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Results for “Experimental validation”

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PubMed1997

Non-Gaussian statistical property of the ultrasonic Doppler signal downstream of a severe stenosis.

The Doppler signal is generally considered as a Gaussian random process. However, to date very little experimental validation of this model has been available. Recently, the K model was proposed to describe the statistical properties of ultrasonic radio frequency signals and their envelopes. The coefficient of kurtosis, which has been shown to be related to the parameters of the K model, was used in the literature as an index to assess the deviations from the Gaussian statistical property of ultrasonic signals obtained from simulations, phantoms and tissues. In the present study, an in vitro flow loop model was used to evaluate the effect of a severe stenosis on the coefficient of kurtosis. Our results showed non-Gaussian statistical properties of the Doppler signal in the jet of the stenosis.

Constriction, Pathologic↗

PubMed2003

Identification of 9 novel transcripts and two RGSL genes within the hereditary prostate cancer region (HPC1) at 1q25.

We applied a systematic bioinformatics approach, followed by careful manual inspection and experimental validation to identify additional expressed sequences located at the Hereditary Prostate Cancer Region (HPC1) between D1S2818 and D1S1642 on chromosome 1q25. All transcripts already described for the 1q25 region were identified and we were able to define 11 additional expressed sequences within this region (three full-length cDNA clone sequences and eight ESTs), increasing the total number of gene count in this region by 38%. Five out of the 11 expressed sequences identified were shown to be expressed in prostate tissue and thus represent novel disease gene candidates for the HPC1 region. Here, we report a detailed characterization of these five novel disease gene candidates, their expression pattern in various tissues, their genomic organization and functional annotation. Two candidates (RGSL1 and RGSL2) correspond to novel members of the RGS family, which is involved in the regulation of G-protein signaling. RGSL1 and RGLS2 expression was detected by real-time polymerase chain reaction in normal prostate tissue, but could not be detected in prostate tumor cell lines, suggesting they might have a role in prostate cancer.

Chromosome Mapping↗

PubMed2002

In vitro and in vivo dose delivery characteristics of large porous particles for inhalation.

The purpose of this study was to evaluate the in vitro and in vivo dose delivery characteristics of two large porous particle placebo formulations with different mass median aerodynamic diameters (MMAD approximately equal to 3 and 5 microm). In vitro dose delivery characteristics were measured using the multistage liquid impinger (MSLI). In vitro lung deposition was predicted by calculating the extrathoracic deposition using the ICRP model, with the remaining fraction assumed to deposit in the lungs. Healthy subjects were trained to inhale through the AIR delivery system at a target peak inspiratory flow rate (PIFR) of 60 l/min, The in vivo dose delivery of large porous particles were obtained by gamma-scintigraphy and was characterized by high ( approximately 90%), reproducible emitted doses for both the small and large MMAD powders. The mean in vivo lung deposition relative to the total metered dose were 59.0 and 37.3% for 3 and 5 microm MMAD powders, respectively. The AIR delivery system produced high in vivo lung deposition and low intersubject CVs (approximately 14%) across the range of PIFRs obtained in the study (50-80 l/min), This is relative to a variety of dry powder inhalers (DPI) that have been published in the literature, with in vivo lung deposition ranging from 13 to 35% with intersubject CVs ranging from 17 to 50%. The ICRP model provided a good estimate of the mean in vivo lung deposition for both powders. Intersubject variability was not captured by the ICRP model due to intersubject differences in the morphology and physiology of the oropharyngeal region. The ICRP model was used to predict the regional lung deposition, although these predictions were only considered speculative in the absence of experimental validation.

Administration, Inhalation↗

PubMed1995

Luminal surface concentration of lipoprotein (LDL) and its effect on the wall uptake of cholesterol by canine carotid arteries.

