Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Computational prediction”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42Linked to original sources
PubMed2005

Using the transcriptome to annotate the genome revisited: application of massively parallel signature sequencing (MPSS).

Transcriptome analysis can provide useful data for refining genome sequence annotation. Application of massively parallel signature sequencing (MPSS) revealed reproducible transcription, in multiple MPSS cycles, from 73% of computationally predicted genes in the Theileria parva schizont lifecycle stage. Signatures spanning consecutive exons confirmed 142 predicted introns. MPSS identified 83 putative genes, >100 codons overlooked by annotation software, and 139 potentially incorrect gene models (with either truncated ORFs or overlooked exons) by interfacing signature locations with stop codon maps. Twenty representative models were confirmed as likely to be incorrect using reverse transcription PCR amplification from independent schizont cDNA preparations. More than 50% of the 60 putative single copy genes in T. parva that were absent from the genome of the closely related T. annulata had MPSS signatures. This study illustrates the utility of MPSS for improving annotation of small, gene-rich microbial eukaryotic genomes.

Animals↗
PubMed2004

Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells.

H2-deleted, HLA-A2, or HLA-B7 transgenic mice were used to identify new human cytomegalovirus (HCMV)-derived major histocompatibility complex class I-restricted epitopes. Three different approaches for mice immunization were compared for their ability to induce a cytotoxic CD8(+) T cell (CTL) response: (1). inoculation of infectious HCMV, (2). injection of immunogenic synthetic peptides, and (3). infection with a newly designed HIV-derived central DNA flap positive lentiviral vector encoding the chimeric IE1-pp65 protein (Trip-IE1-pp65). Targets pulsed with either known immunogenic peptides or computer predicted ones were used to characterize CTL. Most of the mice immunized with the pp65 (495-NLVPMVATV-503) immunodominant peptide responded after one injection whereas only two of six mice responded to two successive inoculations with HCMV. Infection of mice with Trip-IE1-pp65 induced activation and expansion of CTL directed against peptides from both pp65 and IE1 and allowed identification of new epitopes. We further demonstrated the high capacity of monocyte-macrophage cells transduced with Trip-IE1-pp65 to activate and expand CTL directed against pp65 from peripheral blood mononuclear cells of HCMV-seropositive donors. Altogether these results suggest that Trip-IE1-pp65 is a powerful construct both to characterize new epitopes in combination with human leukocyte antigen-transgenic mice immunization and to provide an alternative to the use of known infectious and noninfectious approaches to expand effector T cells for adoptive immunotherapy.

Amino Acid Sequence↗
PubMed2005

Hydrodynamics-induced variability in the USP apparatus II dissolution test.

The USP tablet dissolution test is an analytical tool used for the verification of drug release processes and formulation selection within the pharmaceutical industry. Given the strong impact of this test, it is surprising that operating conditions and testing devices have been selected empirically. In fact, the flow phenomena in the USP test have received little attention in the past. An examination of the hydrodynamics in the USP apparatus II shows that the device is highly vulnerable to mixing problems that can affect testing performance and consistency. Experimental and computational techniques reveal that the flow field within the device is not uniform, and dissolution results can vary dramatically with the position of the tablet within the vessel. Specifically, computations predict sharp variations in the shear along the bottom of the vessel where the tablet is most likely to settle. Experiments in which the tablet location was carefully controlled reveal that the variation of shear within the testing device can affect the measured dissolution rate.

Chemistry, Pharmaceutical↗
PubMed2026

Functional screening of ZIP8 naturally occurring variants identifies pathogenic mutations and trafficking defects.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput (∼150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of ∼20% for AlphaMissense underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular protein trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Journal Article↗
PubMed2006

Determination of limiting mobilities and dissociation constants of 21 amino acids by capillary zone electrophoresis at very low pH.

The dependence of the effective electrophoretic mobility on pH of the background electrolyte was experimentally determined by capillary zone electrophoresis (CZE) for cationic forms of amino acids. The pH of the background electrolytes was in the highly acidic range, 1.6-2.6 pH units, to ensure a high degree of protonation of the amino acids. Poly(vinyl alcohol) was added to the background electrolytes to avoid possible adsorption of the analytes at the inner capillary wall. Non-linear regression of the experimental data was applied to obtain the parameters of the relevant regression functions--the actual mobilities and mixed dissociation constants corresponding to the actual ionic strength. The extended Onsager and Debye-Hückel law was used to calculate the limiting mobilities and thermodynamic dissociation constants. The comparison of the experimental electropherogram with the computer prediction by PeakMaster using the determined data is presented for the selected sample of amino acids.

Amino Acids↗
PubMed2006

Solution structure of a Hck SH3 domain ligand complex reveals novel interaction modes.

