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Results for “Experimental validation”

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At least 559 records · Page 31Linked to original sources

PubMed2026

Multi-omics identification and functional validation of signal regulatory protein gamma as a prognostic biomarker and immune regulator in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) comprises biologically diverse tumors, and durable responses to immune-checkpoint blockade are achieved by only a subset of patients. There remains a need for markers that connect clinical outcome with malignant-cell phenotypes and tissue-level immune organization. METHODS: We integrated The Cancer Genome Atlas HNSCC cohort (TCGA-HNSC), five Gene Expression Omnibus (GEO) validation cohorts, single-cell RNA sequencing, Visium spatial transcriptomics, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq)-informed protein-potential inference, pharmacogenomic screening, genetic-risk analysis and experimental validation. A reconstructed 296-pipeline survival modelling framework was used to prioritize prognostic hub genes across validation-cohort-specific analyses. RESULTS: SIRPG was repeatedly ranked among the top ten selected genes in all five validation cohorts. At single-cell resolution, SIRPG-high tumor cells showed stronger malignant-cell features, immune-inhibitory and metabolic programs, Scissor-positive risk association, CLCA2/P53-related perturbation signals and inferred SIRPG-CD47/signal regulatory protein (SIRP) communication. Spatial analyses placed this axis within an immune-checkpoint-coupled niche, supported by Maxspin/multiview intercellular spatial modelling (MISTy) spatial coupling, communication analysis by optimal transport (COMMOT)-inferred CD47-SIRPG communication and scProTrans-inferred CD47/SIRPG protein-potential overlap. Functionally, SIRPG knockdown reduced HNSCC cell viability and increased apoptosis, whereas re-expression of short hairpin RNA (shRNA)-resistant SIRPG restored the CLCA2-BAX/BCL2 protein response. CONCLUSION: Together, these findings identify SIRPG as an immune-related prognostic hub and context-dependent tumor-cell regulator associated with apoptosis, immune communication and spatial microenvironmental organization in HNSCC.

Humans↗

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↗

PubMed2002

Estimation of the sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks.

Using a combination of experimental and numerical approaches, we have tested two different approaches to calculating the sarcoplasmic reticulum (SR) Ca2+ release flux, which gives rise to cardiac muscle Ca2+ sparks. By using two-photon excited spot photolysis of DM-Nitrophen, known Ca2+ release flux time courses were generated to provide the first experimental validation of spark flux reconstruction algorithms. These artificial Ca2+ sparks show that it is possible to calculate the SR Ca2+ release waveform with reasonable accuracy, provided the flux equations reasonably reflect the properties of the experimental system. Within cardiac muscle cells, we show that Ca2+ flux reconstruction is complicated by the substantial dye binding to proteins, a factor that has not been adequately addressed in previous flux reconstruction algorithms. Furthermore, our numerical experiments suggest that the calculated time course of release flux inactivation based on conventional flux reconstruction algorithms is likely to be in error. We therefore developed novel algorithms based on an explicit dye binding scheme. When these algorithm were applied to evoked Ca2+ sparks in rat cardiac ventricular myocytes, the reconstructed Ca2+ release waveform peaked in ~5 ms and decayed with a halftime of approximately 5 ms. The peak flux magnitude was 7-12 pA, suggesting that sparks must arise from clusters of >15 ryanodine receptors.

Algorithms↗

PubMed2000

Coronary pressure measurement to assess the hemodynamic significance of serial stenoses within one coronary artery: validation in humans.