PURPOSE: The effect of near-wall blood flow velocity and plasma filtration velocity across the arterial wall on luminal surface concentration of low-density lipoproteins (LDL) and the uptake of tritium-cholesterol were investigated. METHODS: A numeric analysis of LDL transport in steady flow, over the range of physiologically relevant flow rates, predicted a surface concentration of LDL of 4% to 16% greater than that in the bulk flow. The LDL surface concentration increased linearly with filtration velocity and inversely with wall shear rate. RESULTS: These were validated experimentally in canine carotid arteries. When the transmural pressure was increased from 100 to 200 mm Hg, the filtration velocity increased from 5.13 x 10(-6) cm/sec to 8.41 x 10(-6) cm/sec, whereas the normalized uptake rate of tritium-cholesterol increased from 3.58 x 10(-4) cm/hour to 7.36 x 10(-4) cm/hour. CONCLUSION: These results indicate that lipids accumulate at the luminal surface in areas where blood flow velocity and wall shear stress are low and where the permeability of the endothelial layer is enhanced. Moreover, the rate of lipid infiltration into the blood vessel walls is affected by the luminal surface concentration. These findings are consistent with chronic hypertension and elevated blood cholesterol concentrations being major risk factors for atherosclerosis.

Animals↗

PubMedDate not supplied

Further evaluation of the multiple-stimulus preference assessment.

Previously researchers have shown that multiple-stimulus preference assessments can produce results comparable to those achieved using the paired-stimulus presentation format. However, extensive experimental validation of this procedure has not yet been accomplished. The purpose of this study was to provide a systematic extension of a study reported by DeLeon and Iwata (1996). We conducted brief stimulus preference assessments with nine participants diagnosed with severe or profound mental retardation. The highest ranked stimuli were then delivered contingent on a target behavior in a multielement format. The stimulus identified as most highly preferred functioned as a reinforcer in six of the nine participants. The results of this study are discussed in the context of current stimulus preference assessment findings.

Adult↗

PubMed1997

A user-friendly method for calibrating a subcutaneous glucose sensor-based hypoglycaemic alarm.

A crucial step in developing a glucose monitoring system using a subcutaneous implanted glucose sensor is the transformation of the sensor signal (a current) into an estimation of a blood glucose concentration. We have developed an Electronic Control Unit (ECU) able to recognize, before and after a glucose load, that the sensor current presents a plateau, thus triggering an alarm asking for blood glucose determination. The system, fed with these results, subsequently transforms the current into an estimation of glucose concentration by linear extrapolation based on the sensor sensitivity and the background current computed from the two sets of current and glycaemia values (two-point calibration). In addition, the system is able to trigger an alarm when this estimation decreases below a threshold that can be set by the user. This system was evaluated in experiments performed in 12 normal rats. The quality of the calibration was assessed by comparing, by error grid analysis, the data displayed on the liquid-crystal display of the ECU to concomitant plasma glucose concentration determined at frequent intervals, 65 +/- 6 and 26 +/- 5% of the values were in zones A (good) and B (acceptable estimation) of the grid, respectively. The system was set to trigger an alarm when the estimation of glucose concentration decreased below 70 mg/dl. Following an insulin administration, the alarm was triggered when the system displayed a 64 +/- 2 mg/dl glucose concentration. The concomitant plasma glucose concentration was 59 +/- 5 mg/dl (NS). In conclusion, this work validates experimentally the new, user-friendly method for calibrating the glucose sensor integrated into the ECU, based on an automatic detection of plateaus. The quality of the sensor calibration performed with this procedure is compatible with the appropriate functioning of this continuous glucose monitoring system, which was demonstrated by its ability to detect mild hypoglycaemia following insulin injection.

Animals↗

PubMed2003

Presymptomatic diagnosis of experimental Parkinsonism with 123I-PE2I SPECT.

Presymptomatic diagnosis of the loss of nigrostriatal neurons that characterises Parkinson's disease, is a crucial issue for future neuroprotective therapies as degeneration exceeds 70 to 80% when symptoms appear. Here we propose an early diagnosis method that utilises single photon emission computerized tomography (SPECT) coupled to the iodine-123-labelled selective dopamine transporter ligand N-(3-ioprop-2E-enyl)-2-beta-(4-methylphenyl)nortropane ((123)I-PE2I), applying Logan's graphical method for quantification. Sequential (123)I-PE2I SPECT acquisitions were performed in nonhuman primates chronically treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine according to a regimen that consistently produces a progressive Parkinsonian state. While classical neurological examination only allows detection of Parkinsonian signs at Day 12 of the protocol of intoxication, the mean distribution volume ratio calculated according to Logan's graphical method is significantly decreased from Day 6 onward, i.e., when animals are clinically normal. (123)I-PE2I SPECT is a very sensitive method to detect presymptomatic lesions of nigrostriatal neurons and the first to be experimentally validated. It could now be used clinically for early diagnosis and follow-up of neuroprotective treatment.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2004

Bioinformatics and cancer target discovery.

