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Results for “target validation”

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At least 757 records · Page 42Linked to original sources

PubMed1998

Preparation of targeted isoniazid microspheres.

Isoniazid (INH) albumin microspheres were prepared by two different stabilization processes: chemical denaturation using glutaraldehyde and heat denaturation. The extent of stabilization was characterized by the solubility of the microspheres. In vitro drug release rates were correlated to the stability of the microspheres and the results showed that the more denatured the albumin by heat stabilization, the slower the drug release rate. A factorial concept has been utilized to synthesize microspheres suitable for passive targeting to the lungs by varying protein concentration, stabilization temperature, time and aqueous volume. These factors significantly affected the sphere size, payload and a release profile of the drug. As the severity of the denaturation conditions increased, the payload decreased and the rate of drug release was slowed. The microspheres carrying isoniazid were followed in experimental animals to validate the targeting process.

Animals↗

PubMed2026

Development of a Droplet-Based RNA Interference Feeding Assay for Neonates of the Citrus Root Weevil Diaprepes abbreviatus.

The citrus root weevil, Diaprepes abbreviatus, is an economically important pest of citrus and ornamental crops whose subterranean larval feeding damages roots and predisposes plants to secondary pathogen infection. Development of efficient RNA interference (RNAi) delivery methods for early larval stages is essential for functional genomics studies and the evaluation of RNAi-based pest management strategies. In this study, we developed a droplet-based feeding assay for oral delivery of double-stranded RNA (dsRNA) to neonates of D. abbreviatus using chitin synthase 2 (DaCHS2) as a model RNAi target to validate the assay. Feeding solutions containing dsRNA were supplemented with sucrose and bromophenol blue dye, with bromophenol blue used to visually confirm ingestion. Across three independent biological replicates, all neonates exposed to DaCHS2-dsRNA, GFP-dsRNA, and water control droplets were confirmed to have ingested the feeding solution (45/45 neonates per treatment; 100% feeding success). Oral delivery of dsRNA targeting DaCHS2 reduced transcript abundance and was associated with developmental abnormalities and mortality, including incomplete molting, abnormal pigmentation, cuticular deformities, defective pupation, and malformed adults. Regression analysis demonstrated moderate and significant relationship between dsRNA concentration and neonate mortality and developmental abnormalities. RT-qPCR further confirmed reduced DaCHS2 transcript abundance following oral dsRNA exposure. The developed assay provides a simple, reproducible, and minimally invasive proof-of-concept platform for oral dsRNA delivery to D. abbreviatus neonates. The assay requires only small dsRNA volumes, provides visual confirmation of ingestion, and may facilitate laboratory-based screening of additional RNAi target genes in D. abbreviatus and other coleopteran pests.

Animals↗

PubMed1996

Bilateral interactions in saccade programming. A saccade-latency study.

Subjects were required to make a saccade to a target appearing randomly 4 degree to the left or right of the current fixation position (1280 trials per experiment). Location cues were used to direct visual attention and start saccade preparation to one of the two locations before target onset. When the cue indicated the target location (valid trials), the generation of express saccades (visually guided saccades with latencies around 100 ms) was strongly facilitated. When the opposite location was cued (invalid trials), express saccades were abolished and replaced by a population of mainly fast-regular saccades (latencies around 150 ms). This was found with a peripheral cue independently of whether the fixation point was removed before target onset (gap condition; experiment 1) or remained on throughout the trial (overlap condition; experiment 2). The same pattern also was observed with a central cue that did not involve any visual stimulation at a peripheral location (experiment 3). In the case where the primary saccade was executed in response to the cue and the target appeared at the opposite location, continuous amplitude transition functions were observed: starting at about 60-70 ms from target onset onward, the amplitude of the cue-elicited saccades continuously decreased from 4 degree to values below 1 degree. The results are explained by a fixation-gating model, according to which the antagonism between fixation and saccade activity gives rise to multimodal distributions of saccade latencies. It is argued that allocation of visual attention and saccade preparation to one location entails a successive disengagement of the fixation system controlling saccade preparation within the hemifield to which the saccade is prepared and a partial engagement of the opposite fixation system.

Adult↗

PubMed1978

The use of category information in perception.