BACKGROUND: When several stenoses are present within 1 coronary artery, the hemodynamic significance of each stenosis is influenced by the presence of the other(s), and the calculation of coronary and fractional flow reserve (CFR and FFR) for each individual stenosis is confounded. Recently, we developed and experimentally validated a method to determine the true FFR of each stenosis as it would be after the removal of the other stenosis; the true FFR can be reliably predicted by coronary pressures measured before treatment at specific locations within the coronary artery using equations accounting for stenosis interaction. The aim of the present study was to test the validity of these equations in humans. METHODS AND RESULTS: In this study of 32 patients with 2 serial stenoses in 1 coronary artery, relevant pressures were measured before the intervention, after the treatment of 1 stenosis, and after the treatment of both stenoses. The true FFR of each stenosis (FFR(true)) was directly measured after the elimination of the other stenosis and compared with the value predicted (FFR(pred)) from the initial pressure measurements before treatment. Although the hyperemic gradient across 1 stenosis increased significantly (from 10+/-7 to 19+/-11 mm Hg after treatment of the other stenosis), FFR(pred) was close to FFR(true) in all patients (0.78+/-0.12 versus 0.78+/-0.11 mm Hg; r=0.92; Delta%=4+/-0%). Without accounting for stenosis interaction, the value of FFR for each stenosis would have been significantly overestimated (0.85+/-0.08; P:<0.01). CONCLUSIONS: Coronary pressure measurements made by a pressure wire at maximum hyperemia provide a simple, practical method for assessing the individual hemodynamic significance of multiple stenoses within the same artery.

Angioplasty, Balloon, Coronary↗

PubMed1996

Validation and assessment of a conversational interaction intervention.

Multiple measures of social behavior were obtained in order to (a) evaluate the effects of a systematic replication of a conversational interaction intervention, (b) identify critical target behaviors, (c) select participants likely to benefit from intervention, and (d) assess the social and experimental validity of the intervention. Combined measures (e.g., performance screening, assessing students' goals, behavioral ratings) corroborated the decision to introduce multiple-exemplar, self-instructional training to 4 students with moderate mental retardation who were observed to have limited social interaction with their peers at school. Findings indicated that the procedure was effective at increasing empirical indicators of participants' generalized conversational interaction and that behavioral change was validated socially by ratings of peers and important others.

Humans↗

PubMed1999

Using rapidly alternating multiple schedules to assess and treat aberrant behavior in natural settings.

The validity of using rapidly alternating multiple schedules (RAMS) as a method for assessing and treating aggression and disruptive behavior was demonstrated by using the naturally occurring reinforces identified in the RAMS to develop treatments that were experimentally tested. The RAMS consists of a series of 2-minute components in which a naturally occurring consequence is applied contingent on the target's behavior, alternated with components in which the consequence is not applied, with no break between components. The aggressive and disruptive behaviors of four 2- to 11-year-old children were analyzed and treated in school and home settings. The RAMS analyses yielded clear results about the reinforcing function of naturally occurring consequences in all cases, and the treatments using the reinforcers identified in the RAMS were all effective. The possible uses of the RAMS as an efficient, ecologically and experimentally valid tool for clinical assessment are discussed.

Affective Symptoms↗

PubMed2003

Temporal response pattern of biochemical analytes in experimental diabetes.

The activities of the enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), creatine kinase (CK), amylase (AMS) and angiotensin converting enzyme (ACE) have been used to assess the toxic effects of xenobiotics that have hypoglycaemic action in hepatic, pancreatic, renal and muscle tissue. Using a validated experimental model of diabetes mellitus in rats, we ascertained whether this syndrome itself affected the serum activities of these enzymes over a 53-day period. Levels of hepatic enzymes AST, ALT and ALP were higher in the streptozotocin (STZ)-diabetic rats (group D), but were controlled by insulin therapy (group DI). AMS was reduced in group D and unchanged in group DI rats. Proteinuria was detected 1 day after STZ administration and partially controlled by insulin (group DI); its early presence in group D rats, and the lack of any change in serum ACE in this group, indicates that proteinuria is the better marker for microangiopathy. Microscopic examination of liver, kidney, heart and skeletal muscles (soleus and extensor digitorum longus) revealed various alterations in group D rat tissues, which were less pronounced in group DI. The liver, pancreas and kidney tissue-damage was consistent with the altered serum levels of AST, ALT, ALP and AMS and proteinuria. We conclude that: (i) rigorous control is required when these serum-enzyme levels are used as indicators of tissue toxicity in experimental diabetes, and (ii) LD, CK and bilirubin serum levels, which are unaffected by diabetes, can be used when testing effects of xenobiotics on tissues.