We studied the interaction of hematopoietic cell kinase SH3 domain (HckSH3) with an artificial 12-residue proline-rich peptide PD1 (HSKYPLPPLPSL) identified as high affinity ligand (K(D)=0.2 muM). PD1 shows an unusual ligand sequence for SH3 binding in type I orientation because it lacks the typical basic anchor residue at position P(-3), but instead has a tyrosine residue at this position. A basic lysine residue, however, is present at position P(-4). The solution structure of the HckSH3:PD1 complex, which is the first HckSH3 complex structure available, clearly reveals that the P(-3) tyrosine residue of PD1 does not take the position of the typical anchor residue but rather forms additional van der Waals interactions with the HckSH3 RT loop. Instead, lysine at position P(-4) of PD1 substitutes the function of the P(-3) anchor residue. This finding expands the well known ligand consensus sequence +xxPpxP by +xxxPpxP. Thus, software tools like iSPOT fail to identify PD1 as a high-affinity HckSH3 ligand so far. In addition, a short antiparallel beta-sheet in the RT loop of HckSH3 is observed upon PD1 binding. The structure of the HckSH3:PD1 complex reveals novel features of SH3 ligand binding and yields new insights into the structural basics of SH3-ligand interactions. Consequences for computational prediction tools adressing SH3-ligand interactions as well as the biological relevance of our findings are discussed.

Amides↗
PubMed2005

On-resonance low B1 pulses for imaging of the effects of PARACEST agents.

Application of the exchange-sensitive, low-power RF pulses positioned on the bulk water resonance for imaging of the effects of PARACEST agents is proposed as an alternative to the standard CW off-resonance irradiation. Specifically, we applied a low-power WALTZ-16 RF train, with the 90 degrees pulse unit replaced by a pulse of the fixed length (WALTZ-16*). Using this sequence, the bulk water signal was found to be sensitive to exchange lifetimes with PARACEST complex bound protons, the transverse relaxation time of bulk water, and longitudinal relaxation time of bound protons. In this report, the concept of using WALTZ-16* to "activate" a PARACEST effect is introduced and some of the salient features of this technique with respect to experimental conditions and performance levels are discussed. Computational predictions are verified and explored by comparison with experimental spectroscopic and imaging data. It is shown that WALTZ-16* can be used to detect PARACEST agents with an RF intensity as low as 200 Hz for concentrations as low as a few tens of microM for lanthanide chelates having appropriate water-exchange rates (Tm,Dy).

Contrast Media↗
PubMed2005

Similar sequence-free amplification of human glyceraldehyde-3-phosphate dehydrogenase for real time RT-PCR applications.

One of the major applications of real time polymerase chain reaction (PCR) is relative quantification, where the expression of a target gene is determined as a ratio to a stably expressed reference gene, the so-called housekeeping gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is a glycolytic enzyme, which is active in all mammalian tissues and is frequently used as housekeeping gene in expression studies. The functional locus maps to human chromosome 12p13, but several GAPD-related sequences, including processed pseudogenes, GenBank homologous sequences and computationally predicted sequences are present along the human genome. Due to the high level of GAPD-related sequences it is very important to avoid genomic DNA amplification when GAPD is used as endogenous control in mRNA quantification. We have outlined a GAPD couple of primers that avoid any genomic DNA amplification for real time reverse transcription PCR applications by SYBR-Green Dye. These new designed primers are an useful and chip alternative to probe technologies, and can carry out specific and reproducible data in mRNA expression studies.

Base Sequence↗
PubMed2026

Sec and Tat Mediated Secretion Safeguards Mycobacterium tuberculosis Membrane Homeostasis.

Protein secretion is essential for the growth and virulence of Mycobacterium tuberculosis, yet the organization and function of its secretion pathways remain poorly understood. We reviewed the existing literature, combined it with systematic queries, and finalized annotations based on experimental data and computational predictions to compile a curated list of 92 secretory components and 198 reactions involved in Sec, twin-arginine translocation (Tat), and ESX pathways. Using CRISPRi, targeted depletion of SecA1 or TatAC impaired both in vitro growth and ex vivo survival. Label-free quantitative secretome analysis revealed decreased export of substrates dependent on SecA1 and TatAC, with enrichment of cytosolic proteins in culture filtrates, indicating increased membrane dysbiosis. Membrane proteomics showed elevated levels of proteins engaged in intermediary and lipid metabolism, while proteins associated with the cell wall and cell processes decreased, suggesting weakened membrane integrity. Loss of SecA1 or TatAC increased membrane permeability, with the effect being more pronounced in the case of TatAC, and caused structural abnormalities seen under electron microscopy. Overall, our integrated multi-omics and functional genetics studies demonstrate that the SecA1 and Tat pathways are essential for maintaining membrane homeostasis in Mycobacterium tuberculosis. These results suggest that essential secretory proteins may be promising targets for therapeutic intervention.