Three tachistoscopic experiments are reported in which presentation of the target stimulus in a letter/digit categorization task was preceded by a briefly exposed priming stimulus (letter or digit). The primer was subject to backward masking from either the target or a pattern mask, and observers were unaware of its occurrence. With a primer duration of 25 ms, when masking was presumed to be at a central level, performance deteriorated when the two items were from different categories. This inhibition effect was reduced when the characters were physically similar. In contrast, there was little evidence of facilitated processing when primers validly cued targets. At shorter primer durations, when masking is presumed to be peripheral in origin, between-condition differences were less marked. An interpretation in terms of an active model of information processing, with utilization of both categorical and physical information extracted from the primer, is proposed.

Adult↗

PubMed2004

Identifying estrogen receptor alpha target genes using integrated computational genomics and chromatin immunoprecipitation microarray.

The estrogen receptor alpha (ERalpha) regulates gene expression by either direct binding to estrogen response elements or indirect tethering to other transcription factors on promoter targets. To identify these promoter sequences, we conducted a genome-wide screening with a novel microarray technique called ChIP-on-chip. A set of 70 candidate ERalpha loci were identified and the corresponding promoter sequences were analyzed by statistical pattern recognition and comparative genomics approaches. We found mouse counterparts for 63 of these loci and classified 42 (67%) as direct ERalpha targets using classification and regression tree (CART) statistical model, which involves position weight matrix and human-mouse sequence similarity scores as model parameters. The remaining genes were considered to be indirect targets. To validate this computational prediction, we conducted an additional ChIP-on-chip assay that identified acetylated chromatin components in active ERalpha promoters. Of the 27 loci upregulated in an ERalpha-positive breast cancer cell line, 20 having mouse counterparts were correctly predicted by CART. This integrated approach, therefore, sets a paradigm in which the iterative process of model refinement and experimental verification will continue until an accurate prediction of promoter target sequences is derived.

Animals↗

PubMed2006

Amplified anticoagulant activity of tissue factor-targeted thrombomodulin: in-vivo validation of a tissue factor-neutralizing antibody fused to soluble thrombomodulin.

Tissue factor (TF) exposure is a potent pro-thrombotic trigger that initiates activation of the coagulation cascade, while thrombomodulin (TM) is a potent anticoagulant protein that limits the extent of activation. Both TF neutralizing antibodies and soluble TM (sTM) are effective anticoagulants. We have developed a novel anticoagulant fusion protein, Ab(TF)-TM, by fusing a TFneutralizing single-chain antibody, Ab(TF), to an active fragment of TM. Ab(TF)-TM is a novel anticoagulant targeting to sites of TF exposure with a dual mechanism of action. The Ab(TF) portion of the molecule inhibitsTF/factorVIIa mediated activation of FIX and FX, and the TM portion of the molecule acts as a cofactor for activation of protein C. In-vitro coagulation assays show that Ab(TF)-TM more potently inhibits TF-initiated coagulation (prothrombin time) than can its individual components, Ab(TF) (20-fold) and sTM (80-fold) alone, or in combination (10-fold). In contrast, the potency of Ab(TF)-TM in the activated partial thromboplastin and thrombin clotting time assays was similar to sTM alone. In a rat model of disseminated intravascular coagulation (DIC), intravenous injection of a human TF-containing thromboplastin reagent (0.5 ml/kg) resulted in an immediate death in approximately 60% of the animals and a clinical score of approximately 2.5. Pre-injection of Ab(TF)-TM or Ab(TF) and sTM, given alone or in combination, showed dose-dependent efficacy. At a dose of 0.7 nmol/kg, Ab(TF)-TM completely prevented death and reduced clinical scores by 79%, while neitherAb(TF) nor sTM, given alone or in combination, showed significant therapeutic effects. Calculated effective doses that reduced mortality by 50% relative to that in the control group (ED(50), nmol/kg) were 0.21 for Ab(TF)-TM, 3.2 for an equimolar mixture of Ab(TF) and sTM, 4.3 for sTM and 20 for Ab(TF). Thus, Ab(TF)-TM presented 10- to 100-fold enhancement of the anticoagulant potency, relative to the ED(50) in Ab(TF) and sTM given either alone or in combination, in a rat DIC model.

Animals↗

PubMed2006

Reconstitution of human hypoxia inducible factor HIF-1 in yeast: a simple in vivo system to identify and characterize HIF-1alpha effectors.