Animals↗

PubMed2005

Small-world network approach to identify key residues in protein-protein interaction.

We show that protein complexes can be represented as small-world networks, exhibiting a relatively small number of highly central amino-acid residues occurring frequently at protein-protein interfaces. We further base our analysis on a set of different biological examples of protein-protein interactions with experimentally validated hot spots, and show that 83% of these predicted highly central residues, which are conserved in sequence alignments and nonexposed to the solvent in the protein complex, correspond to or are in direct contact with an experimentally annotated hot spot. The remaining 17% show a general tendency to be close to an annotated hot spot. On the other hand, although there is no available experimental information on their contribution to the binding free energy, detailed analysis of their properties shows that they are good candidates for being hot spots. Thus, highly central residues have a clear tendency to be located in regions that include hot spots. We also show that some of the central residues in the protein complex interfaces are central in the monomeric structures before dimerization and that possible information relating to hot spots of binding free energy could be obtained from the unbound structures.

Amino Acids↗

PubMed1994

VIP-antiserum inhibits fluid secretion by the inflamed gallbladder mucosa.

The inflammatory fluid secretion by the gallbladder mucosa in experimental cholecystitis is induced by an increased prostaglandin formation and is mediated by intramural nerves. In the present study the effect of VIP-antiserum on the inflammatory fluid secretion in the gallbladder was tested in a validated experimental model in cats. The animals were studied in acute experiments 6 weeks after a procedure when the cystic duct was tied and gallstones were implanted in the gallbladder. During basal conditions there was a continuous secretion of fluid into the lumen of the inflamed gallbladder averaging 0.43 +/- 0.18 ml/h. Injection of VIP antiserum, obtained from immunized rabbits and diluted with saline 1:10 in a bolus of 4 ml into the coeliac artery reversed this secretion into an absorption of 1.72 +/- 0.44 ml h-1 (P < 0.001). VIP-antiserum did not affect the fluid adsorption in control animals with an intact gallbladder and injection of control serum from rabbits not immunized to VIP did not affect fluid secretion in the inflamed gallbladders. The results support the idea that the inflammatory fluid secretion in the gallbladder mucosa is mediated by VIP-ergic nerve fibres.

Animals↗

PubMed2026

Generative artificial intelligence for enzyme design and biocatalysis.

Sparked by innovations in generative artificial intelligence (AI), the field of protein design has undergone a paradigm shift with an explosion of new models for optimizing existing enzymes or creating them from scratch. After more than one decade of low success rates for computationally designed enzymes, generative AI models are now frequently used for designing proficient enzymes. Here, we provide a comprehensive overview and classification of generative AI models for enzyme design, highlighting models with experimental validation relevant to real-world settings and outlining their respective limitations. We argue that generative AI models now have the maturity to create and optimize enzymes for industrial applications. Wider adoption of generative AI models with experimental feedback loops can speed up the development of biocatalysts and serve as a community assessment to inform the next generation of models.

Biocatalysis↗

PubMed2004

Scatterer size estimation in pulse-echo ultrasound using focused sources: calibration measurements and phantom experiments.