Mycobacterium tuberculosis↗
PubMed2005

A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent.

Currently, dengue fever is the most important re-emerging mosquito-borne viral disease, with the major proportion of the target population residing in the developing countries of the world. In endemic areas, potentially fatal secondary dengue infections, characterized by high anti-dengue IgG antibody titers, are most common. Most currently available commercial dengue diagnostic kits rely on the use of whole virus antigens and are consequently associated with false positives due to serologic cross-reactivity, high cost of antigen production, and biohazard risk. This has prompted the need to develop an alternate antigen to replace the whole virus antigen in diagnostic tests. We have designed and expressed a novel recombinant protein antigen by assembling key immunodominant linear IgG-specific dengue virus epitopes, chosen on the basis of pepscan analysis, phage display, and computer predictions. The recombinant dengue multiepitope protein was expressed to high levels in Escherichia coli, purified in a single step, yielding >25 mg pure protein per liter culture. We developed an in-house enzyme-linked immunosorbent assay (ELISA) to detect anti-dengue antibodies in a panel of 20 patient sera using the purified recombinant dengue multiepitope protein as the capture antigen. The ELISA results were in excellent agreement with those obtained using a commercially available diagnostic test, Dengue Duo rapid strip test from PanBio, Australia. The high epitope density, careful choice of epitopes, and the use of E. coli system for expression, coupled to simple purification, jointly have the potential to lead to the development of an inexpensive diagnostic test with a high degree of sensitivity and specificity.

Amino Acid Sequence↗
PubMed2005

Digging deeper into the plant cell wall proteome.

The proteome of the plant cell wall/apoplast is less well characterized than those of other subcellular compartments. This largely reflects the many technical challenges involved in extracting and identifying extracellular proteins, many of which resist isolation and identification, and in capturing a population that is both comprehensive and relatively uncontaminated with intracellular proteins. However, a range of disruptive techniques, involving tissue homogenization and subsequent sequential extraction and non-disruptive approaches has been developed. These approaches have been complemented more recently by other genome-scale screens, such as secretion traps that reveal the genes encoding proteins with N-terminal signal peptides that are targeted to the secretory pathway, many of which are subsequently localized in the wall. While the size and complexity of the wall proteome is still unresolved, the combination of experimental tools and computational prediction is rapidly expanding the catalog of known wall-localized proteins, suggesting the unexpected extracellular localization of other polypeptides and providing the basis for further exploration of plant wall structure and function.

Cell Wall↗
PubMed2006

The anatomy of organogenesis: novel solutions to old problems.

Understanding anatomical aspects of mammalian organ development, in both normal and mutant animals, is important for basic biology and also for regenerative medicine and tissue engineering. The size and complexity of developing organs, together with variations in their detailed anatomy, has made the obtaining of high-resolution time-courses of anatomical change difficult to obtain. The fact that organ development tends to use the same genes as early embryogenesis also makes genetic manipulation difficult, as so many mutant embryos die before organogenesis begins. These problems have seriously hampered the study of organogenesis. Here, we describe three significant advances that promise solutions: (1) the production of GFP-reporter mice that can be used for high-resolution live-imaging of small tissues as they grow, (2) RNA interference, which allows the manipulation of specific genes at any stage of organ development, and (3) optical projection tomography, which allows medium-resolution three-dimensional images of complete embryos to be obtained easily. We finish by looking ahead to the prospects of uniting these three technologies to allow accurate, high-throughput screening of mutants and automated comparison of biological data with computer predictions.

Animals↗
PubMed2026

Expanding the clinical spectrum of ARV1-related disease beyond classical developmental and epileptic encephalopathy.

PURPOSE: Biallelic pathogenic variants in ARV1 are classically associated with developmental and epileptic encephalopathy-38 (DEE38), a severe infantile-onset disorder characterized by drug-resistant epilepsy, profound neurodevelopmental impairment, and early mortality. However, emerging evidence suggests broader phenotypic variability. We aimed to expand the clinical and molecular spectrum of ARV1-related disease through a retrospective case series and structured literature review. METHODS: We retrospectively identified five unrelated Saudi Arabian families with biallelic pathogenic or likely pathogenic ARV1 variants confirmed by whole-exome sequencing. Clinical, neurodevelopmental, neurophysiological, neuroimaging, and multisystem findings were reviewed. A structured literature review was performed to integrate previously reported cases. RESULTS: We identified marked clinical heterogeneity, including a novel ARV1 missense variant (c.214G>T; p.Asp72Tyr), which remains classified as a variant of uncertain significance according to ACMG/AMP criteria despite multiple computational predictions supporting a deleterious effect. Clinical severity ranged from severe developmental and epileptic encephalopathy with drug-resistant epilepsy and early mortality to milder static neurodevelopmental phenotypes with sustained seizure remission and long-term survival into adulthood. Families harboring the same homozygous frameshift variant exhibited markedly different clinical severity, supporting the absence of a strict genotype-phenotype correlation. Multisystem involvement included neurological, cardiac, skeletal, sensory, gastrointestinal, and genitourinary manifestations, and metabolic phenocopies contributed to diagnostic delays. CONCLUSION: Our findings demonstrate that ARV1-related disease represents a broad multisystem clinical spectrum, with classical DEE38 representing its most severe presentation rather than its sole manifestation. Recognition of milder phenotypes, prolonged seizure remission, and marked phenotypic variability has important implications for diagnosis, prognostic counseling, and multidisciplinary long-term surveillance. Early genomic testing should be considered in patients with early-onset epilepsy and multisystem involvement, particularly in consanguineous populations.