The convergence of genomic technologies and the development of drugs designed against specific molecular targets provides many opportunities for using bioinformatics to bridge the gap between biological knowledge and clinical therapy. Identifying genes that have properties similar to known targets is conceptually straightforward. Additionally, genes can be linked to cancer via recurrent genomic or genetic abnormalities. Finally, by integrating large and disparate datasets, gene-level distinctions can be made between the different biological states that the data represents. These bioinformatics approaches and their associated methodologies, which can be applied across a range of technologies, facilitate the rapid identification of new target leads for further experimental validation.

Analysis of Variance↗

PubMed2000

Optimizing initial plasmid copy number distribution for improved protein activity in a recombinant fermentation.

Recombinant bacterial cells in a fermentation broth rarely contain the same number of plasmids, even though this simplification is often used. Recent work has however indicated limitations of the simplified approach. Based on these studies, the distribution of plasmid copy numbers per cell has been represented macroscopically here in a Gaussian form for the fraction of biomass as a function of the copy number. Applying this distribution and an experimentally validated kinetic model to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) synthesis by Escherichia coli containing the plasmid pBR Eco gap, it is seen that GAPDH production in a batch fermentation is maximized by a particular initial (non-zero) copy number variance and an optimal duration. To implement this distribution in a bioreactor, it is suggested that the profile may be discretized, inocula corresponding to the mean copy number of each fraction prepared, and then combined to obtain the seed culture.

Journal Article↗

PubMed1995

The induction of Trypanosoma cruzi trypomastigote to amastigote transformation by low pH.

Following cell invasion, Trypanosoma cruzi trypomastigotes transform into amastigotes, which are the mammalian replicative forms of the parasite. Although amastigotes represent a critical stage in the life-cycle of T. cruzi, little is known of the factors controlling trypomastigote to amastigote transformation. Kanbera et al. (1990) observed that exposure of trypomastigotes to acidic pH induced their transformation into rounded forms resembling amastigotes. We confirm their observation and, using two strains of T. cruzi, establish that these transformants are ultrastructurally and biochemically indistinguishable from natural amastigotes. Incubation of trypomastigotes in medium at pH 5.0 for 2 h was sufficient to trigger their transformation into forms resembling amastigotes. Electron microscopical analysis confirmed that the kinetoplast structure, and general morphological features of the acid-induced, extracellular amastigotes were indistinguishable from those of intracellular-derived amastigotes. The extracellular transformation was accompanied by the acquisition of the stage-specific surface antigen of the naturally transformed amastigotes (Ssp-4), and loss of a stage-specific trypomastigote antigen (Ssp-3). Trypomastigotes incubated at neutral pH did not transform into amastigotes, and did not acquire the Ssp-4 epitope or lose the Ssp-3 epitope. Finally, acid-induced amastigotes subsequently incorporated [3H]thymidine into their DNA, indicating that the important replicative property of intracellular amastigotes is also exhibited by these in vitro transformants. This effect of low pH appears to be of physiological relevance, and acid-induced extracellular transformation appears to represent a valid experimental technique for studies of the molecular mechanisms involved in the differentiation process.

Animals↗

PubMed2003

Electron tunneling through proteins.

Electron transfer processes are vital elements of energy transduction pathways in living cells. More than a half century of research has produced a remarkably detailed understanding of the factors that regulate these 'currents of life'. We review investigations of Ru-modified proteins that have delineated the distance- and driving-force dependences of intra-protein electron-transfer rates. We also discuss electron transfer across protein-protein interfaces that has been probed both in solution and in structurally characterized crystals. It is now clear that electrons tunnel between sites in biological redox chains, and that protein structures tune thermodynamic properties and electronic coupling interactions to facilitate these reactions. Our work has produced an experimentally validated timetable for electron tunneling across specified distances in proteins. Many electron tunneling rates in cytochrome c oxidase and photosynthetic reaction centers agree well with timetable predictions, indicating that the natural reactions are highly optimized, both in terms of thermodynamics and electronic coupling. The rates of some reactions, however, significantly exceed timetable predictions: it is likely that multistep tunneling is responsible for these anomalously rapid charge transfer events.

