with age (p<0.001). No GH dose or gender effect was observed (p=0.5). Distance to target height, severity of GHD and occurrence of multiple pituitary hormone deficiencies had a positive effect (p<0.01) on the calculated HV SDS. When applying the KIGS targets for severe iGHD, mean HV SDS was close to zero (-0.09±0.84). The developed age-specific growth response curves enable rapid identification of poor response to first-year GH treatment in prepubertal iGHD children. Our results validate the published growth targets derived from the KIGS database. © 2014 S. Karger AG, Basel.

Using a web-based, iterative education model to enhance clinical clerkships.

PubMed

Alexander, Erik K; Bloom, Nurit; Falchuk, Kenneth H; Parker, Michael

2006-10-01

Although most clinical clerkship curricula are designed to provide all students consistent exposure to defined course objectives, it is clear that individual students are diverse in their backgrounds and baseline knowledge. Ideally, the learning process should be individualized towards the strengths and weakness of each student, but, until recently, this has proved prohibitively time-consuming. The authors describe a program to develop and evaluate an iterative, Web-based educational model assessing medical students' knowledge deficits and allowing targeted teaching shortly after their identification. Beginning in 2002, a new educational model was created, validated, and applied in a prospective fashion to medical students during an internal medicine clerkship at Harvard Medical School. Using a Web-based platform, five validated questions were delivered weekly and a specific knowledge deficiency identified. Teaching targeted to the deficiency was provided to an intervention cohort of five to seven students in each clerkship, though not to controls (the remaining 7-10 students). Effectiveness of this model was assessed by performance on the following week's posttest question. Specific deficiencies were readily identified weekly using this model. Throughout the year, however, deficiencies varied unpredictably. Teaching targeted to deficiencies resulted in significantly better performance on follow-up questioning compared to the performance of those who did not receive this intervention. This model was easily applied in an additive fashion to the current curriculum, and student acceptance was high. The authors conclude that a Web-based, iterative assessment model can effectively target specific curricular needs unique to each group; focus teaching in a rapid, formative, and highly efficient manner; and may improve the efficiency of traditional clerkship teaching.

Identification and functional analysis of microRNA in myometrium tissue from spontaneous preterm labor

PubMed Central

Tang, Yao; Ji, Hongjing; Liu, Haiyan; Gu, Weirong; Li, Xiaotian; Peng, Ting

2015-01-01

Spontaneous preterm labor is an important complication in perinatology characterized by early onset myometrium contractions leading to labor at preterm. However, the exact mechanism that maintain uterine quiescence and promote increased uterine contractility during labor were incompletely defined. MicroRNAs is a class of short non-coding RNAs that regulate gene expression at the post-transcriptional level by binding the 3’ untranslated region of target mRNAs and play an important role in biological process and cellular functions. We hypothesized we could find differentially expressed microRNAs in the myometrium of women in spontaneous preterm labor. Thus, a microarray analysis of miRNAs of preterm myometrium was performed. 18 out of the 2006 detected microRNAs were found to be significantly dysregulated in myometrium in labor verse not in labor at preterm. Biological validation by quantitative real-time polymerase chain reaction confirms us a consistence rate of 83.3% (5 out of 6) with microarray analysis. The target genes for validated microRNAs were predicted by three algorithms (PicTar, TargetScan, and miRanda). Most of the potential targets of the miRNAs were relevant to positive regulation of cardiac muscle hypertrophy, reduction of cytosolic calcium ion concentration and relaxation of cardiac muscle as well as prostate cancer, adherents junction, regulation of actin cytoskeleton and regulation and other factor-regulated calcium reabsorption. Our result illustrates a characteristic microRNA profile in myometrium tissues and provides a new understanding of the process involved in spontaneous preterm labor. PMID:26722471

Subregional resource allocations in the National Health Service.

PubMed Central

Snaith, A H

1978-01-01

The Resource Allocation Working Party in its report Sharing Resources for Health in England proposes a formula for the identification of both regional and district financial targets (Department of Health and Social Security, 1976). In this paper it is argued that the national formula is not a valid instrument for the latter purpose. Furthermore, research into medical needs and outcomes will not be adequate to bring about real changes in resource distribution at local levels unless it is recognised that the health authorities can meet needs in different ways and that a change in resource management from institutional to service budgeting is required. PMID:262582

Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

PubMed

Kumar, Dhananjay; Dutta, Summi; Singh, Dharmendra; Prabhu, Kumble Vinod; Kumar, Manish; Mukhopadhyay, Kunal

2017-01-01

Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30 % of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust resistance breeding programs.

In Infants' Hands: Identification of Preverbal Infants at Risk for Primary Language Delay

ERIC Educational Resources Information Center

Lüke, Carina; Grimminger, Angela; Rohlfing, Katharina J.; Liszkowski, Ulf; Ritterfeld, Ute

2017-01-01

Early identification of primary language delay is crucial to implement effective prevention programs. Available screening instruments are based on parents' reports and have only insufficient predictive validity. This study employed observational measures of preverbal infants' gestural communication to test its predictive validity for identifying…

Development and validation of a protocol for optimizing the use of paraffin blocks in molecular epidemiological studies: The example from the HPV-AHEAD study.

PubMed

Mena, Marisa; Lloveras, Belen; Tous, Sara; Bogers, Johannes; Maffini, Fausto; Gangane, Nitin; Kumar, Rekha Vijay; Somanathan, Thara; Lucas, Eric; Anantharaman, Devasena; Gheit, Tarik; Castellsagué, Xavier; Pawlita, Michael; de Sanjosé, Silvia; Alemany, Laia; Tommasino, Massimo

2017-01-01

Worldwide use of formalin-fixed paraffin-embedded blocks (FFPE) is extensive in diagnosis and research. Yet, there is a lack of optimized/standardized protocols to process the blocks and verify the quality and presence of the targeted tissue. In the context of an international study on head and neck cancer (HNC)-HPV-AHEAD, a standardized protocol for optimizing the use of FFPEs in molecular epidemiology was developed and validated. First, a protocol for sectioning the FFPE was developed to prevent cross-contamination and distributed between participating centers. Before processing blocks, all sectioning centers underwent a quality control to guarantee a satisfactory training process. The first and last sections of the FFPEs were used for histopathological assessment. A consensus histopathology evaluation form was developed by an international panel of pathologists and evaluated for four indicators in a pilot analysis in order to validate it: 1) presence/type of tumor tissue, 2) identification of other tissue components that could affect the molecular diagnosis and 3) quality of the tissue. No HPV DNA was found in sections from empty FFPE generated in any histology laboratories of HPV-AHEAD consortium and all centers passed quality assurance for processing after quality control. The pilot analysis to validate the histopathology form included 355 HNC cases. The form was filled by six pathologists and each case was randomly assigned to two of them. Most samples (86%) were considered satisfactory. Presence of >50% of invasive carcinoma was observed in all sections of 66% of cases. Substantial necrosis (>50%) was present in <2% of samples. The concordance for the indicators targeted to validate the histopathology form was very high (kappa > 0.85) between first and last sections and fair to high between pathologists (kappa/pabak 0.21-0.72). The protocol allowed to correctly process without signs of contamination all FFPE of the study. The histopathology evaluation of the cases assured the presence of the targeted tissue, identified the presence of other tissues that could disturb the molecular diagnosis and allowed the assessment of tissue quality.

Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the treatment of B-cell lymphoma.

PubMed

Ge, Yang; Wang, Changyuan; Song, Shijie; Huang, Jiaxin; Liu, Zhihao; Li, Yongming; Meng, Qiang; Zhang, Jianbin; Yao, Jihong; Liu, Kexin; Ma, Xiaodong; Sun, Xiuli

2018-01-01

The BTK and JAK3 receptor tyrosine kinases are two validated and therapeutically amenable targets in the treatment of B-cell lymphomas. Here we report the identification of several classes of pyrimidine derivatives as potent BTK and JAK3 dual inhibitors. Among these molecules, approximately two thirds displayed strong inhibitory capacity at less than 10 nM concentration, and four compounds (7e, 7g, 7m and 7n) could significantly inhibit the phosphorylation of BTK and JAK3 enzymes at concentrations lower than 1 nM. Additionally, these pyrimidine derivatives also exhibited enhanced activity to block the proliferation of B-cell lymphoma cells compared with the representative BTK inhibitor ibrutinib. In particular, two structure-specific compounds 7b and 7e displayed stronger activity than reference agents in cell-based evaluation, with IC 50 values lower than 10 μM. Further biological studies, including flow cytometric analysis, and a xenograft model for in vivo evaluation, also indicated their efficacy and low toxicity in the treatment of B-cell lymphoma. These findings provide a new insight for the development of novel anti-B-cell lymphoma drugs with multi-target actions. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Identification of unwanted photoproducts of cosmetic preservatives in personal care products under ultraviolet-light using solid-phase microextraction and micro-matrix solid-phase dispersion.

PubMed

Alvarez-Rivera, Gerardo; Llompart, Maria; Garcia-Jares, Carmen; Lores, Marta

2015-04-17

The photochemical transformation of widely used cosmetic preservatives including benzoates, parabens, BHA, BHT and triclosan has been investigated in this work applying an innovative double-approach strategy: identification of transformation products in aqueous photodegradation experiments (UV-light, 254nm), followed by targeted screening analysis of such photoproducts in UV-irradiated cosmetic samples. Solid-phase microextraction (SPME) was applied, using different fiber coatings, in order to widen the range of detectable photoproducts in water, whereas UV-irradiated personal care products (PCPs) containing the target preservatives were extracted by micro-matrix solid-phase dispersion (micro-MSPD). Both SPME and micro-MSPD-based methodologies were successfully optimized and validated. Degradation kinetics of parent species, and photoformation of their transformation by-products were monitored by gas chromatography coupled to mass spectrometry (GC-MS). Thirty nine photoproducts were detected in aqueous photodegradation experiments, being tentatively identified based on their mass spectra. Transformation pathways between structurally related by-products, consistent with their kinetic behavior were postulated. The photoformation of unexpected photoproducts such as 2- and 4-hydroxybenzophenones, and 2,8-dichlorodibenzo-p-dioxin in PCPs are reported in this work for the first time. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA synthesis inhibitors for the treatment of gastrointestinal cancer.

PubMed

Yasui, Hiroshi; Tsurita, Giichiro; Imai, Kohzoh

2014-11-01

Intensive laboratory, preclinical and clinical studies have identified and validated molecular targets in cancers, leading to a shift toward the development of novel, rationally designed and specific therapeutic agents. However, gastrointestinal cancers continue to have a poor prognosis, largely due to drug resistance. Here, we discuss the current understanding of DNA synthesis inhibitors and their mechanisms of action for the treatment of gastrointestinal malignancies. Conventional agents, including DNA synthesis inhibitors such as fluoropyrimidines and platinum analogs, remain the most effective therapeutics and are the standards against which new drugs are compared. Novel DNA synthesis inhibitors for the treatment of gastrointestinal malignancies include a combination of the antimetabolite TAS-102, which consists of trifluorothymidine with a thymidine phosphorylase inhibitor, and a novel micellar formulation of cisplatin NC-6004 that uses a nanotechnology-based drug delivery system. The challenges of translational cancer research using DNA synthesis inhibitors include the identification of drugs that are specific to tumor cells to reduce toxicity and increase antitumor efficacy, biomarkers to predict pharmacological responses to chemotherapeutic drugs, identification of ways to overcome drug resistance and development of novel combination therapies with DNA synthesis inhibitors and other cancer therapies, such as targeted molecular therapeutics. Here, we discuss the current understanding of DNA synthesis inhibitors and their mechanisms of action for the treatment of gastrointestinal malignancies.

Reprint of: Nutrition in the Management of Cirrhosis and its Neurological Complications☆

PubMed Central

Bémeur, Chantal; Butterworth, Roger F.

2015-01-01

Malnutrition is a common feature of chronic liver diseases that is often associated with a poor prognosis including worsening of clinical outcome, neuropsychiatric complications as well as outcome following liver transplantation. Nutritional assessment in patients with cirrhosis is challenging owing to confounding factors related to liver failure. The objectives of nutritional intervention in cirrhotic patients are the support of liver regeneration, the prevention or correction of specific nutritional deficiencies and the prevention and/or treatment of the complications of liver disease per se and of liver transplantation. Nutritional recommendations target the optimal supply of adequate substrates related to requirements linked to energy, protein, carbohydrates, lipids, vitamins and minerals. Some issues relating to malnutrition in chronic liver disease remain to be addressed including the development of an appropriate well-validated nutritional assessment tool, the identification of mechanistic targets or therapy for sarcopenia, the development of nutritional recommendations for obese cirrhotic patients and liver-transplant recipients and the elucidation of the roles of vitamin A hepatotoxicity, as well as the impact of deficiencies in riboflavin and zinc on clinical outcomes. Early identification and treatment of malnutrition in chronic liver disease has the potential to lead to better disease outcome as well as prevention of the complications of chronic liver disease and improved transplant outcomes. PMID:26041952

FilmArray, an Automated Nested Multiplex PCR System for Multi-Pathogen Detection: Development and Application to Respiratory Tract Infection

PubMed Central

Poritz, Mark A.; Blaschke, Anne J.; Byington, Carrie L.; Meyers, Lindsay; Nilsson, Kody; Jones, David E.; Thatcher, Stephanie A.; Robbins, Thomas; Lingenfelter, Beth; Amiott, Elizabeth; Herbener, Amy; Daly, Judy; Dobrowolski, Steven F.; Teng, David H. -F.; Ririe, Kirk M.

2011-01-01

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the “FilmArray”, which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon. PMID:22039434

Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise.

PubMed

Alves, Cíntia; Pereira, Rui; Prieto, Lourdes; Aler, Mercedes; Amaral, Cesar R L; Arévalo, Cristina; Berardi, Gabriela; Di Rocco, Florencia; Caputo, Mariela; Carmona, Cristian Hernandez; Catelli, Laura; Costa, Heloísa Afonso; Coufalova, Pavla; Furfuro, Sandra; García, Óscar; Gaviria, Anibal; Goios, Ana; Gómez, Juan José Builes; Hernández, Alexis; Hernández, Eva Del Carmen Betancor; Miranda, Luís; Parra, David; Pedrosa, Susana; Porto, Maria João Anjos; Rebelo, Maria de Lurdes; Spirito, Matteo; Torres, María Del Carmen Villalobos; Amorim, António; Pereira, Filipe

2017-05-01

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of linear energy transfers and quality factors of charged particles produced by spontaneous fission neutrons from 252Cf and 244Pu in the human body.