ARV1↗
PubMed2006

Topological determinants of protein domain swapping.

Protein domain swapping has been repeatedly observed in a variety of proteins and is believed to result from destabilization due to mutations or changes in environment. Based on results from our studies and others, we propose that structures of the domain-swapped proteins are mainly determined by their native topologies. We performed molecular dynamics simulations of seven different proteins, known to undergo domain swapping experimentally, under mildly denaturing conditions and found in all cases that the domain-swapped structures can be recapitulated by using protein topology in a simple protein model. Our studies further indicated that, in many cases, domain swapping occurs at positions around which the protein tends to unfold prior to complete unfolding. This, in turn, enabled prediction of protein structural elements that are responsible for domain swapping. In particular, two distinct domain-swapped dimer conformations of the focal adhesion targeting domain of focal adhesion kinase were predicted computationally and were supported experimentally by data obtained from NMR analyses.

Animals↗
PubMed2005

Systems biotechnology for strain improvement.

Various high-throughput experimental techniques are routinely used for generating large amounts of omics data. In parallel, in silico modelling and simulation approaches are being developed for quantitatively analyzing cellular metabolism at the systems level. Thus informative high-throughput analysis and predictive computational modelling or simulation can be combined to generate new knowledge through iterative modification of an in silico model and experimental design. On the basis of such global cellular information we can design cells that have improved metabolic properties for industrial applications. This article highlights the recent developments in these systems approaches, which we call systems biotechnology, and discusses future prospects.

Genome↗
PubMed2005

In vitro models for the blood-brain barrier.

The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.

Animals↗
PubMed2006

Analysis of the temporal expression of Trichoplusia ni single nucleopolyhedrovirus genes following transfection of BT1-Tn-5B1-4 cells.

Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), a 134,394-bp double-stranded DNA group II Nucleopolyhedrovirus, is pathogenic to the lepidopteran T. ni. TnSNPV transcription is temporally regulated and divided into three promoter sequence-dependent classes (early, late and very late genes). A viral oligonucleotide DNA microarray containing all potential (144) viral genes of TnSNPV was designed to investigate global viral gene expression during cell infection. Total BT1-Tn-5B1-4 cellular mRNAs extracted between 0 and 72 h posttransfection with TnSNPV genomic DNA were hybridized to the microarray. Initial average expression of early genes was detected between 12 and 24 h posttransfection while late genes were mainly detected between 24 and 72 h posttransfection. The microarray expression profiling data verified many computer predicted promoter assignments. K-means clustering was used to sort the 144 genes based on their temporal expression pattern similarities. This clustering resulted in the confirmation and temporal class assignment of previously unidentified genes and promoters.

Animals↗
PubMed2004

Bridging expressed sequence alignments through targeted cDNA sequencing.

One of the major challenges in genome research is the identification of the complete set of genes in a genome. Alignments of expressed sequences (RNA and EST) with genomic sequences have been used to characterize genes. However, the number of alignments far exceeds the likely number of genes in a genome, suggesting that, for many genes, two or more alignments can be joined through overlapping sequences to yield accurate gene structures. High-throughput EST sequencing becomes less efficient in closing those alignment gaps due to its nonselective nature. We sought to bridge these alignments through a novel approach: targeted cDNA sequencing. Human expressed sequences from GenBank version 124 were aligned with the genomic sequence from NCBI build 24 using LEADS, Compugen's EST and RNA clustering and assembly software system. Nine hundred forty-eight pairs of alignments were selected based on EST clone information and/or their homology to the same known proteins. Reverse transcriptase PCR and sequencing yielded sequences for 363 of those pairs. These sequences helped characterize over 60 novel or otherwise incomplete genes in the recent UniGene build 153, which included over 1 million additional ESTs. These results indicate that this integrated and targeted strategy, combining computational prediction and experimental cDNA sequencing, can efficiently generate the overlapping sequences and enable the full characterization of genomes. Additional information about the contig pairs, the resultant overlapping sequences, tissue sources, and tissue profiles are available in a supplemental file.

Cloning, Molecular↗