Azurin↗

PubMed1990

Children use syntax to learn verb meanings.

Verb learning is clearly a function of observation of real-world contingencies; however, it is argued that such observational information is insufficient to account fully for vocabulary acquisition. This paper provides an experimental validation of Landau & Gleitman's (1985) syntactic bootstrapping procedure; namely, that children may use syntactic information to learn new verbs. Pairs of actions were presented simultaneously with a nonsense verb in one of two syntactic structures. The actions were subsequently separated, and the children (MA = 2;1) were asked to select which action was the referent for the verb. The children's choice of referent was found to be a function of the syntactic structure in which the verb had appeared.

Attention↗

PubMed2001

Complete 5' and 3' end maturation of group II intron-containing tRNA precursors.

Higher plant chloroplasts provide the only experimentally validated example of functional tRNA genes that are disrupted by group II introns. Here, precursor transcripts for tRNA(Gly)(UCC), tRNA(Val)(UAC), and tRNA(Ala)(UGC) were investigated for processing of 5' leader and 3' trailer sequences in vivo. Use of intron-specific primer pairs and inclusion of a barley chloroplast splicing mutant specifically allowed us to evaluate the potential effect of intervening sequences that disrupt tRNA secondary and tertiary structures. The data suggest that (1) neither integrity of the dihydrouridine nor the anticodon domain is required for the nucleotidyltransferase-mediated addition of 3'-terminal CCA; (2) interruption of these two structural elements by group II introns does not interfere with nucleotide-specific 5' maturation by RNase P; (3) processing intermediates of chloroplast tRNAs can be 3' polyadenylated; and (4) plastid DNA-encoded proteins are not required for 3' and 5' maturation of plastid tRNAs.

Base Pairing↗

PubMed2002

Patterning flows using grooved surfaces.

Through a simple analytical description we quantify how pressure-driven flows over grooved surfaces develop transverse components, which, for shallow grooves, can be modeled with simple anisotropic effective boundary conditions. Helical recirculation results in channels or capillaries with grooved walls. An experimental validation of our model is presented. Our analysis provides a workable guide for the design of 3D flows with simple patterns of grooved regions, e.g., to control the position of streams in the cross section of a channel or to promote mixing. Potential applications in microfluidics are outlined.

Algorithms↗

PubMed2005

Electrokinetic transport in nanochannels. 1. Theory.

Electrokinetic transport in fluidic channels facilitates control and separation of ionic species. In nanometer-scale electrokinetic systems, the electric double layer thickness is comparable to characteristic channel dimensions, and this results in nonuniform velocity profiles and strong electric fields transverse to the flow. In such channels, streamwise and transverse electromigration fluxes contribute to the separation and dispersion of analyte ions. In this paper, we report on analytical and numerical models for nanochannel electrophoretic transport and separation of neutral and charged analytes. We present continuum-based theoretical studies in nanoscale channels with characteristic depths on the order of the Debye length. Our model yields analytical expressions for electroosmotic flow, species transport velocity, streamwise-transverse concentration field distribution, and ratio of apparent electrophoretic mobility for a nanochannel to (standard) ion mobility. The model demonstrates that the effective mobility governing electrophoretic transport of charged species in nanochannels depends not only on electrolyte mobility values but also on zeta potential, ion valence, and background electrolyte concentration. We also present a method we term electrokinetic separation by ion valence (EKSIV) whereby both ion valence and ion mobility may be determined independently from a comparison of micro- and nanoscale transport measurements. In the second of this two-paper series, we present experimental validation of our models.

Electrochemistry↗

PubMed2006

Probabilistic enrichment of phosphopeptides by their mass defect.