PubMed

Endo, Akira; Sato, Tatsuhiko

2013-04-01

Absorbed doses, linear energy transfers (LETs) and quality factors of secondary charged particles in organs and tissues, generated via the interactions of the spontaneous fission neutrons from (252)Cf and (244)Pu within the human body, were studied using the Particle and Heavy Ion Transport Code System (PHITS) coupled with the ICRP Reference Phantom. Both the absorbed doses and the quality factors in target organs generally decrease with increasing distance from the source organ. The analysis of LET distributions of secondary charged particles led to the identification of the relationship between LET spectra and target-source organ locations. A comparison between human body-averaged mean quality factors and fluence-averaged radiation weighting factors showed that the current numerical conventions for the radiation weighting factors of neutrons, updated in ICRP103, and the quality factors for internal exposure are valid.

Demonstration of cardiac rotor and source mapping techniques in embryonic chick monolayers

NASA Astrophysics Data System (ADS)

You, Min Ju; Langfield, Peter; Campanari, Lucas; Dobbs, Matt; Shrier, Alvin; Glass, Leon

2017-09-01

Excitable media, such as the heart, display propagating waves with different geometries including target patterns and rotors (spiral waves). Collision of two waves leads to annihilation of both. We present algorithms for data processing and analysis to identify the core of rotors. In this work, we show that as the spatial sampling resolution decreases it becomes increasingly difficult to identify rotors—there are instances of false negatives and false positives. These observations are relevant to current controversies concerning the role of rotors in the initiation, maintenance, and treatment of cardiac arrhythmias, especially atrial fibrillation. Currently some practitioners target the core of rotors for ablation, but the effectiveness of this procedure has been questioned. In view of the difficulties inherent in the identification of rotors, we conclude that methods to identify rotors need to first be validated prior to assessing the efficacy of ablation.

Hypoxia inducible factor (HIF) as a model for studying inhibition of protein–protein interactions

PubMed Central

Burslem, George M.; Kyle, Hannah F.; Nelson, Adam; Edwards, Thomas A.

2017-01-01

The modulation of protein–protein interactions (PPIs) represents a major challenge in modern chemical biology. Current approaches (e.g. high-throughput screening, computer aided ligand design) are recognised as having limitations in terms of identification of hit matter. Considerable success has been achieved in terms of developing new approaches to PPI modulator discovery using the p53/hDM2 and Bcl-2 family of PPIs. However these important targets in oncology might be considered as “low-hanging-fruit”. Hypoxia inducible factor (HIF) is an emerging, but not yet fully validated target for cancer chemotherapy. Its role is to regulate the hypoxic response and it does so through a plethora of protein–protein interactions of varying topology, topography and complexity: its modulation represents an attractive approach to prevent development of new vasculature by hypoxic tumours. PMID:28878873

Feasibility of CRISPR-Cas9-Based In Vitro Drug Target Identification for Personalized Prostate Cancer Medicine

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-16-1-0502 TITLE: Feasibility of CRISPR -Cas9-Based In Vitro Drug Target Identification for Personalized Prostate Cancer Medicine...CONTRACT NUMBER Feasibility of CRISPR -Cas9-Based In Vitro Drug Target Identification for Personalized Prostate Cancer Medicine 5b. GRANT NUMBER...Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT This study tests the feasibility of using CRISPR -Cas9 to

Identification of predictive markers of cytarabine response in AML by integrative analysis of gene-expression profiles with multiple phenotypes

PubMed Central

Lamba, Jatinder K; Crews, Kristine R; Pounds, Stanley B; Cao, Xueyuan; Gandhi, Varsha; Plunkett, William; Razzouk, Bassem I; Lamba, Vishal; Baker, Sharyn D; Raimondi, Susana C; Campana, Dario; Pui, Ching-Hon; Downing, James R; Rubnitz, Jeffrey E; Ribeiro, Raul C

2011-01-01

Aim To identify gene-expression signatures predicting cytarabine response by an integrative analysis of multiple clinical and pharmacological end points in acute myeloid leukemia (AML) patients. Materials & methods We performed an integrated analysis to associate the gene expression of diagnostic bone marrow blasts from acute myeloid leukemia (AML) patients treated in the discovery set (AML97; n = 42) and in the independent validation set (AML02; n = 46) with multiple clinical and pharmacological end points. Based on prior biological knowledge, we defined a gene to show a therapeutically beneficial (detrimental) pattern of association of its expression positively (negatively) correlated with favorable phenotypes such as intracellular cytarabine 5´-triphosphate levels, morphological response and event-free survival, and negatively (positively) correlated with unfavorable end points such as post-cytarabine DNA synthesis levels, minimal residual disease and cytarabine LC50. Results We identified 240 probe sets predicting a therapeutically beneficial pattern and 97 predicting detrimental pattern (p ≤ 0.005) in the discovery set. Of these, 60 were confirmed in the independent validation set. The validated probe sets correspond to genes involved in PIK3/PTEN/AKT/mTOR signaling, G-protein-coupled receptor signaling and leukemogenesis. This suggests that targeting these pathways as potential pharmacogenomic and therapeutic candidates could be useful for improving treatment outcomes in AML. Conclusion This study illustrates the power of integrated data analysis of genomic data as well as multiple clinical and pharmacologic end points in the identification of genes and pathways of biological relevance. PMID:21449673

Expansion of the Scope of AOAC First Action Method 2012.25--Single-Laboratory Validation of Triphenylmethane Dye and Leuco Metabolite Analysis in Shrimp, Tilapia, Catfish, and Salmon by LC-MS/MS.

PubMed

Andersen, Wendy C; Casey, Christine R; Schneider, Marilyn J; Turnipseed, Sherri B

2015-01-01

Prior to conducting a collaborative study of AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood, a single-laboratory validation of method 2012.25 was performed to expand the scope of the method to other seafood matrixes including salmon, catfish, tilapia, and shrimp. The validation included the analysis of fortified and incurred residues over multiple weeks to assess analyte stability in matrix at -80°C, a comparison of calibration methods over the range 0.25 to 4 μg/kg, study of matrix effects for analyte quantification, and qualitative identification of targeted analytes. Method accuracy ranged from 88 to 112% with 13% RSD or less for samples fortified at 0.5, 1.0, and 2.0 μg/kg. Analyte identification and determination limits were determined by procedures recommended both by the U. S. Food and Drug Administration and the European Commission. Method detection limits and decision limits ranged from 0.05 to 0.24 μg/kg and 0.08 to 0.54 μg/kg, respectively. AOAC First Action Method 2012.25 with an extracted matrix calibration curve and internal standard correction is suitable for the determination of triphenylmethane dyes and leuco metabolites in salmon, catfish, tilapia, and shrimp by LC-MS/MS at a residue determination level of 0.5 μg/kg or below.

A High Throughput Model of Post-Traumatic Osteoarthritis using Engineered Cartilage Tissue Analogs

PubMed Central

Mohanraj, Bhavana; Meloni, Gregory R.; Mauck, Robert L.; Dodge, George R.

2014-01-01

(1) Objective A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. (2) Method We utilized a high throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. (3) Results Engineered cartilage response to injury was strain dependent, with a 2-fold increase in GAG loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in LDH release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor (ZVF) was effective at reducing cell death, while the amphiphilic polymer (P188) and the free-radical scavenger (NAC) reduced GAG loss as compared to injury alone. (4) Conclusions The injury response in this engineered cartilage model replicated key features of the response from cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA. PMID:24999113

Development and Validation of Targeted Next-Generation Sequencing Panels for Detection of Germline Variants in Inherited Diseases.

PubMed

Santani, Avni; Murrell, Jill; Funke, Birgit; Yu, Zhenming; Hegde, Madhuri; Mao, Rong; Ferreira-Gonzalez, Andrea; Voelkerding, Karl V; Weck, Karen E

2017-06-01

- The number of targeted next-generation sequencing (NGS) panels for genetic diseases offered by clinical laboratories is rapidly increasing. Before an NGS-based test is implemented in a clinical laboratory, appropriate validation studies are needed to determine the performance characteristics of the test. - To provide examples of assay design and validation of targeted NGS gene panels for the detection of germline variants associated with inherited disorders. - The approaches used by 2 clinical laboratories for the development and validation of targeted NGS gene panels are described. Important design and validation considerations are examined. - Clinical laboratories must validate performance specifications of each test prior to implementation. Test design specifications and validation data are provided, outlining important steps in validation of targeted NGS panels by clinical diagnostic laboratories.

Computational Acoustic Beamforming for Noise Source Identification for Small Wind Turbines.

PubMed

Ma, Ping; Lien, Fue-Sang; Yee, Eugene

2017-01-01

This paper develops a computational acoustic beamforming (CAB) methodology for identification of sources of small wind turbine noise. This methodology is validated using the case of the NACA 0012 airfoil trailing edge noise. For this validation case, the predicted acoustic maps were in excellent conformance with the results of the measurements obtained from the acoustic beamforming experiment. Following this validation study, the CAB methodology was applied to the identification of noise sources generated by a commercial small wind turbine. The simulated acoustic maps revealed that the blade tower interaction and the wind turbine nacelle were the two primary mechanisms for sound generation for this small wind turbine at frequencies between 100 and 630 Hz.

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) – a class-I KNOX gene in potato

PubMed Central

Mahajan, Ameya S.; Kondhare, Kirtikumar R.; Rajabhoj, Mohit P.; Kumar, Amit; Ghate, Tejashree; Ravindran, Nevedha; Habib, Farhat; Siddappa, Sundaresha; Banerjee, Anjan K.

2016-01-01

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato. PMID:27217546

Systematic screening identifies dual PI3K and mTOR inhibition as a conserved therapeutic vulnerability in osteosarcoma

PubMed Central

Gupte, Ankita; Baker, Emma K.; Wan, Soo-San; Stewart, Elizabeth; Loh, Amos; Shelat, Anang A.; Gould, Cathryn M.; Chalk, Alistair M.; Taylor, Scott; Lackovic, Kurt; Karlström, Åsa; Mutsaers, Anthony J.; Desai, Jayesh; Madhamshettiwar, Piyush B.; Zannettino, Andrew CW.; Burns, Chris; Huang, David CS.; Dyer, Michael A.; Simpson, Kaylene J.; Walkley, Carl R.

2015-01-01

Purpose Osteosarcoma (OS) is the most common cancer of bone occurring mostly in teenagers. Despite rapid advances in our knowledge of the genetics and cell biology of OS, significant improvements in patient survival have not been observed. The identification of effective therapeutics has been largely empirically based. The identification of new therapies and therapeutic targets are urgently needed to enable improved outcomes for OS patients. Experimental Design We have used genetically engineered murine models of human OS in a systematic, genome wide screen to identify new candidate therapeutic targets. We performed a genome wide siRNA screen, with or without doxorubicin. In parallel a screen of therapeutically relevant small molecules was conducted on primary murine and primary human OS derived cell cultures. All results were validated across independent cell cultures and across human and mouse OS. Results The results from the genetic and chemical screens significantly overlapped, with a profound enrichment of pathways regulated by PI3K and mTOR pathways. Drugs that concurrently target both PI3K and mTOR were effective at inducing apoptosis in primary OS cell cultures in vitro in both human and mouse OS, while specific PI3K or mTOR inhibitors were not effective. The results were confirmed with siRNA and small molecule approaches. Rationale combinations of specific PI3K and mTOR inhibitors could recapitulate the effect on OS cell cultures. Conclusions The approaches described here have identified dual inhibition of the PI3K/mTOR pathway as a sensitive, druggable target in OS and provide rationale for translational studies with these agents. PMID:25862761

Safety Lead Optimization and Candidate Identification: Integrating New Technologies into Decision-Making.

PubMed

Dambach, Donna M; Misner, Dinah; Brock, Mathew; Fullerton, Aaron; Proctor, William; Maher, Jonathan; Lee, Dong; Ford, Kevin; Diaz, Dolores

2016-04-18

Discovery toxicology focuses on the identification of the most promising drug candidates through the development and implementation of lead optimization strategies and hypothesis-driven investigation of issues that enable rational and informed decision-making. The major goals are to [a] identify and progress the drug candidate with the best overall drug safety profile for a therapeutic area, [b] remove the most toxic drugs from the portfolio prior to entry into humans to reduce clinical attrition due to toxicity, and [c] establish a well-characterized hazard and translational risk profile to enable clinical trial designs. This is accomplished through a framework that balances the multiple considerations to identify a drug candidate with the overall best drug characteristics and provides a cogent understanding of mechanisms of toxicity. The framework components include establishing a target candidate profile for each program that defines the qualities of a successful candidate based on the intended therapeutic area, including the risk tolerance for liabilities; evaluating potential liabilities that may result from engaging the therapeutic target (pharmacology-mediated or on-target) and that are chemical structure-mediated (off-target); and characterizing identified liabilities. Lead optimization and investigation relies upon the integrated use of a variety of technologies and models (in silico, in vitro, and in vivo) that have achieved a sufficient level of qualification or validation to provide confidence in their use. We describe the strategic applications of various nonclinical models (established and new) for a holistic and integrated risk assessment that is used for rational decision-making. While this review focuses on strategies for small molecules, the overall concepts, approaches, and technologies are generally applicable to biotherapeutics.

Signal peptide discrimination and cleavage site identification using SVM and NN.

PubMed

Kazemian, H B; Yusuf, S A; White, K

2014-02-01

About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model. © 2013 Published by Elsevier Ltd.