Hypoxia inducible factor 1 (HIF-1), the master regulator of hypoxia-activated genes, is involved in many diseases and is a valid drug target. In order to develop a simple and genetically tractable in vivo system for HIF-1 analysis, we tested the inducible expression of both human HIF-1 subunits (HIF-1alpha and ARNT) in the yeast Saccharomyces cerevisiae and showed the formation of transcriptionally active HIF-1. The use of this system for the identification and characterization of HIF-1 effectors was first validated by showing that two chemical Hsp90 inhibitors, geldanamycin and radicicol, impaired the activity of HIF-1 in yeast. By applying this system in mutant yeast strains, we then identified Hsp90 co-chaperones, which were required for HIF-1 activity. Furthermore, using yeast strains co-expressing truncated forms of HIF-1alpha with ARNT or both HIF-1alpha and ARNT, we characterized fragments of HIF-1alpha that acted as dominant negative mutants and suppressed HIF-1 activity.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed2005

Systems and integrative biology as alternative guises for pharmacology: prime time for an iPharm concept?

Understanding the molecular basis of human disease pathophysiology is critical to accurate disease diagnosis, defining disease progression and to identifying new drugs that more specifically address target diseases. Advances in the understanding of tissue function (including draft maps of the human genome) coupled with industrial-scale, 'enabling' technologies like high throughput screening, combinatorial chemistry, proteomics, etc., have generated data on a scale never before possible. Despite this, there continues to be a dearth of new drug approvals ascribed to: (i) the challenges of working with novel, often non-validated disease targets; (ii) targeting diseases (stroke, Alzheimer's) with limited (if any) treatment and ill-defined clinical trial endpoints; (iii) enhanced regulatory hurdles for drug approval; (iv) insufficient time for the new knowledge and enabling technologies to have reached a productive level. An alternate viewpoint is that unfettered access to such technologies, where exclusion rather than integration has been the hallmark, has markedly reduced the intellectual competent of the biomedical research endeavor, with perceived technological 'quick fixes' displacing the integrative, hierarchical approach of pharmacology, that with medicinal chemistry, represents the core of the drug discovery process. After two decades of profound neglect, pharmacology has re-emerged as the key discipline in providing context to the drug discovery process, facilitating more timely, context-relevant and data-driven outcomes in the search for new drugs. Rather than viewing the future of drug discovery in terms of the 'new' biologies, systems and integrative, a rubric along the lines of iPharm, integrating both established and new technologies, is required.

Animals↗

PubMed2026

Integrative Cross-platform Analysis of Kinase Inhibitor Effects on Statin-relevant Cardioprotective Pathways in Human Cardiomyocytes.

BACKGROUND/AIM: Kinase inhibitors (KIs) can cause cardiotoxicity through mechanisms overlapping with statin cardioprotective pathways, yet their effects on these pathways in cardiomyocytes remain uncertain. We evaluated six literature-defined statin-relevant gene sets using transcriptomic and proteomic data. MATERIALS AND METHODS: Pre-ranked gene set enrichment analysis was performed for 23 KIs in primary cardiac cells (GSE146096; n=319) and iPSC-derived cardiomyocytes (GSE217421; n=541), with cross-platform analysis of 21 KIs by shotgun proteomics (PXD014791; n=300). Pathway-specific concordance was assessed by Spearman correlation with Benjamini-Hochberg correction; protein scores were estimated after adjustment for cell line. RESULTS: KI effects were heterogeneous. The anti-fibrotic pathway showed nominal concordance across the two transcriptomic datasets (ρ=0.495, p=0.016, q=0.098; 91% direction concordance) and significant cell-line-adjusted transcriptomic-proteomic concordance (ρ=0.644, p=0.0016, q=0.0081). Nilotinib reproducibly upregulated NF-κB pathway genes [normalized enrichment score (NES)=+2.29 and +2.18 in discovery and validation], with targeted inter-gene-correlation-adjusted testing supporting higher NF-κB expression than under rosuvastatin (CAMERA p=3.54×10-8). No global cross-omics summary remained significant after harmonizing pathway universes and accounting for repeated pathways. CONCLUSION: KI effects on statin-relevant pathways were pathway-specific. Anti-fibrotic concordance and nilotinib-associated NF-κB upregulation are hypothesis-generating candidates for experimental validation.

Humans↗

PubMed2001

Spatial attention modulates sound localization in barn owls.

Attentional influence on sound-localization behavior of barn owls was investigated in a cross-modal spatial cuing paradigm. After being cued to the most probable target side with a visual cuing stimulus, owls localized upcoming auditory target stimuli with a head turn toward the position of the sound source. In 80% of the trials, cuing stimuli pointed toward the side of the upcoming target stimulus (valid configuration), and in 20% they pointed toward the opposite side (invalid configuration). We found that owls initiated the head turns by a mean of 37.4 ms earlier in valid trials, i.e., mean response latencies of head turns were reduced by 16% after a valid cuing stimulus. Thus auditory stimuli appearing at the cued side were processed faster than stimuli appearing at the uncued side, indicating the influence of a spatial-selective attention mechanism. Turning angles were not different when owls turned their head toward a cued or an uncued location. Other types of attention influencing sound localization, e.g., a reduction of response latency as a function of the duration of cue-target delay, could not be observed. This study is the first attempt to investigate attentional influences on sound localization in an animal model.