The mass defect, that is, the difference between the nominal and actual monoisotopic masses, of a phosphorus in a phosphate group is greater than for most other atoms present in proteins. When the mass defects of tryptic peptides derived from the human proteome are plotted against their masses, phosphopeptides tend to fall off the regression line. By calculating the masses of all potential tryptic peptides from the human proteome, we show that regions of higher phosphorylation probability exist on such a plot. We developed a transformation function to estimate the mass defect of a peptide from its monoisotopic mass and empirically defined a simple formula for a user-selectable discriminant line that categorizes a peptide mass according to its probability of being phosphorylated. Our method performs similarly well on phosphopeptides derived from a database of experimentally validated phosphoproteins. The method is relatively insensitive to mass measurement error of up to 20 ppm. The approach can be used with a tandem mass spectrometer in real time to rapidly select and rank order the possible phosphopeptides from a mixture of unmodified peptides for subsequent phosphorylation site mapping and peptide sequence analysis.

Peptide Mapping↗

PubMed2026

Structural Basis for Stepwise Substrate Transport and Disease Phenotypic Heterogeneity of the Mitochondrial ADP/ATP Carrier.

The mitochondrial ADP/ATP carrier (AAC) is essential for cellular energy metabolism and responsible for exchanging ADP for ATP across the inner mitochondrial membrane. However, the precise molecular determinants of substrate binding and the mechanisms underlying the phenotypic heterogeneity of AAC-related diseases remain poorly understood. Here, we combined AlphaFold3 predictions, molecular dynamics simulations, and experimental validation to identify and characterize a previously unrecognized ADP-binding site in AAC (site S2), which is distinct from the canonical bottom site (site S1). AlphaFold3 predictions on AAC variants with disrupted site S1 consistently placed ADP at site S2, interacting with residues R188, K92, and K96, a finding that was independently corroborated by our prior MD simulations. Systematic mutagenesis and functional analysis revealed distinct roles for site S2 residues: R188 serves as the primary phosphate-specific anchor; K92 and K96 facilitate initial recruitment and stabilization; and the aromatic ladder (Y187/Y191/F192/Y195) assists the conformational transition of ADP from anti to syn, a critical step enabling downward translocation. Functional characterization demonstrated that mutations in site S2 significantly impaired ADP transport and oxidative phosphorylation. Notably, the spatial distribution of AAC disease mutations correlates with this bipartite architecture: mild PEOA2-associated mutations cluster near site S2 and perturb the local conformation without abolishing binding, whereas lethal mutations cluster near site S1 and disrupt both ADP binding and structural integrity of the m-gate. Our findings provide new mechanistic insights into stepwise substrate transport and potential therapeutic targets for AAC-related diseases.

Mitochondrial ADP, ATP Translocases↗

PubMed2026

Comprehensive Landscape of Post-Translational Modification Alterations in Nephrolithiasis Revealing Activation of Multiple Cell Death Pathways.

Nephrolithiasis is a common urinary disorder characterized by high prevalence and recurrence, but the molecular mechanisms underlying calcium oxalate (CaOx)-crystal-induced renal injury remain incompletely understood. We applied integrated proteomic, phosphoproteomic, acetylomic, and lactylomic analyses to kidney tissues from a mouse model of CaOx nephrolithiasis followed by bioinformatic analysis and experimental validation. We identified 658 differentially expressed proteins, 735 differential phosphorylation sites, 335 differential acetylation sites, and 113 differential lactylation sites. Functional enrichment indicated immune activation, fibrotic remodeling, and alterations in PI3K-Akt, NOD-like receptor, p53, and Toll-like receptor signaling together with changes in fatty acid degradation, the tricarboxylic acid cycle, and glycolysis. Kinase activity prediction suggested the relative activation of multiple cyclin-dependent kinases. Proteins associated with ferroptosis, autophagy, necroptosis, and pyroptosis, including ACSL4, BNIP3, RIPK3, and GSDMD, showed coordinated abundance and modification changes. Several candidate sites, including MTOR_S1849, GCLM_K94, GCLM_S59, and GSS_K172, were also dysregulated. These data provide a multiomics resource for CaOx nephrolithiasis and identify candidate PTM events and regulatory pathways for future mechanistic validation.

Animals↗