Meeting Report: Using Stem Cells for Biological and Therapeutics Discovery in Mental Illness, April 2012

PubMed Central

2013-01-01

This report synthesizes the discussions during a workshop convened April 24–25, 2012, by the National Institute of Mental Health and the Foundation for the NIH in Bethesda, Maryland, that focused on progress and challenges in the use of patient-derived reprogrammed cells for basic biological discovery, target identification, screening, and drug development for mental illnesses such as schizophrenia, bipolar disorder, and autism spectrum disorders. The workshop revealed that the greatest progress has been made in reprogramming methods and agreed-upon standards for validating the resulting induced pluripotent stem cell lines. However, challenges remain in several areas, including efficiently generating and validating specific neural cell types with respect to regional identity, establishing assays with predictive validity to mental illness pathophysiology, and generating sufficient statistical power and data reproducibility across laboratories. A brainstorming session yielded a number of suggestions, including calls to (a) facilitate the replication of results by standardizing protocols and samples used across laboratories; (b) improve technology by generating cheaper/faster targeting methods, reporters, and assays; and (c) improve resource sharing and collaboration, with an emphasis on rapid sharing of new cell lines, technologies, and best practices, possibly incorporated into a public-private partnership. The meeting provided an important venue for academic, government, and private sector scientists to address potential opportunities for translational and clinical applications of reprogrammed cell research. A number of activities since the workshop have reflected the feedback from meeting participants. PMID:23408104

Meeting report: using stem cells for biological and therapeutics discovery in mental illness, April 2012.

PubMed

Panchision, David M

2013-03-01

This report synthesizes the discussions during a workshop convened April 24-25, 2012, by the National Institute of Mental Health and the Foundation for the NIH in Bethesda, Maryland, that focused on progress and challenges in the use of patient-derived reprogrammed cells for basic biological discovery, target identification, screening, and drug development for mental illnesses such as schizophrenia, bipolar disorder, and autism spectrum disorders. The workshop revealed that the greatest progress has been made in reprogramming methods and agreed-upon standards for validating the resulting induced pluripotent stem cell lines. However, challenges remain in several areas, including efficiently generating and validating specific neural cell types with respect to regional identity, establishing assays with predictive validity to mental illness pathophysiology, and generating sufficient statistical power and data reproducibility across laboratories. A brainstorming session yielded a number of suggestions, including calls to (a) facilitate the replication of results by standardizing protocols and samples used across laboratories; (b) improve technology by generating cheaper/faster targeting methods, reporters, and assays; and (c) improve resource sharing and collaboration, with an emphasis on rapid sharing of new cell lines, technologies, and best practices, possibly incorporated into a public-private partnership. The meeting provided an important venue for academic, government, and private sector scientists to address potential opportunities for translational and clinical applications of reprogrammed cell research. A number of activities since the workshop have reflected the feedback from meeting participants.

Flanking sequence determination and event-specific detection of genetically modified wheat B73-6-1.

PubMed

Xu, Junyi; Cao, Jijuan; Cao, Dongmei; Zhao, Tongtong; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2013-05-01

In order to establish a specific identification method for genetically modified (GM) wheat, exogenous insert DNA and flanking sequence between exogenous fragment and recombinant chromosome of GM wheat B73-6-1 were successfully acquired by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. Newly acquired exogenous fragment covered the full-length sequence of transformed genes such as transformed plasmid and corresponding functional genes including marker uidA, herbicide-resistant bar, ubiquitin promoter, and high-molecular-weight gluten subunit. The flanking sequence between insert DNA revealed high similarity with Triticum turgidum A gene (GenBank: AY494981.1). A specific PCR detection method for GM wheat B73-6-1 was established on the basis of primers designed according to the flanking sequence. This specific PCR method was validated by GM wheat, GM corn, GM soybean, GM rice, and non-GM wheat. The specifically amplified target band was observed only in GM wheat B73-6-1. This method is of high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of GM wheat B73-6-1.

Haplotype-Based Genotyping in Polyploids.

PubMed

Clevenger, Josh P; Korani, Walid; Ozias-Akins, Peggy; Jackson, Scott

2018-01-01

Accurate identification of polymorphisms from sequence data is crucial to unlocking the potential of high throughput sequencing for genomics. Single nucleotide polymorphisms (SNPs) are difficult to accurately identify in polyploid crops due to the duplicative nature of polyploid genomes leading to low confidence in the true alignment of short reads. Implementing a haplotype-based method in contrasting subgenome-specific sequences leads to higher accuracy of SNP identification in polyploids. To test this method, a large-scale 48K SNP array (Axiom Arachis2) was developed for Arachis hypogaea (peanut), an allotetraploid, in which 1,674 haplotype-based SNPs were included. Results of the array show that 74% of the haplotype-based SNP markers could be validated, which is considerably higher than previous methods used for peanut. The haplotype method has been implemented in a standalone program, HAPLOSWEEP, which takes as input bam files and a vcf file and identifies haplotype-based markers. Haplotype discovery can be made within single reads or span paired reads, and can leverage long read technology by targeting any length of haplotype. Haplotype-based genotyping is applicable in all allopolyploid genomes and provides confidence in marker identification and in silico-based genotyping for polyploid genomics.

Forecasting the ocean optical environment in support of Navy mine warfare operations

NASA Astrophysics Data System (ADS)

Ladner, S. D.; Arnone, R.; Jolliff, J.; Casey, B.; Matulewski, K.

2012-06-01

A 3D ocean optical forecast system called TODS (Tactical Ocean Data System) has been developed to determine the performance of underwater LIDAR detection/identification systems. TODS fuses optical measurements from gliders, surface satellite optical properties, and 3D ocean forecast circulation models to extend the 2-dimensional surface satellite optics into a 3-dimensional optical volume including subsurface optical layers of beam attenuation coefficient (c) and diver visibility. Optical 3D nowcast and forecasts are combined with electro-optical identification (EOID) models to determine the underwater LIDAR imaging performance field used to identify subsurface mine threats in rapidly changing coastal regions. TODS was validated during a recent mine warfare exercise with Helicopter Mine Countermeasures Squadron (HM-14). Results include the uncertainties in the optical forecast and lidar performance and sensor tow height predictions that are based on visual detection and identification metrics using actual mine target images from the EOID system. TODS is a new capability of coupling the 3D optical environment and EOID system performance and is proving important for the MIW community as both a tactical decision aid and for use in operational planning, improving timeliness and efficiency in clearance operations.

5 CFR 2100.4 - Requirements for identification.

Code of Federal Regulations, 2010 CFR

2010-01-01

... must provide their full name and as much information as possible in order that a proper search for..., military or annuitant identification card, or any official document as acceptable identification validation...

C-mii: a tool for plant miRNA and target identification.

PubMed

Numnark, Somrak; Mhuantong, Wuttichai; Ingsriswang, Supawadee; Wichadakul, Duangdao

2012-01-01

MicroRNAs (miRNAs) have been known to play an important role in several biological processes in both animals and plants. Although several tools for miRNA and target identification are available, the number of tools tailored towards plants is limited, and those that are available have specific functionality, lack graphical user interfaces, and restrict the number of input sequences. Large-scale computational identifications of miRNAs and/or targets of several plants have been also reported. Their methods, however, are only described as flow diagrams, which require programming skills and the understanding of input and output of the connected programs to reproduce. To overcome these limitations and programming complexities, we proposed C-mii as a ready-made software package for both plant miRNA and target identification. C-mii was designed and implemented based on established computational steps and criteria derived from previous literature with the following distinguishing features. First, software is easy to install with all-in-one programs and packaged databases. Second, it comes with graphical user interfaces (GUIs) for ease of use. Users can identify plant miRNAs and targets via step-by-step execution, explore the detailed results from each step, filter the results according to proposed constraints in plant miRNA and target biogenesis, and export sequences and structures of interest. Third, it supplies bird's eye views of the identification results with infographics and grouping information. Fourth, in terms of functionality, it extends the standard computational steps of miRNA target identification with miRNA-target folding and GO annotation. Fifth, it provides helper functions for the update of pre-installed databases and automatic recovery. Finally, it supports multi-project and multi-thread management. C-mii constitutes the first complete software package with graphical user interfaces enabling computational identification of both plant miRNA genes and miRNA targets. With the provided functionalities, it can help accelerate the study of plant miRNAs and targets, especially for small and medium plant molecular labs without bioinformaticians. C-mii is freely available at http://www.biotec.or.th/isl/c-mii for both Windows and Ubuntu Linux platforms.

C-mii: a tool for plant miRNA and target identification

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) have been known to play an important role in several biological processes in both animals and plants. Although several tools for miRNA and target identification are available, the number of tools tailored towards plants is limited, and those that are available have specific functionality, lack graphical user interfaces, and restrict the number of input sequences. Large-scale computational identifications of miRNAs and/or targets of several plants have been also reported. Their methods, however, are only described as flow diagrams, which require programming skills and the understanding of input and output of the connected programs to reproduce. Results To overcome these limitations and programming complexities, we proposed C-mii as a ready-made software package for both plant miRNA and target identification. C-mii was designed and implemented based on established computational steps and criteria derived from previous literature with the following distinguishing features. First, software is easy to install with all-in-one programs and packaged databases. Second, it comes with graphical user interfaces (GUIs) for ease of use. Users can identify plant miRNAs and targets via step-by-step execution, explore the detailed results from each step, filter the results according to proposed constraints in plant miRNA and target biogenesis, and export sequences and structures of interest. Third, it supplies bird's eye views of the identification results with infographics and grouping information. Fourth, in terms of functionality, it extends the standard computational steps of miRNA target identification with miRNA-target folding and GO annotation. Fifth, it provides helper functions for the update of pre-installed databases and automatic recovery. Finally, it supports multi-project and multi-thread management. Conclusions C-mii constitutes the first complete software package with graphical user interfaces enabling computational identification of both plant miRNA genes and miRNA targets. With the provided functionalities, it can help accelerate the study of plant miRNAs and targets, especially for small and medium plant molecular labs without bioinformaticians. C-mii is freely available at http://www.biotec.or.th/isl/c-mii for both Windows and Ubuntu Linux platforms. PMID:23281648

Early Identification System: Year Two. Research Report 80-15.

ERIC Educational Resources Information Center

Stennett, R. G.; Earl, L. M.

During the academic year 1978-79, school teams implemented a newly developed early identification system in all kindergarten and grade one classes in London, Ontario schools. After analysis and revision of the system, the internal consistency and concurrent validity of the process and a test of its short-term predictive validity were investigated.…

Identification of cutting force coefficients in machining process considering cutter vibration

NASA Astrophysics Data System (ADS)

Yao, Qi; Luo, Ming; Zhang, Dinghua; Wu, Baohai

2018-03-01

Among current cutting force models, cutting force coefficients still are the foundation of predicting calculation combined with consideration of geometry engagement variation, equipment characteristics, material properties and so on. Attached with unimpeachable significance, the traditional and some novel identification methods of cutting force coefficient are still faced with trouble, including repeated onerous work, over ideal measuring condition, variation of value due to material divergence, interference from measuring units. To utilize the large amount of data from real manufacturing section, enlarge data sources and enrich cutting data base for former prediction task, a novel identification method is proposed by considering stiffness properties of the cutter-holder-spindle system in this paper. According to previously proposed studies, the direct result of cutter vibration is the form of dynamic undeformed chip thickness. This fluctuation is considered in two stages of this investigation. Firstly, a cutting force model combined with cutter vibration is established in detailed way. Then, on the foundation of modeling, a novel identification method is developed, in which the dynamic undeformed chip thickness could be obtained by using collected data. In a carefully designed experiment procedure, the reliability of model is validated by comparing predicted and measured results. Under different cutting condition and cutter stiffness, data is collected for the justification of identification method. The results showed divergence in calculated coefficients is acceptable confirming the possibility of accomplishing targets by applying this new method. In discussion, the potential directions of improvement are proposed.

Mining, identification and function analysis of microRNAs and target genes in peanut (Arachis hypogaea L.).

PubMed

Zhang, Tingting; Hu, Shuhao; Yan, Caixia; Li, Chunjuan; Zhao, Xiaobo; Wan, Shubo; Shan, Shihua

2017-02-01

In the present investigation, a total of 60 conserved peanut (Arachis hypogaea L.) microRNA (miRNA) sequences, belonging to 16 families, were identified using bioinformatics methods. There were 392 target gene sequences, identified from 58 miRNAs with Target-align software and BLASTx analyses. Gene Ontology (GO) functional analysis suggested that these target genes were involved in mediating peanut growth and development, signal transduction and stress resistance. There were 55 miRNA sequences, verified employing a poly (A) tailing test, with a success rate of up to 91.67%. Twenty peanut target gene sequences were randomly selected, and the 5' rapid amplification of the cDNA ends (5'-RACE) method were used to validate the cleavage sites of these target genes. Of these, 14 (70%) peanut miRNA targets were verified by means of gel electrophoresis, cloning and sequencing. Furthermore, functional analysis and homologous sequence retrieval were conducted for target gene sequences, and 26 target genes were chosen as the objects for stress resistance experimental study. Real-time fluorescence quantitative PCR (qRT-PCR) technology was applied to measure the expression level of resistance-associated miRNAs and their target genes in peanut exposed to Aspergillus flavus (A. flavus) infection and drought stress, respectively. In consequence, 5 groups of miRNAs & targets were found accorded with the mode of miRNA negatively controlling the expression of target genes. This study, preliminarily determined the biological functions of some resistance-associated miRNAs and their target genes in peanut. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Neuromorphic Optical Signal Processing and Image Understanding for Automated Target Recognition

DTIC Science & Technology

1989-12-01

34 Stochastic Learning Machine " Neuromorphic Target Identification * Cognitive Networks 3. Conclusions ..... ................ .. 12 4. Publications...16 5. References ...... ................... . 17 6. Appendices ....... .................. 18 I. Optoelectronic Neural Networks and...Learning Machines. II. Stochastic Optical Learning Machine. III. Learning Network for Extrapolation AccesFon For and Radar Target Identification

Biased lineup instructions and face identification from video images.