In a companion paper [T. A. Bigelow and W. D. O'Brien Jr., J. Acoust. Soc. Am. 116, 578 (2004)], theory, supported by simulations, showed that accurate scatterer size estimates could be obtained using highly focused sources provided that the derived generalized attenuation-compensation function was used and the velocity potential field near the focus could be approximated as a three-dimensional Gaussian. Herein, the theory is further evaluated via experimental studies. A calibration technique is developed to find the necessary equivalent Gaussian dimensions for a focused source using reflections obtained from a rigid plane scanned through the focus. Then, the theoretical analysis of focused sources is validated experimentally using three spherically focused ultrasound transducers to estimate the radius of glass beads imbedded in tissue mimicking phantoms. Both the impact of focusing (f/1, f/2, and f/4) and the effect of scatterer type (comparing glass bead results to simulation results that used scatterers with Gaussian impedance distributions) were tested. The simulated differences agree with the measured differences to within 2.5% provided that the comparison is made between the same scatterer type and sources with the same equivalent Gaussian dimensions. The improvement provided by the generalized attenuation-compensation function is greatly influenced by the type of scatterer whose size is being estimated and decreases as the wavelength dependence of the Gaussian depth of focus is reduced.

Calibration↗

PubMed2004

Strengths and weaknesses of EST-based prediction of tissue-specific alternative splicing.

BACKGROUND: Alternative splicing contributes significantly to the complexity of the human transcriptome and proteome. Computational prediction of alternative splice isoforms are usually based on EST sequences that also allow to approximate the expression pattern of the related transcripts. However, the limited number of tissues represented in the EST data as well as the different cDNA construction protocols may influence the predictive capacity of ESTs to unravel tissue-specifically expressed transcripts. METHODS: We predict tissue and tumor specific splice isoforms based on the genomic mapping (SpliceNest) of the EST consensus sequences and library annotation provided in the GeneNest database. We further ascertain the potentially rare tissue specific transcripts as the ones represented only by ESTs derived from normalized libraries. A subset of the predicted tissue and tumor specific isoforms are then validated via RT-PCR experiments over a spectrum of 40 tissue types. RESULTS: Our strategy revealed 427 genes with at least one tissue specific transcript as well as 1120 genes showing tumor specific isoforms. While our experimental evaluation of computationally predicted tissue-specific isoforms revealed a high success rate in confirming the expression of these isoforms in the respective tissue, the strategy frequently failed to detect the expected restricted expression pattern. The analysis of putative lowly expressed transcripts using normalized cDNA libraries suggests that our ability to detect tissue-specific isoforms strongly depends on the expression level of the respective transcript as well as on the sensitivity of the experimental methods. Especially splice isoforms predicted to be disease-specific tend to represent transcripts that are expressed in a set of healthy tissues rather than novel isoforms. CONCLUSIONS: We propose to combine the computational prediction of alternative splice isoforms with experimental validation for efficient delineation of an accurate set of tissue-specific transcripts.