Acoustic Stimulation↗

PubMed2003

A new paradigm for drug discovery: integrating clinical, genetic, genomic and molecular phenotype data to identify drug targets.

Application of statistical genetics approaches to variations in mRNA transcript abundances in segregating populations can be used to identify genes and pathways associated with common human diseases. The combination of this genetic information with gene expression and clinical trait data can also be used to identify subtypes of a disease and the genetic loci specific to each subtype. Here we highlight results from some of our recent work in this area and further explore the many possibilities that exist in employing a more comprehensive genetics and functional genomics approach to the functional annotation of genomes, and in applying such methods to the validation of targets for complex traits in the drug discovery process.

Animals↗

PubMed2004

Computational analysis of Plasmodium falciparum metabolism: organizing genomic information to facilitate drug discovery.

Identification of novel targets for the development of more effective antimalarial drugs and vaccines is a primary goal of the Plasmodium genome project. However, deciding which gene products are ideal drug/vaccine targets remains a difficult task. Currently, a systematic disruption of every single gene in Plasmodium is technically challenging. Hence, we have developed a computational approach to prioritize potential targets. A pathway/genome database (PGDB) integrates pathway information with information about the complete genome of an organism. We have constructed PlasmoCyc, a PGDB for Plasmodium falciparum 3D7, using its annotated genomic sequence. In addition to the annotations provided in the genome database, we add 956 additional annotations to proteins annotated as "hypothetical" using the GeneQuiz annotation system. We apply a novel computational algorithm to PlasmoCyc to identify 216 "chokepoint enzymes." All three clinically validated drug targets are chokepoint enzymes. A total of 87.5% of proposed drug targets with biological evidence in the literature are chokepoint reactions. Therefore, identifying chokepoint enzymes represents one systematic way to identify potential metabolic drug targets.

Algorithms↗

PubMed2006

Alcoholism and neuropeptides: an update.

As with other addictions, human alcoholism is characterised as a chronically relapsing condition. Consequently, the "holy grail" from a therapeutic viewpoint is the development of clinically effective, safe drugs that promote high compliance rates and prevent relapse. Here we discuss the potential of therapeutics targeting neuropeptide systems implicated in aberrant alcohol-seeking behaviour. Clearly, much of the data so far available comes from pre-clinical studies; however, one of the first effective therapeutic strategies for alcoholism (still in use today) was the use of non-selective opioid receptor antagonists, such as naltrexone (Revia). In addition to opioid receptors, other neuropeptide receptors including those for corticotrophin releasing factor (CRF), neuropeptide Y and nociceptin may represent valid therapeutic targets to regulate alcohol consumption and the affective consequences of alcohol withdrawal.

Alcoholism↗

PubMed2007

Interactions between peptides containing nucleobase amino acids and T7 phages displaying S. cerevisiae proteins.

The importance of high-throughput analyses of protein abundances and functions is interestingly increasing in genomic/proteomic studies. In such postgenome sequencing era, a protein-detecting chip, in which a large number of molecules specifically capturing target proteins (capturing agents) such as antibodies, recombinant proteins, and small molecules are arrayed onto solid, wet, or semi-wet substrates, enables comprehensive analysis of proteomes by a single experiment. However, whole proteomes are generally complicated for comprehensive analyses so that alternative approaches to subproteome analysis categorized by protein functions and binding properties (focused proteome) would be effective. Approaching the goal of development of designed peptide chip for protein analysis, diversity increases in peptide structures and validation of target proteins are needed. We herein describe design and synthesis of nucleobase amino acid (NBA)-containing peptides, selection of nucleic acid-related proteins derived from S. cerevisiae, and detection of interactions between NBA-containing peptides and T7 phages displaying proteins by both enzyme-linked immunosorbent assays (ELISA) and label-free anomalous reflection of gold (AR) measurements. Twenty-eight phage clones were obtained by the phage-display method and sequenced. Ten of 28 clones were expected to be nucleic acid-related proteins including initiation factor, TYB protein, ribosomal proteins, elongation factor, ATP synthase subunit, GTP-binding protein, and ribonuclease. Other phage clones encoded several classes of enzymes such as reductase, oxidase, aldolase, metalloprotease, and hexokinase. Both ELISA and AR measurements suggested that the methodology of in vitro selection for recognition of the NBA-containing peptide presented in this study was successfully established. Such a combination of NBA and phage display technologies would be potential to efficiently confirm valuable target proteins binding specifically to capturing agents, to be arrayed onto solid surfaces to develop the designed peptide chip.