PubMed

Thompson, W Burt; Johnson, Jaime

2008-01-01

Previous eyewitness memory research has shown that biased lineup instructions reduce identification accuracy, primarily by increasing false-positive identifications in target-absent lineups. Because some attempts at identification do not rely on a witness's memory of the perpetrator but instead involve matching photos to images on surveillance video, the authors investigated the effects of biased instructions on identification accuracy in a matching task. In Experiment 1, biased instructions did not affect the overall accuracy of participants who used video images as an identification aid, but nearly all correct decisions occurred with target-present photo spreads. Both biased and unbiased instructions resulted in high false-positive rates. In Experiment 2, which focused on video-photo matching accuracy with target-absent photo spreads, unbiased instructions led to more correct responses (i.e., fewer false positives). These findings suggest that investigators should not relax precautions against biased instructions when people attempt to match photos to an unfamiliar person recorded on video.

Effects of Resolution, Range, and Image Contrast on Target Acquisition Performance.

PubMed

Hollands, Justin G; Terhaar, Phil; Pavlovic, Nada J

2018-05-01

We sought to determine the joint influence of resolution, target range, and image contrast on the detection and identification of targets in simulated naturalistic scenes. Resolution requirements for target acquisition have been developed based on threshold values obtained using imaging systems, when target range was fixed, and image characteristics were determined by the system. Subsequent work has examined the influence of factors like target range and image contrast on target acquisition. We varied the resolution and contrast of static images in two experiments. Participants (soldiers) decided whether a human target was located in the scene (detection task) or whether a target was friendly or hostile (identification task). Target range was also varied (50-400 m). In Experiment 1, 30 participants saw color images with a single target exemplar. In Experiment 2, another 30 participants saw monochrome images containing different target exemplars. The effects of target range and image contrast were qualitatively different above and below 6 pixels per meter of target for both tasks in both experiments. Target detection and identification performance were a joint function of image resolution, range, and contrast for both color and monochrome images. The beneficial effects of increasing resolution for target acquisition performance are greater for closer (larger) targets.

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells

PubMed Central

Salopiata, Florian; Depner, Sofia; Wäsch, Marvin; Böhm, Martin E.; Mücke, Oliver; Plass, Christoph; Lehmann, Wolf D.; Kreutz, Clemens; Timmer, Jens; Klingmüller, Ursula

2016-01-01

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types. PMID:27494133

A new disaster victim identification management strategy targeting "near identification-threshold" cases: Experiences from the Boxing Day tsunami.

PubMed

Wright, Kirsty; Mundorff, Amy; Chaseling, Janet; Forrest, Alexander; Maguire, Christopher; Crane, Denis I

2015-05-01

The international disaster victim identification (DVI) response to the Boxing Day tsunami, led by the Royal Thai Police in Phuket, Thailand, was one of the largest and most complex in DVI history. Referred to as the Thai Tsunami Victim Identification operation, the group comprised a multi-national, multi-agency, and multi-disciplinary team. The traditional DVI approach proved successful in identifying a large number of victims quickly. However, the team struggled to identify certain victims due to incomplete or poor quality ante-mortem and post-mortem data. In response to these challenges, a new 'near-threshold' DVI management strategy was implemented to target presumptive identifications and improve operational efficiency. The strategy was implemented by the DNA Team, therefore DNA kinship matches that just failed to reach the reporting threshold of 99.9% were prioritized, however the same approach could be taken by targeting, for example, cases with partial fingerprint matches. The presumptive DNA identifications were progressively filtered through the Investigation, Dental and Fingerprint Teams to add additional information necessary to either strengthen or conclusively exclude the identification. Over a five-month period 111 victims from ten countries were identified using this targeted approach. The new identifications comprised 87 adults, 24 children and included 97 Thai locals. New data from the Fingerprint Team established nearly 60% of the total near-threshold identifications and the combined DNA/Physical method was responsible for over 30%. Implementing the new strategy, targeting near-threshold cases, had positive management implications. The process initiated additional ante-mortem information collections, and established a much-needed, distinct "end-point" for unresolved cases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A universal and reliable assay for molecular sex identification of three-spined sticklebacks (Gasterosteus aculeatus).

PubMed

Toli, E-A; Calboli, F C F; Shikano, T; Merilä, J

2016-11-01

In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex-related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three-spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex-linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single-marker-based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost-efficient tool for universal sex identification of three-spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species. © 2016 John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffith, David A.; Kung, Daniel W.; Esler, William P.

We found that Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. Here, we disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 asmore » a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffith, David A.; Kung, Daniel W.; Esler, William P.

Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. We disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate formore » the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. In conclusion, this demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease.« less

Development of small-molecule inhibitors of the group I p21-activated kinases, emerging therapeutic targets in cancer.

PubMed

Yi, Chunling; Maksimoska, Jasna; Marmorstein, Ronen; Kissil, Joseph L

2010-09-01

The p21-activated kinases (PAKs), immediate downstream effectors of the small G-proteins of the Rac/cdc42 family, are critical mediators of signaling pathways regulating cellular behaviors and as such, have been implicated in pathological conditions including cancer. Recent studies have validated the requirement for PAKs in promoting tumorigenesis in breast carcinoma and neurofibromatosis. Thus, there has been considerable interest in the development of inhibitors to the PAKs, as biological markers and leads for the development of therapeutics. While initial approaches were based on screening for competitive organic inhibitors, more recent efforts have focused on the identification of allosteric inhibitors, organometallic ATP-competitive inhibitors and the use of PAK1/inhibitor crystal structures for inhibitor optimization. This has led to the identification of highly selective and potent inhibitors, which will serve as a basis for further development of inhibitors for therapeutic applications. Copyright 2010 Elsevier Inc. All rights reserved.

High-throughput gene mapping in Caenorhabditis elegans.

PubMed

Swan, Kathryn A; Curtis, Damian E; McKusick, Kathleen B; Voinov, Alexander V; Mapa, Felipa A; Cancilla, Michael R

2002-07-01

Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 +/- 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18.

High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.

PubMed

Zhou, Yuexin; Zhu, Shiyou; Cai, Changzu; Yuan, Pengfei; Li, Chunmei; Huang, Yanyi; Wei, Wensheng

2014-05-22

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

Real-time identification of vehicle motion-modes using neural networks

NASA Astrophysics Data System (ADS)

Wang, Lifu; Zhang, Nong; Du, Haiping

2015-01-01

A four-wheel ground vehicle has three body-dominated motion-modes, that is, bounce, roll, and pitch motion-modes. Real-time identification of these motion-modes can make vehicle suspensions, in particular, active suspensions, target on the dominant motion-mode and apply appropriate control strategies to improve its performance with less power consumption. Recently, a motion-mode energy method (MEM) was developed to identify the vehicle body motion-modes. However, this method requires the measurement of full vehicle states and road inputs, which are not always available in practice. This paper proposes an alternative approach to identify vehicle primary motion-modes with acceptable accuracy by employing neural networks (NNs). The effectiveness of the trained NNs is verified on a 10-DOF full-car model under various types of excitation inputs. The results confirm that the proposed method is effective in determining vehicle primary motion-modes with comparable accuracy to the MEM method. Experimental data is further used to validate the proposed method.

DNA microarray-based detection and identification of Burkholderia mallei, Burkholderia pseudomallei and Burkholderia spp.

PubMed

Schmoock, Gernot; Ehricht, Ralf; Melzer, Falk; Rassbach, Astrid; Scholz, Holger C; Neubauer, Heinrich; Sachse, Konrad; Mota, Rinaldo Aparecido; Saqib, Muhammad; Elschner, Mandy

2009-01-01

We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains.

Personal Background Preparation Survey for early identification of nursing students at risk for attrition.

PubMed

Johnson, Craig W; Johnson, Ronald; Kim, Mira; McKee, John C

2009-11-01

During 2004 and 2005 orientations, all 187 and 188 new matriculates, respectively, in two southwestern U.S. nursing schools completed Personal Background and Preparation Surveys (PBPS) in the first predictive validity study of a diagnostic and prescriptive instrument for averting adverse academic status events (AASE) among nursing or health science professional students. One standard deviation increases in PBPS risks (p < 0.05) multiplied odds of first-year or second-year AASE by approximately 150%, controlling for school affiliation and underrepresented minority student (URMS) status. AASE odds one standard deviation above mean were 216% to 250% those one standard deviation below mean. Odds of first-year or second-year AASE for URMS one standard deviation above the 2004 PBPS mean were 587% those for non-URMS one standard deviation below mean. The PBPS consistently and significantly facilitated early identification of nursing students at risk for AASE, enabling proactive targeting of interventions for risk amelioration and AASE or attrition prevention. Copyright 2009, SLACK Incorporated.

Computer applications making rapid advances in high throughput microbial proteomics (HTMP).

PubMed

Anandkumar, Balakrishna; Haga, Steve W; Wu, Hui-Fen

2014-02-01

The last few decades have seen the rise of widely-available proteomics tools. From new data acquisition devices, such as MALDI-MS and 2DE to new database searching softwares, these new products have paved the way for high throughput microbial proteomics (HTMP). These tools are enabling researchers to gain new insights into microbial metabolism, and are opening up new areas of study, such as protein-protein interactions (interactomics) discovery. Computer software is a key part of these emerging fields. This current review considers: 1) software tools for identifying the proteome, such as MASCOT or PDQuest, 2) online databases of proteomes, such as SWISS-PROT, Proteome Web, or the Proteomics Facility of the Pathogen Functional Genomics Resource Center, and 3) software tools for applying proteomic data, such as PSI-BLAST or VESPA. These tools allow for research in network biology, protein identification, functional annotation, target identification/validation, protein expression, protein structural analysis, metabolic pathway engineering and drug discovery.

Beyond radio-displacement techniques for Identification of CB1 Ligands: The First Application of a Fluorescence-quenching Assay

PubMed Central

Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

2014-01-01

Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

Development and validation of a real-time TaqMan assay for the detection and enumeration of Pseudomonas fluorescens ATCC 13525 used as a challenge organism in testing of food equipments.

PubMed

Saha, Ratul; Bestervelt, Lorelle L; Donofrio, Robert S

2012-02-01

Pseudomonas fluorescens ATCC 13525 is used as the challenge organism to evaluate the efficacy of the clean-in-place (CIP) process of food equipment (automatic ice-maker) as per NSF/ANSI Standard 12. Traditional culturing methodology is presently used to determine the concentration of the challenge organism, which takes 48 h to confirm the cell density. Storage of the challenge preparation in the refrigerator might alter the cell density as P. fluorescens is capable of growing at 4 °C. Also, background organism can grow on the Pseudomonas F agar (PFA) used for the recovery of P. fluorescens thus affecting the results of the test. Real-time TaqMan assay targeting the cpn60 gene was developed for the enumeration and the identification of P. fluorescens because of its specificity, accuracy, and shorter turnaround time. The TaqMan primer-probe pair developed using the Allele ID® 7.0 probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10 colony forming units (CFU)/mL. The assay was also successful in determining the concentration of the challenge preparation within 2 h. Based on these observations, TaqMan assay targeting the cpn60 gene can be efficiently used for strain level identification and enumeration of bacteria. Pseudomonas fluorescens ATCC 13525 is used as a challenge organism in the efficacy testing of clean-in-place process of food equipments. Currently, culturing technique is used for its identification and estimation, which is not only time-consuming but also prone to error. Real-time TaqMan assay is more specific, sensitive, and accurate along with a shorter turnaround time compared to culturing techniques, thereby increasing the overall quality of the testing methodology to evaluate the clean-in-place process critical for the food industry to protect public health and safety. © 2012 Institute of Food Technologists®

Rapid identification and validation of novel targeted approaches for Glioblastoma: A combined ex vivo-in vivo pharmaco-omic model.

PubMed

Daher, Ahmad; de Groot, John

2018-01-01

Tumor heterogeneity is a major factor in glioblastoma's poor response to therapy and seemingly inevitable recurrence. Only two glioblastoma drugs have received Food and Drug Administration approval since 1998, highlighting the urgent need for new therapies. Profiling "omics" analyses have helped characterize glioblastoma molecularly and have thus identified multiple molecular targets for precision medicine. These molecular targets have influenced clinical trial design; many "actionable" mutation-focused trials are underway, but because they have not yet led to therapeutic breakthroughs, new strategies for treating glioblastoma, especially those with a pharmacological functional component, remain in high demand. In that regard, high-throughput screening that allows for expedited preclinical drug testing and the use of GBM models that represent tumor heterogeneity more accurately than traditional cancer cell lines is necessary to maximize the successful translation of agents into the clinic. High-throughput screening has been successfully used in the testing, discovery, and validation of potential therapeutics in various cancer models, but it has not been extensively utilized in glioblastoma models. In this report, we describe the basic aspects of high-throughput screening and propose a modified high-throughput screening model in which ex vivo and in vivo drug testing is complemented by post-screening pharmacological, pan-omic analysis to expedite anti-glioma drugs' preclinical testing and develop predictive biomarker datasets that can aid in personalizing glioblastoma therapy and inform clinical trial design. Copyright © 2017 Elsevier Inc. All rights reserved.

Deep sequencing and in silico analysis of small RNA library reveals novel miRNA from leaf Persicaria minor transcriptome.

PubMed

Samad, Abdul Fatah A; Nazaruddin, Nazaruddin; Murad, Abdul Munir Abdul; Jani, Jaeyres; Zainal, Zamri; Ismail, Ismanizan

2018-03-01

In current era, majority of microRNA (miRNA) are being discovered through computational approaches which are more confined towards model plants. Here, for the first time, we have described the identification and characterization of novel miRNA in a non-model plant, Persicaria minor ( P . minor ) using computational approach. Unannotated sequences from deep sequencing were analyzed based on previous well-established parameters. Around 24 putative novel miRNAs were identified from 6,417,780 reads of the unannotated sequence which represented 11 unique putative miRNA sequences. PsRobot target prediction tool was deployed to identify the target transcripts of putative novel miRNAs. Most of the predicted target transcripts (mRNAs) were known to be involved in plant development and stress responses. Gene ontology showed that majority of the putative novel miRNA targets involved in cellular component (69.07%), followed by molecular function (30.08%) and biological process (0.85%). Out of 11 unique putative miRNAs, 7 miRNAs were validated through semi-quantitative PCR. These novel miRNAs discoveries in P . minor may develop and update the current public miRNA database.