Alternative Splicing↗

PubMed2026

SERPINE1-centric inflammatory signature associates with treatment resistance and survival in laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) prognosis remains poor despite treatment advances. More accurate prognostic assessment models can help guide individualized treatment and improve prognosis. Chronic inflammation contributes to tumorigenesis, yet inflammatory response-related genes (IRGs) in LSCC prognosis are underexplored. This study aimed to construct an IRG prognostic signature for LSCC and further dissect core IRG-mediated mechanisms of immune escape and chemoresistance. METHODS: Transcriptional profiles and clinical data from LSCC patients were retrieved from The Cancer Genome Atlas (TCGA). IRGs were sourced from Gene Set Enrichment Analysis (GSEA) hallmark gene set. We identified differentially expressed IRGs linked to survival outcomes in LSCC. Key IRGs were subsequently selected using least absolute shrinkage and selection operator (LASSO) Cox regression analysis to establish an inflammatory risk score model. This model underwent internal validation within the TCGA cohort and external validation using independent Gene Expression Omnibus (GEO) datasets. We further assessed the model's association with the tumor immune microenvironment and the impact of IRGs on chemotherapy response. Finally, the functional roles of interested signature IRG were experimentally validated in LSCC cell lines. RESULTS: Four significant IRGs (AQP9, ITGA5, LCK, SERPINE1) were identified to build the risk score model. The model stratified LSCC patients into distinct prognostic groups: TCGA cohort: 5-year area under the curve (AUC) =0.836, P<0.001; GSE25727 cohort: 5-year AUC =0.706, P=0.02; GSE27020 cohort: 5-year AUC =0.798, P<0.01. Multivariate analysis confirmed the risk score as an independent prognostic factor (P<0.05). High-risk patients showed reduced immune cell infiltration (CD8+ T cells, dendritic cells) and suppressed immune pathways. Multi-algorithm immune analysis further revealed defective antigen presentation and reduced anti-tumor immune infiltration in high-risk LSCC, promoting tumor immune escape. GSEA/Gene Ontology (GO) enrichment combined with drug sensitivity prediction further revealed that high-risk tumors activate invasive signaling and acquire broad chemoresistance alongside impaired anti-tumor immunity. SERPINE1 might be associated with chemotherapy resistance and exhibited the highest alteration frequency (predominantly amplification) and overexpression in LSCC tissues. Its knockdown significantly suppressed proliferation, migration, invasion and chemoresistance in LSCC cells. Immunohistochemistry (IHC) confirmed tumor SERPINE1 overexpression (P=0.002 vs. normal tissues), correlating with poor survival (P<0.001). CONCLUSIONS: The 4-IRG risk signature is a reliable prognostic indicator reflecting immune dysfunction in LSCC. SERPINE1 is validated as a therapeutic target and biomarker, enriching our understanding of gene regulation dynamics in LSCC.

Laryngeal cancer↗

PubMed2026

Microglial PICALM: A novel genetic driver and therapeutic target in vascular dementia.

BACKGROUND: Vascular dementia (VaD) lacks well-defined genetic mechanisms. Cell-type-specific effects of GWAS loci remain unexplored. METHODS: We integrated single‑cell eQTL data (183 donors, eight cell types) with VaD GWAS (3624 cases, 475,484 controls) using Mendelian randomization and Bayesian colocalization, replicated in an independent cohort (2074 cases, 456,366 controls). Subtype, snRNA‑seq, cell‑cell communication, PheWAS, expression profiling, and drug prediction with BBB permeability assessment were performed. RESULTS: Microglial PICALM was the only robustly replicated signal (OR = 0.8334, p = 5.3 × 10⁻⁴; colocalization PP.H4 > 0.75). The effect was strongest in multiple infarctions dementia (OR = 0.7746). Exploratory snRNA-seq analysis (4 VaD vs. 4 controls; GSE282111) provided supporting evidence for microglial PICALM enrichment and downregulation (p < 0.001). PICALM‑high microglia showed enhanced neurovascular‑ and phagocytosis‑related communication (e.g., SPP1, GAS6, GRN). PheWAS revealed no pleiotropy. In silico drug repurposing prioritised three FDA-approved BBB-penetrant compounds (disopyramide, benzocaine, amantadine) as candidates warranting further mechanistic validation. CONCLUSIONS: Microglial PICALM is identified as a likely genetic determinant of VaD, especially in the multiple infarctions subtype. Upregulating PICALM may be associated with a neuroprotective microglial phenotype, highlighting PICALM as a candidate therapeutic target warranting further experimental validation.

Humans↗

PubMed2004

Design of highly specific ligands of fibrin for therapeutic applications.