Bacteriophage T7↗

PubMed2006

Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster.

RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, we examined mRNA expression profiles in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. We also profiled cells depleted of the miRNA-processing enzyme Drosha. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought.

Animals↗

PubMed2003

The NMDA receptor glycine modulatory site: a therapeutic target for improving cognition and reducing negative symptoms in schizophrenia.

Numerous clinical studies demonstrate that subanaesthetic doses of dissociative anaesthetics, which are non-competitive antagonists at the NMDA receptor, replicate in normal subjects the cognitive impairments, negative symptoms and brain functional abnormalities of schizophrenia. Post-mortem and genetic studies have identified several abnormalities associated with schizophrenia that would interfere with the activation of the glycine modulatory site on the NMDA receptor. Placebo controlled clinical trials with agents that directly or indirectly activate the glycine modulatory site consistently reduce negative symptoms and frequently improve cognition in patients with chronic schizophrenia, who are receiving concurrent typical antipsychotics. Thus, there is convincing evidence that the glycine modulatory site on the NMDA receptor is a valid therapeutic target for improving cognition and associated negative symptoms in schizophrenia.

Animals↗

PubMed2006

Systematic identification of microRNA functions by combining target prediction and expression profiling.

Target predictions and validations are major obstacles facing microRNA (miRNA) researchers. Animal miRNA target prediction is challenging because of limited miRNA sequence complementarity to the targets. In addition, only a small number of predicted targets have been experimentally validated and the miRNA mechanism is poorly understood. Here we present a novel algorithm for animal miRNA target prediction. The algorithm combines relevant parameters for miRNA target recognition and heuristically assigns different weights to these parameters according to their relative importance. A score calculation scheme is introduced to reflect the strength of each parameter. We also performed microarray time course experiments to identify downregulated genes due to miRNA overexpression. The computational target prediction is combined with the miRNA transfection experiment to systematically identify the gene targets of human miR-124. miR-124 overexpression led to a significant downregulation of many cell cycle related genes. This may be the result of direct suppression of a few cell growth inhibitors at the early stage of miRNA overexpression, and these targeted genes were continuously suppressed over a long period of time. Our high-throughput approach can be generalized to globally identify the targets and functions of other miRNAs.

Algorithms↗

PubMed2006

p53 and its downstream proteins as molecular targets of cancer.

The p53 tumor suppressor gene plays a key role in prevention of tumor formation through transcriptional dependent and independent mechanisms. Transcriptional-dependent mechanisms are mainly mediated by p53 regulation of downstream targets, leading to growth arrest and apoptosis. Mutational inactivation of the p53 gene is detected in more than 50% of human cancers. Mutation of p53 renders cancer cells more resistant to current cancer therapies due to lack of p53-mediated apoptosis. Extensive studies have been conducted to identify small molecules that manipulate p53, including restoration of mutant p53 conformation to wild-type, disruption of murine double minute-2 (Mdm2)-p53 binding to increase p53 level and inhibition of Mdm2 E3 ubiquitin ligase activity to prevent p53 degradation. Another approach was to identify and validate "drugable" target(s) in p53 signaling pathways that modulate p53-induced apoptosis. We profiled a p53 temperature-sensitive lung cancer cell model with the Affymetrix human HG-U133 GeneChip, covering the entire human transcriptome. We identified thousands of unique genes that were either induced or repressed in response to p53-induced apoptosis. A follow-up study characterized a p53-repressed gene, SAK, a polo-like kinase (PLK) family member, as an appealing cancer drug target. Snk/Plk-akin kinase (SAK) silencing via small interfering RNA (siRNA) induced apoptosis, whereas SAK overexpression attenuated p53-induced apoptosis. Thus, SAK repression by p53 contributes to p53-induced apoptosis. Future work is directed at determining the normal cell response to SAK silencing. If a therapeutic window is obtained, a SAK inhibitor identified from high throughput screening (HTS) could serve as a lead compound for development of a novel class of apoptosis-inducing anticancer drugs.

Humans↗