Identification of Submergence-Responsive MicroRNAs and Their Targets Reveals Complex MiRNA-Mediated Regulatory Networks in Lotus (Nelumbo nucifera Gaertn)

PubMed Central

Jin, Qijiang; Xu, Yingchun; Mattson, Neil; Li, Xin; Wang, Bei; Zhang, Xiao; Jiang, Hongwei; Liu, Xiaojing; Wang, Yanjie; Yao, Dongrui

2017-01-01

MicroRNAs (miRNAs) are endogenous non-coding RNAs with important regulatory functions in plant development and stress responses. However, their population abundance in lotus (Nelumbo nucifera Gaertn) has so far been poorly described, particularly in response to stresses. In this work, submergence-related miRNAs and their target genes were systematically identified, compared, and validated at the transcriptome-wide level using high-throughput sequencing data of small RNA, Mrna, and the degradome. A total of 128 known and 20 novel miRNAs were differentially expressed upon submergence. We identified 629 target transcripts for these submergence-responsive miRNAs. Based on the miRNA expression profiles and GO and KEGG annotation of miRNA target genes, we suggest possible molecular responses and physiological changes of lotus in response to submergence. Several metabolic, physiological and morphological adaptations-related miRNAs, i.e., NNU_far-miR159, NNU_gma-miR393h, and NNU_aly-miR319c-3p, were found to play important regulatory roles in lotus response to submergence. This work will contribute to a better understanding of miRNA-regulated adaption responses of lotus to submergence stress. PMID:28149304

Identification of alsterpaullone as a novel small molecule inhibitor to target group 3 medulloblastoma.

PubMed

Faria, Claudia C; Agnihotri, Sameer; Mack, Stephen C; Golbourn, Brian J; Diaz, Roberto J; Olsen, Samantha; Bryant, Melissa; Bebenek, Matthew; Wang, Xin; Bertrand, Kelsey C; Kushida, Michelle; Head, Renee; Clark, Ian; Dirks, Peter; Smith, Christian A; Taylor, Michael D; Rutka, James T

2015-08-28

Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma.

Targeting IL-17 AND IL-17D receptors of rheumatoid arthritis using phytocompounds: A Molecular Docking study

NASA Astrophysics Data System (ADS)

Thabitha, A.; Thoufic Ali, A. M. Mohamed; Shweta Kumari, Singh; Rakhi; Swami, Varsha; Mohana Priya, A.; Sajitha Lulu, S.

2017-11-01

Rheumatoid arthritis (RA) is a chronic autoimmune condition of the connective tissue in synovial joints, characterized by inflammation which can lead to bone and cartilage destruction. IL-17 and IL-17D cytokines produced by a number of cell types, primarily promote pro-inflammatory immune responses and negative regulator in fibroblast growth factor signalling. Thus, the promising therapeutic strategies focus on targeting these cytokines, which has led to the identification of effective inhibitors. However, several studies focused on identifying the anti-arthritic potential of natural compounds. Therefore, in the present study we undertook in silico investigations to decipher the anti-inflammatory prospective of phytocompounds by targeting IL-17 and IL-17D cytokines using Patch Dock algorithm. Additionally, IL-17 and IL-17D proteins structure were modelled and validated for molecular docking study. Further, phytocompounds based on anti-inflammatory property were subjected to Lipinski filter and ADMET properties indicated that all of these compounds showed desirable drug-like criteria. The outcome of this investigation sheds light on the anti-inflammatory mechanism of phytocompounds by targeting IL-17 and IL-D for effective treatment of RA.

Kepler Data Validation I—Architecture, Diagnostic Tests, and Data Products for Vetting Transiting Planet Candidates

NASA Astrophysics Data System (ADS)

Twicken, Joseph D.; Catanzarite, Joseph H.; Clarke, Bruce D.; Girouard, Forrest; Jenkins, Jon M.; Klaus, Todd C.; Li, Jie; McCauliff, Sean D.; Seader, Shawn E.; Tenenbaum, Peter; Wohler, Bill; Bryson, Stephen T.; Burke, Christopher J.; Caldwell, Douglas A.; Haas, Michael R.; Henze, Christopher E.; Sanderfer, Dwight T.

2018-06-01

The Kepler Mission was designed to identify and characterize transiting planets in the Kepler Field of View and to determine their occurrence rates. Emphasis was placed on identification of Earth-size planets orbiting in the Habitable Zone of their host stars. Science data were acquired for a period of four years. Long-cadence data with 29.4 min sampling were obtained for ∼200,000 individual stellar targets in at least one observing quarter in the primary Kepler Mission. Light curves for target stars are extracted in the Kepler Science Data Processing Pipeline, and are searched for transiting planet signatures. A Threshold Crossing Event is generated in the transit search for targets where the transit detection threshold is exceeded and transit consistency checks are satisfied. These targets are subjected to further scrutiny in the Data Validation (DV) component of the Pipeline. Transiting planet candidates are characterized in DV, and light curves are searched for additional planets after transit signatures are modeled and removed. A suite of diagnostic tests is performed on all candidates to aid in discrimination between genuine transiting planets and instrumental or astrophysical false positives. Data products are generated per target and planet candidate to document and display transiting planet model fit and diagnostic test results. These products are exported to the Exoplanet Archive at the NASA Exoplanet Science Institute, and are available to the community. We describe the DV architecture and diagnostic tests, and provide a brief overview of the data products. Transiting planet modeling and the search for multiple planets on individual targets are described in a companion paper. The final revision of the Kepler Pipeline code base is available to the general public through GitHub. The Kepler Pipeline has also been modified to support the Transiting Exoplanet Survey Satellite (TESS) Mission which is expected to commence in 2018.

Kepler Data Validation I: Architecture, Diagnostic Tests, and Data Products for Vetting Transiting Planet Candidates

NASA Technical Reports Server (NTRS)

Twicken, Joseph D.; Catanzarite, Joseph H.; Clarke, Bruce D.; Giroud, Forrest; Jenkins, Jon M.; Klaus, Todd C.; Li, Jie; McCauliff, Sean D.; Seader, Shawn E.; Tennenbaum, Peter;

A Study of the Validity of Language Usage as an Indicator of Ethnic Identification.

ERIC Educational Resources Information Center

Patella, Victoria Morrow

In determining whether Spanish usage is a valid indicator of ethnic identification, 669 Texas high school sophomores (3 Negroes, 70 Anglos, and 596 Mexican Americans) were interviewed. Mexican American respondents were then isolated on the basis of response to 4 questions, and their responses regarding family were grouped under 2 headings: (1)…

Computational Acoustic Beamforming for Noise Source Identification for Small Wind Turbines

PubMed Central

Lien, Fue-Sang

2017-01-01

This paper develops a computational acoustic beamforming (CAB) methodology for identification of sources of small wind turbine noise. This methodology is validated using the case of the NACA 0012 airfoil trailing edge noise. For this validation case, the predicted acoustic maps were in excellent conformance with the results of the measurements obtained from the acoustic beamforming experiment. Following this validation study, the CAB methodology was applied to the identification of noise sources generated by a commercial small wind turbine. The simulated acoustic maps revealed that the blade tower interaction and the wind turbine nacelle were the two primary mechanisms for sound generation for this small wind turbine at frequencies between 100 and 630 Hz. PMID:28378012

Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings.

PubMed

Loi, Monica; Di Paolo, Daniela; Soster, Marco; Brignole, Chiara; Bartolini, Alice; Emionite, Laura; Sun, Jessica; Becherini, Pamela; Curnis, Flavio; Petretto, Andrea; Sani, Monica; Gori, Alessandro; Milanese, Marco; Gambini, Claudio; Longhi, Renato; Cilli, Michele; Allen, Theresa M; Bussolino, Federico; Arap, Wadih; Pasqualini, Renata; Corti, Angelo; Ponzoni, Mirco; Marchiò, Serena; Pastorino, Fabio

2013-09-10

Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB. By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Real-Time Motion Tracking for Indoor Moving Sphere Objects with a LiDAR Sensor.

PubMed

Huang, Lvwen; Chen, Siyuan; Zhang, Jianfeng; Cheng, Bang; Liu, Mingqing

2017-08-23

Object tracking is a crucial research subfield in computer vision and it has wide applications in navigation, robotics and military applications and so on. In this paper, the real-time visualization of 3D point clouds data based on the VLP-16 3D Light Detection and Ranging (LiDAR) sensor is achieved, and on the basis of preprocessing, fast ground segmentation, Euclidean clustering segmentation for outliers, View Feature Histogram (VFH) feature extraction, establishing object models and searching matching a moving spherical target, the Kalman filter and adaptive particle filter are used to estimate in real-time the position of a moving spherical target. The experimental results show that the Kalman filter has the advantages of high efficiency while adaptive particle filter has the advantages of high robustness and high precision when tested and validated on three kinds of scenes under the condition of target partial occlusion and interference, different moving speed and different trajectories. The research can be applied in the natural environment of fruit identification and tracking, robot navigation and control and other fields.

No arousal-biased competition in focused visuospatial attention.

PubMed

Ásgeirsson, Árni Gunnar; Nieuwenhuis, Sander

2017-11-01

Arousal sometimes enhances and sometimes impairs perception and memory. A recent theory attempts to reconcile these findings by proposing that arousal amplifies the competition between stimulus representations, strengthening already strong representations and weakening already weak representations. Here, we report a stringent test of this arousal-biased competition theory in the context of focused visuospatial attention. Participants were required to identify a briefly presented target in the context of multiple distractors, which varied in the degree to which they competed for representation with the target, as revealed by psychophysics. We manipulated arousal using emotionally arousing pictures (Experiment 1), alerting tones (Experiment 2) and white-noise stimulation (Experiment 3), and validated these manipulations with electroencephalography and pupillometry. In none of the experiments did we find evidence that arousal modulated the effect of distractor competition on the accuracy of target identification. Bayesian statistics revealed moderate to strong evidence against arousal-biased competition. Modeling of the psychophysical data based on Bundesen's (1990) theory of visual attention corroborated the conclusion that arousal does not bias competition in focused visuospatial attention. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-Time Motion Tracking for Indoor Moving Sphere Objects with a LiDAR Sensor

PubMed Central

Chen, Siyuan; Zhang, Jianfeng; Cheng, Bang; Liu, Mingqing

2017-01-01

Object tracking is a crucial research subfield in computer vision and it has wide applications in navigation, robotics and military applications and so on. In this paper, the real-time visualization of 3D point clouds data based on the VLP-16 3D Light Detection and Ranging (LiDAR) sensor is achieved, and on the basis of preprocessing, fast ground segmentation, Euclidean clustering segmentation for outliers, View Feature Histogram (VFH) feature extraction, establishing object models and searching matching a moving spherical target, the Kalman filter and adaptive particle filter are used to estimate in real-time the position of a moving spherical target. The experimental results show that the Kalman filter has the advantages of high efficiency while adaptive particle filter has the advantages of high robustness and high precision when tested and validated on three kinds of scenes under the condition of target partial occlusion and interference, different moving speed and different trajectories. The research can be applied in the natural environment of fruit identification and tracking, robot navigation and control and other fields. PMID:28832520

A real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens.

PubMed

De Luca, Eliana; Crisi, Paolo Emidio; Di Domenico, Marco; Malatesta, Daniela; Vincifori, Giacomo; Di Tommaso, Morena; Di Guardo, Giovanni; Di Francesco, Gabriella; Petrini, Antonio; Savini, Giovanni; Boari, Andrea; Lorusso, Alessio

2018-05-03

The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed. RNA titres were low in all tested samples. Results also indicated the absence of cross-reaction with related morbilliviruses and existing pathogens of cats. In tissues with low levels of FeMV RNA, the presence of viral antigen was also evidenced by immunohistochemistry targeting the N viral protein. This newly described assay allows for a rapid, accurate and reliable quantitative detection of FeMV RNA that can be applied for diagnostics and research studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Appearance-change instruction effects on eyewitness lineup identification accuracy are not moderated by amount of appearance change.

PubMed

Molinaro, Peter F; Arndorfer, Andrea; Charman, Steve D

2013-12-01

Instructing witnesses that a criminal may have changed appearance prior to showing them a lineup has been shown to increase false identifications without increasing correct identifications (S. D. Charman & G. L. Wells, 2007, Is the appearance-change instruction a good idea? Law and Human Behavior, Vol. 31, pp. 3-22). However, the generalizability of the effects of this appearance-change instruction (ACI) is unknown, and there are reasons to believe that the instruction's effects may be moderated by the amount of appearance change the criminal has actually undergone. The current study tested this hypothesis. Undergraduate students (N = 315) were exposed to a series of target faces and lineups, some of which contained the target and some of which did not, and made identification decisions. Half of the participants received a standard ACI prior to each lineup; the other half did not. The targets varied with respect to the amount to which their appearance had changed. Results indicated that the ACI inflated false identifications without inflating correct identifications, and that these effects did not depend on the amount of appearance change the target had undergone. Current recommendations to administer the ACI seem to be unfounded and may be harmful to the reliability of identification evidence. (c) 2013 APA, all rights reserved

Test-retest reliability and validity of the Sniffin' TOM odor memory test.

PubMed

Croy, Ilona; Zehner, Cora; Larsson, Maria; Zucco, Gesualdo M; Hummel, Thomas

2015-03-01

Few attempts have been made to develop an olfactory test that captures episodic retention of olfactory information. Assessment of episodic odor memory is of particular interest in aging and in the cognitively impaired as both episodic memory deficits and olfactory loss have been targeted as reliable hallmarks of cognitive decline and impending dementia. Here, 96 healthy participants (18-92 years) and an additional 19 older people with mild cognitive impairment were tested (73-82 years). Participants were presented with 8 common odors with intentional encoding instructions that were followed by a yes-no recognition test. After recognition completion, participants were asked to identify all odors by means of free or cued identification. A retest of the odor memory test (Sniffin' TOM = test of odor memory) took place 17 days later. The results revealed satisfactory test-retest reliability (0.70) of odor recognition memory. Both recognition and identification performance were negatively affected by age and more pronounced among the cognitively impaired. In conclusion, the present work presents a reliable, valid, and simple test of episodic odor recognition memory that may be used in clinical groups where both episodic memory deficits and olfactory loss are prevalent preclinically such as Alzheimer's disease. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

False discovery rates in spectral identification.