The work presented here is aimed at designing high-affinity ligands for the fibrin gamma (312-324) epitope. This epitope is specific for fibrin recognition, as it is exposed only on the fibrin surface, while in fibrinogen it is buried in the protein bulk. This property makes it very useful for therapeutic applications. In fact, it may be exploited in driving systems for targeted delivery of thrombolytic drugs toward the specific compartment where they are needed. It will then allow avoidance of serious unwanted side effects produced by a conventional systemic administration. The approach chosen for designing putative ligands is based on the known three-dimensional (3D) structure of the epitope. A wide virtual library made up of oligo-peptides and analogues designed by a combinatorial approach, on the basis of chemical complementarity criteria, has been screened by means of a docking/scoring approach (DOCK program). The peculiarity of the problem under study required a considerable effort in finding a method enabling the experimental validation of the design work results. In fact the selected biological target is absolutely new, so that neither a endogenous, nor a synthetic high-affinity ligand is known up to now. It does not allow for the validation of computational results by means of classical binding tests based on the use of known labeled high-affinity ligands. Preliminary binding essayes were so carried out by means of the Plasmon Surface Resonance (PSR) technique. The experimental results suggested that most of the molecules predicted to be good ligands by means of the selected computational tools, could carry the wanted affinity toward the selected target.

Amino Acid Sequence↗

PubMed1997

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. I. Invariants and validation of electron microscopy models.

Solutions of selectively deuterated 70 S Escherichia coli ribosomes and of free 30 S and 50 S subunits were studied by neutron scattering using contrast variation. The integrity of the partially deuterated particles was controlled by parallel X-ray measurements. Integral parameters of the entire ribosome, of its subunits and of the protein and rRNA moieties were evaluated. The data allow an experimental validation of the two most recent electron microscopy reconstructions of the 70 S ribosome presented by the groups of J. Frank (Albany) and of M. van Heel & R. Brimacombe (Berlin). For each reconstruction, integral parameters and theoretical scattering curves from the 70 S and its subunits were calculated and compared with the experimental data. Although neither of the two models yields a comprehensive agreement with the experimental data, Frank's model provides a better fit. For the 50 S subunit of van Heel & Brimacombe's model the fit with the experimental data improves significantly when the internal channels and tunnels are filled up. The poorer fit of the latter model is thus caused by its "sponge"-like structure which may partly be due to an enhancement of high frequency contributions in some of the steps of the three-dimensional image reconstruction. It seems therefore unlikely that the ribosome has a "sponge"-like structure with a pronounced network of channels.

Deuterium↗

PubMed2004

Programmed field decay thermal field flow fractionation of polymers: a calibration method.

The universal calibration procedure typical of thermal field flow fractionation (ThFFF) under constant thermal field operation was extended to thermal field programming (TFP) operation. The method requires knowledge of the following: (a) the programming function, which only depends on the thermal field decay function, (b) the physicochemical properties of the solvent, and (c) the calibration plot under varying channel cold wall temperatures (T(c)). Two field flow fractionation field programming conditions, with either a constant or a variable in time carrier flow velocity, are exploited. The method is based on determination, for each retention time position, of the average lambda retention value typical of TFP ThFFF. This parameter is then used to obtain the calibration plot (i.e., the molecular weight of the species as a function of the retention time position) by using the programming function and the calibration plot under varying T(c) values. The procedure approximation errors are also derived as a function of the programming type and solute-solvent system. To properly test the procedure, the calibration plot for the system constituted by polystyrene (PS) in cis-trans Decalin was determined, under varying conditions T(c) and thermal gradients, by using a set of monodisperse PS standards of different molecular weights (M). The procedure was first validated by simulation under two typical cases of TFP ThFFF operation. The approximation errors were found acceptable (in the worse cases, the accuracy in M prediction was 3%) and are in agreement with the theory. The procedure was then experimentally validated under varying programming decay function conditions. The reproducibility and accuracy of the M determination are both better than 2%.

Journal Article↗

PubMed2002

Metallic inductive and capacitive grids: theory and experiment.

We present theoretical modeling and experimental validation of both capacitive (dot) and inductive (hole) metallic crossed gratings in the mid-infrared (2-5 microm). The gratings are fabricated by use of interferometric lithography and modeled by use of rigorous coupled-wave analysis. Our experimental and numerical investigations of the transmittance spectra of these gratings suggest that, as in inductive grids, the behavior of capacitive grids is described by the coupling of the incident light into surface plasma waves.

Journal Article↗