PubMed

Jeong, Kyowon; Kim, Sangtae; Bandeira, Nuno

2012-01-01

Automated database search engines are one of the fundamental engines of high-throughput proteomics enabling daily identifications of hundreds of thousands of peptides and proteins from tandem mass (MS/MS) spectrometry data. Nevertheless, this automation also makes it humanly impossible to manually validate the vast lists of resulting identifications from such high-throughput searches. This challenge is usually addressed by using a Target-Decoy Approach (TDA) to impose an empirical False Discovery Rate (FDR) at a pre-determined threshold x% with the expectation that at most x% of the returned identifications would be false positives. But despite the fundamental importance of FDR estimates in ensuring the utility of large lists of identifications, there is surprisingly little consensus on exactly how TDA should be applied to minimize the chances of biased FDR estimates. In fact, since less rigorous TDA/FDR estimates tend to result in more identifications (at higher 'true' FDR), there is often little incentive to enforce strict TDA/FDR procedures in studies where the major metric of success is the size of the list of identifications and there are no follow up studies imposing hard cost constraints on the number of reported false positives. Here we address the problem of the accuracy of TDA estimates of empirical FDR. Using MS/MS spectra from samples where we were able to define a factual FDR estimator of 'true' FDR we evaluate several popular variants of the TDA procedure in a variety of database search contexts. We show that the fraction of false identifications can sometimes be over 10× higher than reported and may be unavoidably high for certain types of searches. In addition, we further report that the two-pass search strategy seems the most promising database search strategy. While unavoidably constrained by the particulars of any specific evaluation dataset, our observations support a series of recommendations towards maximizing the number of resulting identifications while controlling database searches with robust and reproducible TDA estimation of empirical FDR.

Target-decoy Based False Discovery Rate Estimation for Large-scale Metabolite Identification.

PubMed

Wang, Xusheng; Jones, Drew R; Shaw, Timothy I; Cho, Ji-Hoon; Wang, Yuanyuan; Tan, Haiyan; Xie, Boer; Zhou, Suiping; Li, Yuxin; Peng, Junmin

2018-05-23

Metabolite identification is a crucial step in mass spectrometry (MS)-based metabolomics. However, it is still challenging to assess the confidence of assigned metabolites. In this study, we report a novel method for estimating false discovery rate (FDR) of metabolite assignment with a target-decoy strategy, in which the decoys are generated through violating the octet rule of chemistry by adding small odd numbers of hydrogen atoms. The target-decoy strategy was integrated into JUMPm, an automated metabolite identification pipeline for large-scale MS analysis, and was also evaluated with two other metabolomics tools, mzMatch and mzMine 2. The reliability of FDR calculation was examined by false datasets, which were simulated by altering MS1 or MS2 spectra. Finally, we used the JUMPm pipeline coupled with the target-decoy strategy to process unlabeled and stable-isotope labeled metabolomic datasets. The results demonstrate that the target-decoy strategy is a simple and effective method for evaluating the confidence of high-throughput metabolite identification.

Is facial skin tone sufficient to produce a cross-racial identification effect?

PubMed

Alley, T R; Schultheis, J A

2001-06-01

Research clearly supports the existence of an other race effect for human faces whereby own-race faces are more accurately perceived and recognized. Why this occurs remains unclear. A computerized program (Mac-a-Mug Pro) for face composition was used to create pairs of target and distractor faces that differed only in skin tone. The six target faces were rated on honesty and aggressiveness by 72 university students, with just one 'Black' and one 'White' face viewed by each student. One week later, they attempted to identify these faces in four lineups: two with target-present and two with target-absent. The order of presentation of targets, lineups, and faces within lineups was varied. Own-race identification was slightly better than cross-racial identification. There was no significant difference in the confidence of responses to own- versus other-race faces. These results indicate that neither morphological variation nor differential confidence is necessary for a cross-racial identification effect.

Identification of (antioxidative) plants in herbal pharmaceutical preparations and dietary supplements.

PubMed

Deconinck, Eric; Custers, Deborah; De Beer, Jacques Omer

2015-01-01

The standard procedures for the identification, authentication, and quality control of medicinal plants and herbs are nowadays limited to pure herbal products. No guidelines or procedures, describing the detection or identification of a targeted plant or herb in pharmaceutical preparations or dietary supplements, can be found. In these products the targeted plant is often present together with other components of herbal or synthetic origin. This chapter describes a strategy for the fast development of a chromatographic fingerprint approach that allows the identification of a targeted plant in herbal preparations and dietary supplements. The strategy consists of a standard chromatographic gradient that is tested for the targeted plant with different extraction solvents and different mobile phases. From the results obtained, the optimal fingerprint is selected. Subsequently the samples are analyzed according to the selected methodological parameters, and the obtained fingerprints can be compared with the one obtained for the pure herbal product or a standard preparation. Calculation of the dissimilarity between these fingerprints will result in a probability of presence of the targeted plant. Optionally mass spectrometry can be used to improve specificity, to confirm identification, or to identify molecules with a potential medicinal or antioxidant activity.

A post-gene silencing bioinformatics protocol for plant-defence gene validation and underlying process identification: case study of the Arabidopsis thaliana NPR1.

PubMed

Yocgo, Rosita E; Geza, Ephifania; Chimusa, Emile R; Mazandu, Gaston K

2017-11-23

Advances in forward and reverse genetic techniques have enabled the discovery and identification of several plant defence genes based on quantifiable disease phenotypes in mutant populations. Existing models for testing the effect of gene inactivation or genes causing these phenotypes do not take into account eventual uncertainty of these datasets and potential noise inherent in the biological experiment used, which may mask downstream analysis and limit the use of these datasets. Moreover, elucidating biological mechanisms driving the induced disease resistance and influencing these observable disease phenotypes has never been systematically tackled, eliciting the need for an efficient model to characterize completely the gene target under consideration. We developed a post-gene silencing bioinformatics (post-GSB) protocol which accounts for potential biases related to the disease phenotype datasets in assessing the contribution of the gene target to the plant defence response. The post-GSB protocol uses Gene Ontology semantic similarity and pathway dataset to generate enriched process regulatory network based on the functional degeneracy of the plant proteome to help understand the induced plant defence response. We applied this protocol to investigate the effect of the NPR1 gene silencing to changes in Arabidopsis thaliana plants following Pseudomonas syringae pathovar tomato strain DC3000 infection. Results indicated that the presence of a functionally active NPR1 reduced the plant's susceptibility to the infection, with about 99% of variability in Pseudomonas spore growth between npr1 mutant and wild-type samples. Moreover, the post-GSB protocol has revealed the coordinate action of target-associated genes and pathways through an enriched process regulatory network, summarizing the potential target-based induced disease resistance mechanism. This protocol can improve the characterization of the gene target and, potentially, elucidate induced defence response by more effectively utilizing available phenotype information and plant proteome functional knowledge.

ARI Image Interpretation Research: 1970-1980

DTIC Science & Technology

1980-07-01

28 3. Index marks on data base stereo pair ... .......... .. 41 4. Identification learning curves for three methods used by interpreters of high...it may be impractical in operational units (but not in the school). Team consensus feedback can increase target identification proficiency and...in target identification can be provided with a minimum of instructor participation using operational imagery as the basic instructional material

Identification of Loci Associated with Drought Resistance Traits in Heterozygous Autotetraploid Alfalfa (Medicago sativa L.) Using Genome-Wide Association Studies with Genotyping by Sequencing.

PubMed

Zhang, Tiejun; Yu, Long-Xi; Zheng, Ping; Li, Yajun; Rivera, Martha; Main, Dorrie; Greene, Stephanie L

2015-01-01

Drought resistance is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. Identification of genes involved in drought tolerance will facilitate breeding for improving drought resistance and water use efficiency in alfalfa. Our objective was to use a diversity panel of alfalfa accessions comprised of 198 cultivars and landraces to identify genes involved in drought tolerance. The panel was selected from the USDA-ARS National Plant Germplasm System alfalfa collection and genotyped using genotyping by sequencing. A greenhouse procedure was used for phenotyping two important traits associated with drought tolerance: drought resistance index (DRI) and relative leaf water content (RWC). Marker-trait association identified nineteen and fifteen loci associated with DRI and RWC, respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. Markers associated with DRI are located on all chromosomes while markers associated with RWC are located on chromosomes 1, 2, 3, 4, 5, 6 and 7. Co-localizations of significant markers between DRI and RWC were found on chromosomes 3, 5 and 7. Most loci associated with DRI in this work overlap with the reported QTLs associated with biomass under drought in alfalfa. Additional significant markers were targeted to several contigs with unknown chromosomal locations. BLAST search using their flanking sequences revealed homology to several annotated genes with functions in stress tolerance. With further validation, these markers may be used for marker-assisted breeding new alfalfa varieties with drought resistance and enhanced water use efficiency.

The Statistical Analysis Techniques to Support the NGNP Fuel Performance Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bihn T. Pham; Jeffrey J. Einerson

2010-06-01

This paper describes the development and application of statistical analysis techniques to support the AGR experimental program on NGNP fuel performance. The experiments conducted in the Idaho National Laboratory’s Advanced Test Reactor employ fuel compacts placed in a graphite cylinder shrouded by a steel capsule. The tests are instrumented with thermocouples embedded in graphite blocks and the target quantity (fuel/graphite temperature) is regulated by the He-Ne gas mixture that fills the gap volume. Three techniques for statistical analysis, namely control charting, correlation analysis, and regression analysis, are implemented in the SAS-based NGNP Data Management and Analysis System (NDMAS) for automatedmore » processing and qualification of the AGR measured data. The NDMAS also stores daily neutronic (power) and thermal (heat transfer) code simulation results along with the measurement data, allowing for their combined use and comparative scrutiny. The ultimate objective of this work includes (a) a multi-faceted system for data monitoring and data accuracy testing, (b) identification of possible modes of diagnostics deterioration and changes in experimental conditions, (c) qualification of data for use in code validation, and (d) identification and use of data trends to support effective control of test conditions with respect to the test target. Analysis results and examples given in the paper show the three statistical analysis techniques providing a complementary capability to warn of thermocouple failures. It also suggests that the regression analysis models relating calculated fuel temperatures and thermocouple readings can enable online regulation of experimental parameters (i.e. gas mixture content), to effectively maintain the target quantity (fuel temperature) within a given range.« less

A targeted metabolomics approach for clinical diagnosis of inborn errors of metabolism.

PubMed

Jacob, Minnie; Malkawi, Abeer; Albast, Nour; Al Bougha, Salam; Lopata, Andreas; Dasouki, Majed; Abdel Rahman, Anas M

2018-09-26

Metabolome, the ultimate functional product of the genome, can be studied through identification and quantification of small molecules. The global metabolome influences the individual phenotype through clinical and environmental interventions. Metabolomics has become an integral part of clinical research and allowed for another dimension of better understanding of disease pathophysiology and mechanism. More than 95% of the clinical biochemistry laboratory routine workload is based on small molecular identification, which can potentially be analyzed through metabolomics. However, multiple challenges in clinical metabolomics impact the entire workflow and data quality, thus the biological interpretation needs to be standardized for a reproducible outcome. Herein, we introduce the establishment of a comprehensive targeted metabolomics method for a panel of 220 clinically relevant metabolites using Liquid chromatography-tandem mass spectrometry (LC-MS/MS) standardized for clinical research. The sensitivity, reproducibility and molecular stability of each targeted metabolite (amino acids, organic acids, acylcarnitines, sugars, bile acids, neurotransmitters, polyamines, and hormones) were assessed under multiple experimental conditions. The metabolic tissue distribution was determined in various rat organs. Furthermore, the method was validated in dry blood spot (DBS) samples collected from patients known to have various inborn errors of metabolism (IEMs). Using this approach, our panel appears to be sensitive and robust as it demonstrated differential and unique metabolic profiles in various rat tissues. Also, as a prospective screening method, this panel of diverse metabolites has the ability to identify patients with a wide range of IEMs who otherwise may need multiple, time-consuming and expensive biochemical assays causing a delay in clinical management. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of Molecular Targets for 4,5-Dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) in Teleosts: New Insight into Mechanism of Toxicity.

PubMed

Chen, Lianguo; Au, Doris W T; Hu, Chenyan; Peterson, Drew R; Zhou, Bingsheng; Qian, Pei-Yuan

2017-02-07

Environmental pollutants are capable of concomitantly inducing diverse toxic effects. However, it is largely unknown which effects are directly induced and which effects are secondary, thus calling for definitive identification of the initiating molecular event for a pollutant to elucidate the mechanism of toxicity. In the present study, affinity pull-down assays were used to identify target proteins for 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT), a costal pollutant of emerging concern, in various tissues (e.g., brain, liver, plasma, and gonad) from marine medaka (Oryzias melastigma) and zebrafish (Danio rerio). Pull-down results showed that, in male and female brains from medaka and zebrafish, DCOIT had a consistently high affinity for G protein alpha subunits (Gα), suggesting the targeted effects of DCOIT on signaling transduction from G protein-coupled receptors (GPCRs) and an extrapolatable mode of action in teleost brains. Validation using recombinant proteins and molecular docking analysis confirmed that binding of DCOIT to Gα protein competitively inhibited its activation by substrate. Considering the involvement of GPCRs in the regulation of myriad biological processes, including the hypothalamus-pituitary-gonadal-liver axis, binding of DCOIT to upstream Gα proteins in the brain may provide a plausible explanation for the diversity of toxic effects resulting from DCOIT challenge, especially abnormal hormonal production through the mitogen-activated protein kinase pathway. A new mechanism of action based on GPCR signaling is thus hypothesized for endocrine disrupting chemicals and warrants further research to clearly elucidate the link between GPCR signaling and endocrine disruption.

The Effects of Signal Erosion and Core Genome Reduction on the Identification of Diagnostic Markers

PubMed Central

Sahl, Jason W.; Vazquez, Adam J.; Hall, Carina M.; Busch, Joseph D.; Tuanyok, Apichai; Mayo, Mark; Schupp, James M.; Lummis, Madeline; Pearson, Talima; Shippy, Kenzie; Allender, Christopher J.; Theobald, Vanessa; Hutcheson, Alex; Korlach, Jonas; LiPuma, John J.; Ladner, Jason; Lovett, Sean; Koroleva, Galina; Palacios, Gustavo; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Wongsuwan, Gumphol; Currie, Bart J.

2016-01-01

ABSTRACT Whole-genome sequence (WGS) data are commonly used to design diagnostic targets for the identification of bacterial pathogens. To do this effectively, genomics databases must be comprehensive to identify the strict core genome that is specific to the target pathogen. As additional genomes are analyzed, the core genome size is reduced and there is erosion of the target-specific regions due to commonality with related species, potentially resulting in the identification of false positives and/or false negatives. PMID:27651357

System Identification Methods for Aircraft Flight Control Development and Validation

DOT National Transportation Integrated Search

1995-10-01

System-identification methods compose a mathematical model, or series of models, : from measurements of inputs and outputs of dynamic systems. This paper : discusses the use of frequency-domain system-identification methods for the : development and ...

Electro-Optic Identification Research Program

DTIC Science & Technology

2002-04-01

Electro - optic identification (EOID) sensors provide photographic quality images that can be used to identify mine-like contacts provided by long...tasks such as validating existing electro - optic models, development of performance metrics, and development of computer aided identification and

Measuring Critical Care Providers' Attitudes About Controlled Donation After Circulatory Death.

PubMed

Rodrigue, James R; Luskin, Richard; Nelson, Helen; Glazier, Alexandra; Henderson, Galen V; Delmonico, Francis L

2018-06-01

Unfavorable attitudes and insufficient knowledge about donation after cardiac death among critical care providers can have important consequences for the appropriate identification of potential donors, consistent implementation of donation after cardiac death policies, and relative strength of support for this type of donation. The lack of reliable and valid assessment measures has hampered research to capture providers' attitudes. Design and Research Aims: Using stakeholder engagement and an iterative process, we developed a questionnaire to measure attitudes of donation after cardiac death in critical care providers (n = 112) and examined its psychometric properties. Exploratory factor analysis, internal consistency, and validity analyses were conducted to examine the measure. A 34-item questionnaire consisting of 4 factors (Personal Comfort, Process Satisfaction, Family Comfort, and System Trust) provided the most parsimonious fit. Internal consistency was acceptable for each of the subscales and the total questionnaire (Cronbach α > .70). A strong association between more favorable attitudes overall and knowledge ( r = .43, P < .001) provides evidence of convergent validity. Multivariable regression analyses showed that white race ( P = .002) and more experience with donation after cardiac death ( P < .001) were significant predictors of more favorable attitudes. Study findings support the utility, reliability, and validity of a questionnaire for measuring attitudes in critical care providers and for isolating targets for additional education on donation after cardiac death.

Food for Thought ... Mechanistic Validation

PubMed Central

Hartung, Thomas; Hoffmann, Sebastian; Stephens, Martin

2013-01-01

Summary Validation of new approaches in regulatory toxicology is commonly defined as the independent assessment of the reproducibility and relevance (the scientific basis and predictive capacity) of a test for a particular purpose. In large ring trials, the emphasis to date has been mainly on reproducibility and predictive capacity (comparison to the traditional test) with less attention given to the scientific or mechanistic basis. Assessing predictive capacity is difficult for novel approaches (which are based on mechanism), such as pathways of toxicity or the complex networks within the organism (systems toxicology). This is highly relevant for implementing Toxicology for the 21st Century, either by high-throughput testing in the ToxCast/ Tox21 project or omics-based testing in the Human Toxome Project. This article explores the mostly neglected assessment of a test's scientific basis, which moves mechanism and causality to the foreground when validating/qualifying tests. Such mechanistic validation faces the problem of establishing causality in complex systems. However, pragmatic adaptations of the Bradford Hill criteria, as well as bioinformatic tools, are emerging. As critical infrastructures of the organism are perturbed by a toxic mechanism we argue that by focusing on the target of toxicity and its vulnerability, in addition to the way it is perturbed, we can anchor the identification of the mechanism and its verification. PMID:23665802

Colorimetric detection with aptamer-gold nanoparticle conjugates coupled to an android-based color analysis application for use in the field.

PubMed

Smith, Joshua E; Griffin, Daniel K; Leny, Juliann K; Hagen, Joshua A; Chávez, Jorge L; Kelley-Loughnane, Nancy

2014-04-01

The feasibility of using aptamer-gold nanoparticle conjugates (Apt-AuNPs) to design colorimetric assays for in the field detection of small molecules was investigated. An assay to detect cocaine was designed using two clones of a known cocaine-binding aptamer. The assay was based on the AuNPs difference in affinity for single-stranded DNA (non-binding) and double stranded DNA (target bound). In the first assay, a commonly used design was followed, in which the aptamer and target were incubated to allow binding followed by exposure to the AuNPs. Interactions between the non-bound analytes and the AuNPs surface resulted in a number of false positives. The assay was redesigned by incubating the AuNPs and the aptamer prior to target addition to passivate the AuNPs surface. The adsorbed aptamer was able to bind the target while preventing non-specific interactions. The assay was validated with a number of masking and cutting agents and other controlled substances showing minimal false positives. Studies to improve the assay performance in the field were performed, showing that assay activity could be preserved for up to 2 months. To facilitate the assay analysis, an android application for automatic colorimetric characterization was developed. The application was validated by challenging the assay with cocaine standards of different concentrations, and comparing the results to a conventional plate reader, showing outstanding agreement. Finally, the rapid identification of cocaine in mixtures mimicking street samples was demonstrated. This work established that Apt-AuNPs can be used to design robust assays to be used in the field. Copyright © 2013 Elsevier B.V. All rights reserved.

Computational exploration of microRNAs from expressed sequence tags of Humulus lupulus, target predictions and expression analysis.

PubMed

Mishra, Ajay Kumar; Duraisamy, Ganesh Selvaraj; Týcová, Anna; Matoušek, Jaroslav

2015-12-01

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental-in silico computational based approach was adopted for the identification and characterization of miRNAs in Humulus lupulus (hop), which is widely cultivated for use by the brewing industry and apart from, used as a medicinal herb. A total of 22 miRNAs belonging to 17 miRNA families were identified in hop following comparative computational approach and EST-based homology search according to a series of filtering criteria. Selected miRNAs were validated by end-point PCR and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), confirmed the existence of conserved miRNAs in hop. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 47 potential miRNA targets were identified in hop. Strikingly, the majority of predicted targets were belong to transcriptional factors which could regulate hop growth and development, including leaf, root and even cone development. Moreover, the identified miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and other physiological processes. The cis-regulatory elements relevant to biotic and abiotic stress, plant hormone response, flavonoid biosynthesis were identified in the promoter regions of those miRNA genes. Overall, findings from this study will accelerate the way for further researches of miRNAs, their functions in hop and shows a path for the prediction and analysis of miRNAs to those species whose genomes are not available. Copyright © 2015 Elsevier Ltd. All rights reserved.

Computational drug discovery

PubMed Central

Ou-Yang, Si-sheng; Lu, Jun-yan; Kong, Xiang-qian; Liang, Zhong-jie; Luo, Cheng; Jiang, Hualiang

2012-01-01

Computational drug discovery is an effective strategy for accelerating and economizing drug discovery and development process. Because of the dramatic increase in the availability of biological macromolecule and small molecule information, the applicability of computational drug discovery has been extended and broadly applied to nearly every stage in the drug discovery and development workflow, including target identification and validation, lead discovery and optimization and preclinical tests. Over the past decades, computational drug discovery methods such as molecular docking, pharmacophore modeling and mapping, de novo design, molecular similarity calculation and sequence-based virtual screening have been greatly improved. In this review, we present an overview of these important computational methods, platforms and successful applications in this field. PMID:22922346

The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology.

PubMed

McDonald, Peter R; Roy, Anuradha; Chaguturu, Rathnam

2011-07-01

The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory.

Screening and confirmation of microRNA markers for forensic body fluid identification.

PubMed

Wang, Zheng; Zhang, Ji; Luo, Haibo; Ye, Yi; Yan, Jing; Hou, Yiping

2013-01-01

MicroRNAs (miRNAs, ∼22 nucleotides) are small, non-protein coding RNAs that regulate gene expression at the post-transcriptional level. MiRNAs can express in a tissue-specific manner, and have been introduced to forensic body fluid identification. In this study, we employed the qPCR-array (TaqMan(®) Array Human MicroRNA Cards) to screen the body fluid-specific miRNAs. Seven candidate miRNAs were identified as potentially body fluid-specific and could be used as forensically relevant body fluid markers: miR16 and miR486 for venous blood, miR888 and miR891a for semen, miR214 for menstrual blood, miR124a for vaginal secretions, and miR138-2 for saliva. The candidate miRNA markers were then validated via hydrolysis probes quantitative real-time polymerase chain reaction (TaqMan-qPCR). In addition, BestKeeper software was used to validate the expression stability of four genes, RNU44, RNU48, U6 and U6b, regularly used as reference genes (RGs) for studies involving forensic body fluids. The current study suggests that U6 could be used as a proper RG of miRNAs in forensic body fluid identification. The relative expression ratios (R) of miR486, miR888, miR214, miR16 and miR891a can differentiate the target body fluid from other body fluids that were tested in this study. The detection limit of TaqMan-qPCR of the five confirmed miRNA markers was 10pg of total RNA. The effect of time-wise degradation of blood stains and semen stains for 1 month under normal laboratory conditions was tested and did not significantly affect the detection results. Herein, this study proposes five body fluid-specific miRNAs for the forensic identification of venous blood, semen, and menstrual blood, of which miR486, miR888, and miR214 may be used as new markers for body fluid identification. Additional work remains necessary in search for suitable miRNA markers and stable RGs for forensic body fluid identification. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

A Scalable Approach for Protein False Discovery Rate Estimation in Large Proteomic Data Sets

PubMed Central

Savitski, Mikhail M.; Wilhelm, Mathias; Hahne, Hannes; Kuster, Bernhard; Bantscheff, Marcus

2015-01-01

Calculating the number of confidently identified proteins and estimating false discovery rate (FDR) is a challenge when analyzing very large proteomic data sets such as entire human proteomes. Biological and technical heterogeneity in proteomic experiments further add to the challenge and there are strong differences in opinion regarding the conceptual validity of a protein FDR and no consensus regarding the methodology for protein FDR determination. There are also limitations inherent to the widely used classic target–decoy strategy that particularly show when analyzing very large data sets and that lead to a strong over-representation of decoy identifications. In this study, we investigated the merits of the classic, as well as a novel target–decoy-based protein FDR estimation approach, taking advantage of a heterogeneous data collection comprised of ∼19,000 LC-MS/MS runs deposited in ProteomicsDB (https://www.proteomicsdb.org). The “picked” protein FDR approach treats target and decoy sequences of the same protein as a pair rather than as individual entities and chooses either the target or the decoy sequence depending on which receives the highest score. We investigated the performance of this approach in combination with q-value based peptide scoring to normalize sample-, instrument-, and search engine-specific differences. The “picked” target–decoy strategy performed best when protein scoring was based on the best peptide q-value for each protein yielding a stable number of true positive protein identifications over a wide range of q-value thresholds. We show that this simple and unbiased strategy eliminates a conceptual issue in the commonly used “classic” protein FDR approach that causes overprediction of false-positive protein identification in large data sets. The approach scales from small to very large data sets without losing performance, consistently increases the number of true-positive protein identifications and is readily implemented in proteomics analysis software. PMID:25987413

Human target acquisition performance

NASA Astrophysics Data System (ADS)

Teaney, Brian P.; Du Bosq, Todd W.; Reynolds, Joseph P.; Thompson, Roger; Aghera, Sameer; Moyer, Steven K.; Flug, Eric; Espinola, Richard; Hixson, Jonathan

2012-06-01

The battlefield has shifted from armored vehicles to armed insurgents. Target acquisition (identification, recognition, and detection) range performance involving humans as targets is vital for modern warfare. The acquisition and neutralization of armed insurgents while at the same time minimizing fratricide and civilian casualties is a mounting concern. U.S. Army RDECOM CERDEC NVESD has conducted many experiments involving human targets for infrared and reflective band sensors. The target sets include human activities, hand-held objects, uniforms & armament, and other tactically relevant targets. This paper will define a set of standard task difficulty values for identification and recognition associated with human target acquisition performance.

Simulation of target interpretation based on infrared image features and psychology principle

NASA Astrophysics Data System (ADS)

Lin, Wei; Chen, Yu-hua; Gao, Hong-sheng; Wang, Zhan-feng; Wang, Ji-jun; Su, Rong-hua; Huang, Yan-ping

2009-07-01

It's an important and complicated process in target interpretation that target features extraction and identification, which effect psychosensorial quantity of interpretation person to target infrared image directly, and decide target viability finally. Using statistical decision theory and psychology principle, designing four psychophysical experiment, the interpretation model of the infrared target is established. The model can get target detection probability by calculating four features similarity degree between target region and background region, which were plotted out on the infrared image. With the verification of a great deal target interpretation in practice, the model can simulate target interpretation and detection process effectively, get the result of target interpretation impersonality, which can provide technique support for target extraction, identification and decision-making.

Inter-Rater Reliability and Validity of the Australian Football League’s Kicking and Handball Tests

PubMed Central

Cripps, Ashley J.; Hopper, Luke S.; Joyce, Christopher

2015-01-01

Talent identification tests used at the Australian Football League’s National Draft Combine assess the capacities of athletes to compete at a professional level. Tests created for the National Draft Combine are also commonly used for talent identification and athlete development in development pathways. The skills tests created by the Australian Football League required players to either handball (striking the ball with the hand) or kick to a series of 6 randomly generated targets. Assessors subjectively rate each skill execution giving a 0-5 score for each disposal. This study aimed to investigate the inter-rater reliability and validity of the skills tests at an adolescent sub-elite level. Male Australian footballers were recruited from sub-elite adolescent teams (n = 121, age = 15.7 ± 0.3 years, height = 1.77 ± 0.07 m, mass = 69.17 ± 8.08 kg). The coaches (n = 7) of each team were also recruited. Inter-rater reliability was assessed using Inter-class correlations (ICC) and Limits of Agreement statistics. Both the kicking (ICC = 0.96, p < .01) and handball tests (ICC = 0.89, p < .01) demonstrated strong reliability and acceptable levels of absolute agreement. Content validity was determined by examining the test scores sensitivity to laterality and distance. Concurrent validity was assessed by comparing coaches’ perceptions of skill to actual test outcomes. Multivariate analysis of variance (MANOVA) examined the main effect of laterality, with scores on the dominant hand (p = .04) and foot (p < .01) significantly higher compared to the non-dominant side. Follow-up univariate analysis reported significant differences at every distance in the kicking test. A poor correlation was found between coaches’ perceptions of skill and testing outcomes. The results of this study demonstrate both skill tests demonstrate acceptable inter-rater reliable. Partial content validity was confirmed for the kicking test, however further research is required to confirm validity of the handball test. Key points The skill tests created by the AFL demonstrated acceptable levels of relative and absolute inter-rater reliability. Both the AFL’s skills tests are able to differentiate between athletes dominant and non-dominant limbs. However, only the kicking test could consistently differentiated between score outcomes over a range of Australian Football specific disposal distances. Both tests demonstrated poor concurrent validity, with no correlation found between coaches’ perceptions of technical skills and actual skill outcomes measured. PMID:26336356

Improved Targeting Through Collaborative Decision-Making and Brain Computer Interfaces

NASA Technical Reports Server (NTRS)

Stoica, Adrian; Barrero, David F.; McDonald-Maier, Klaus

2013-01-01

This paper reports a first step toward a brain-computer interface (BCI) for collaborative targeting. Specifically, we explore, from a broad perspective, how the collaboration of a group of people can increase the performance on a simple target identification task. To this end, we requested a group of people to identify the location and color of a sequence of targets appearing on the screen and measured the time and accuracy of the response. The individual results are compared to a collective identification result determined by simple majority voting, with random choice in case of drawn. The results are promising, as the identification becomes significantly more reliable even with this simple voting and a small number of people (either odd or even number) involved in the decision. In addition, the paper briefly analyzes the role of brain-computer interfaces in collaborative targeting, extending the targeting task by using a BCI instead of a mechanical response.

Introducing a brief measure of cultural and religious identification in American Jewish identity.

PubMed

Friedlander, Myrna L; Friedman, Michelle L; Miller, Matthew J; Ellis, Michael V; Friedlander, Lee K; Mikhaylov, Vadim G

2010-07-01

The authors conducted 3 studies to develop and investigate the psychometric properties of the American Jewish Identity Scales (AJIS), a brief self-report measure that assesses cultural identification and religious identification. Study 1 assessed the content validity of the item pool using an expert panel. In Study 2, 1,884 Jewish adults completed the initial AJIS and various measures of ethnic identity, collective self-esteem, and religiosity. Using confirmatory factor analyses, the authors selected and cross-validated 33 items that loaded highly and differentially on the 2 theorized latent factors. Study 3 assessed the AJIS's short-term stability and its relation to social desirability. Tests of reliability and construct validity provided initial psychometric support for the measure and confirmed the theorized primary salience of cultural identification. Participants reported significantly more private than public collective self-esteem, and the most Jewish-identified participants reported greater private self-esteem, acculturative stress, and perceived discrimination than did their more assimilated counterparts. (c) 2010 APA, all rights reserved.

Detection and identification of human targets in radar data

NASA Astrophysics Data System (ADS)

Gürbüz, Sevgi Z.; Melvin, William L.; Williams, Douglas B.

2007-04-01

Radar offers unique advantages over other sensors, such as visual or seismic sensors, for human target detection. Many situations, especially military applications, prevent the placement of video cameras or implantment seismic sensors in the area being observed, because of security or other threats. However, radar can operate far away from potential targets, and functions during daytime as well as nighttime, in virtually all weather conditions. In this paper, we examine the problem of human target detection and identification using single-channel, airborne, synthetic aperture radar (SAR). Human targets are differentiated from other detected slow-moving targets by analyzing the spectrogram of each potential target. Human spectrograms are unique, and can be used not just to identify targets as human, but also to determine features about the human target being observed, such as size, gender, action, and speed. A 12-point human model, together with kinematic equations of motion for each body part, is used to calculate the expected target return and spectrogram. A MATLAB simulation environment is developed including ground clutter, human and non-human targets for the testing of spectrogram-based detection and identification algorithms. Simulations show that spectrograms have some ability to detect and identify human targets in low noise. An example gender discrimination system correctly detected 83.97% of males and 91.11% of females. The problems and limitations of spectrogram-based methods in high clutter environments are discussed. The SNR loss inherent to spectrogram-based methods is quantified. An alternate detection and identification method that will be used as a basis for future work is proposed.

A PCR primer bank for quantitative gene expression analysis.

PubMed

Wang, Xiaowei; Seed, Brian

2003-12-15

Although gene expression profiling by microarray analysis is a useful tool for assessing global levels of transcriptional activity, variability associated with the data sets usually requires that observed differences be validated by some other method, such as real-time quantitative polymerase chain reaction (real-time PCR). However, non-specific amplification of non-target genes is frequently observed in the latter, confounding the analysis in approximately 40% of real-time PCR attempts when primer-specific labels are not used. Here we present an experimentally validated algorithm for the identification of transcript-specific PCR primers on a genomic scale that can be applied to real-time PCR with sequence-independent detection methods. An online database, PrimerBank, has been created for researchers to retrieve primer information for their genes of interest. PrimerBank currently contains 147 404 primers encompassing most known human and mouse genes. The primer design algorithm has been tested by conventional and real-time PCR for a subset of 112 primer pairs with a success rate of 98.2%.

Using Experimental Paradigms to Examine Alcohol’s Role in Men’s Sexual Aggression: Opportunities and Challenges in Proxy Development

PubMed Central

Abbey, Antonia; Wegner, Rhiana

2015-01-01

The goals of this article are to review the major findings from alcohol administration studies that use sexual aggression proxies and to encourage additional experimental research that evaluates hypotheses about the role of alcohol in the etiology of men’s sexual aggression. Experiments allow participants to be randomly assigned to drink conditions, therefore ensuring that any differences between drinkers and nondrinkers can be attributed to their alcohol consumption. One of the biggest challenges faced by experimental researchers is the identification of valid operationalizations of key constructs. The tension between internal and external validity is particularly problematic for violence researchers because they cannot allow participants to engage in the target behavior in the laboratory. The strengths and limitations associated with written vignettes, audiotapes, videotapes, and confederate proxies for sexual aggression are described. Suggestions are made for future research to broaden the generalizability of the findings from experimental research. PMID:26048214

Elastic light scattering for clinical pathogens identification: application to early screening of Staphylococcus aureus on specific medium

NASA Astrophysics Data System (ADS)

Schultz, E.; Genuer, V.; Marcoux, P.; Gal, O.; Belafdil, C.; Decq, D.; Maurin, Max; Morales, S.

2018-02-01

Elastic Light Scattering (ELS) is an innovative technique to identify bacterial pathogens directly on culture plates. Compelling results have already been reported for agri-food applications. Here, we have developed ELS for clinical diagnosis, starting with Staphylococcus aureus early screening. Our goal is to bring a result (positive/negative) after only 6 h of growth to fight surgical-site infections. The method starts with the acquisition of the scattering pattern arising from the interaction between a laser beam and a single bacterial colony growing on a culture medium. Then, the resulting image, considered as the bacterial species signature, is analyzed using statistical learning techniques. We present a custom optical setup able to target bacterial colonies with various sizes (30-500 microns). This system was used to collect a reference dataset of 38 strains of S. aureus and other Staphyloccocus species (5459 images) on ChromIDSAID/ MRSA bi-plates. A validation set from 20 patients has then been acquired and clinically-validated according to chromogenic enzymatic tests. The best correct-identification rate between S. aureus and S. non-aureus (94.7%) has been obtained using a support vector machine classifier trained on a combination of Fourier-Bessel moments and Local- Binary-Patterns extracted features. This statistical model applied to the validation set provided a sensitivity and a specificity of 90.0% and 56.9%, or alternatively, a positive predictive value of 47% and a negative predictive value of 93%. From a clinical point of view, the results head in the right direction and pave the way toward the WHO's requirements for rapid, low-cost, and automated diagnosis tools.

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples.

PubMed

Lewandowska, Dagmara W; Zagordi, Osvaldo; Geissberger, Fabienne-Desirée; Kufner, Verena; Schmutz, Stefan; Böni, Jürg; Metzner, Karin J; Trkola, Alexandra; Huber, Michael

2017-08-08

Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples. In order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs. The workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.

Molecular Elucidation of Disease Biomarkers at the Interface of Chemistry and Biology.

PubMed

Zhang, Liqin; Wan, Shuo; Jiang, Ying; Wang, Yanyue; Fu, Ting; Liu, Qiaoling; Cao, Zhijuan; Qiu, Liping; Tan, Weihong

2017-02-22

Disease-related biomarkers are objectively measurable molecular signatures of physiological status that can serve as disease indicators or drug targets in clinical diagnosis and therapy, thus acting as a tool in support of personalized medicine. For example, the prostate-specific antigen (PSA) biomarker is now widely used to screen patients for prostate cancer. However, few such biomarkers are currently available, and the process of biomarker identification and validation is prolonged and complicated by inefficient methods of discovery and few reliable analytical platforms. Therefore, in this Perspective, we look at the advanced chemistry of aptamer molecules and their significant role as molecular probes in biomarker studies. As a special class of functional nucleic acids evolved from an iterative technology termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX), these single-stranded oligonucleotides can recognize their respective targets with selectivity and affinity comparable to those of protein antibodies. Because of their fast turnaround time and exceptional chemical properties, aptamer probes can serve as novel molecular tools for biomarker investigations, particularly in assisting identification of new disease-related biomarkers. More importantly, aptamers are able to recognize biomarkers from complex biological environments such as blood serum and cell surfaces, which can provide direct evidence for further clinical applications. This Perspective highlights several major advancements of aptamer-based biomarker discovery strategies and their potential contribution to the practice of precision medicine.

Identification of Escherichia coli O157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

PubMed Central

Swimley, Michelle S.; Taylor, Amber W.; Dawson, Erica D.

2011-01-01

Abstract Shiga toxin–producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 76.71 ± 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100–1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains. PMID:21288130

Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

PubMed Central

Madhunapantula, SubbaRao V.; Sharma, Arati; Gowda, Raghavendra; Robertson, Gavin P.

2014-01-01

Summary Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA-mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis-inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR-A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma. PMID:24034838

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters.

PubMed

Kraft, Thomas E; Heitmeier, Monique R; Putanko, Marina; Edwards, Rachel L; Ilagan, Ma Xenia G; Payne, Maria A; Autry, Joseph M; Thomas, David D; Odom, Audrey R; Hruz, Paul W

2016-12-01

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC 50 ) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

275 Candidates and 149 Validated Planets Orbiting Bright Stars in K2 Campaigns 0–10

NASA Astrophysics Data System (ADS)

Mayo, Andrew W.; Vanderburg, Andrew; Latham, David W.; Bieryla, Allyson; Morton, Timothy D.; Buchhave, Lars A.; Dressing, Courtney D.; Beichman, Charles; Berlind, Perry; Calkins, Michael L.; Ciardi, David R.; Crossfield, Ian J. M.; Esquerdo, Gilbert A.; Everett, Mark E.; Gonzales, Erica J.; Hirsch, Lea A.; Horch, Elliott P.; Howard, Andrew W.; Howell, Steve B.; Livingston, John; Patel, Rahul; Petigura, Erik A.; Schlieder, Joshua E.; Scott, Nicholas J.; Schumer, Clea F.; Sinukoff, Evan; Teske, Johanna; Winters, Jennifer G.

2018-03-01

Since 2014, NASA’s K2 mission has observed large portions of the ecliptic plane in search of transiting planets and has detected hundreds of planet candidates. With observations planned until at least early 2018, K2 will continue to identify more planet candidates. We present here 275 planet candidates observed during Campaigns 0–10 of the K2 mission that are orbiting stars brighter than 13 mag (in Kepler band) and for which we have obtained high-resolution spectra (R = 44,000). These candidates are analyzed using the vespa package in order to calculate their false-positive probabilities (FPP). We find that 149 candidates are validated with an FPP lower than 0.1%, 39 of which were previously only candidates and 56 of which were previously undetected. The processes of data reduction, candidate identification, and statistical validation are described, and the demographics of the candidates and newly validated planets are explored. We show tentative evidence of a gap in the planet radius distribution of our candidate sample. Comparing our sample to the Kepler candidate sample investigated by Fulton et al., we conclude that more planets are required to quantitatively confirm the gap with K2 candidates or validated planets. This work, in addition to increasing the population of validated K2 planets by nearly 50% and providing new targets for follow-up observations, will also serve as a framework for validating candidates from upcoming K2 campaigns and the Transiting Exoplanet Survey Satellite, expected to launch in 2018.

Model Identification in Time-Series Analysis: Some Empirical Results.

ERIC Educational Resources Information Center

Padia, William L.

Model identification of time-series data is essential to valid statistical tests of intervention effects. Model identification is, at best, inexact in the social and behavioral sciences where one is often confronted with small numbers of observations. These problems are discussed, and the results of independent identifications of 130 social and…

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

PubMed

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren; Nielsen, Morten

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

PubMed Central

Cattenoz, Pierre B.; Popkova, Anna; Southall, Tony D.; Aiello, Giuseppe; Brand, Andrea H.; Giangrande, Angela

2016-01-01

High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

PubMed Central

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

Validation of the Electromagnetic Code FACETS for Numerical Simulation of Radar Target Images

DTIC Science & Technology

2009-12-01

Validation of the electromagnetic code FACETS for numerical simulation of radar target images S. Wong...Validation of the electromagnetic code FACETS for numerical simulation of radar target images S. Wong DRDC Ottawa...for simulating radar images of a target is obtained, through direct simulation-to-measurement comparisons. A 3-dimensional computer-aided design