Regulation of PDH in human arm and leg muscles at rest and during intense exercise

To test the hypothesis that pyruvate dehydrogenase (PDH) is differentially regulated in specific human muscles, regulation of PDH was examined in triceps, deltoid, and vastus lateralis at rest and during intense exercise. To elicit considerable glycogen use, subjects performed 30 min of exhaustive arm cycling on two occasions and leg cycling exercise on a third day. Muscle biopsies were obtained from deltoid or triceps on the arm exercise days and from vastus lateralis on the leg cycling day. Resting PDH protein content and phosphorylation on PDH-E1 alpha sites 1 and 2 were higher (P < or = 0.05) in vastus lateralis than in triceps and deltoid as was the activity of oxidative enzymes. Net muscle glycogen utilization was similar in vastus lateralis and triceps ( approximately 50%) but less in deltoid (likely reflecting less recruitment of deltoid), while muscle lactate accumulation was approximately 55% higher (P < or = 0.05) in triceps than vastus lateralis. Exercise induced (P < or = 0.05) dephosphorylation of both PDH-E1 alpha site 1 and site 2 in all three muscles, but it was more pronounced at PDH-E1 alpha site 1 in triceps than in vastus lateralis (P < or = 0.05). The increase in activity of the active form of PDH (PDHa) after 10 min of exercise was more marked in vastus lateralis ( approximately 246%) than in triceps ( approximately 160%), but when it was related to total PDH-E1 alpha protein content, no difference was evident. In conclusion, PDH protein content seems to be related to metabolic enzyme profile, rather than myosin heavy chain composition, and less PDH capacity in triceps is a likely contributing factor to higher lactate accumulation in triceps than in vastus lateralis.     

Changes in neurodynamic parameters of the human brain during an individual annual cycle

Neurodynamic parameters of the human brain and their changes depending on the trimester of an individual annual cycle (IAC), which lasts from one birthday to the next, were studied. In the course of a year, fourfold examination (in February, April, July, and October) of 124 men and 242 women aged from 18 to 20 years was carried out. According to psychological characteristics, the examined subjects were divided into introverts and extroverts. The strength and mobility of nervous processes in extrovert men tended to decrease in the fourth trimester of the IAC, simultaneously with a decrease in the overall state of health. Introvert men exhibited a lower brain efficiency than extrovert men; changes in their brain functional mobility during an IAC were more pronounced than in the case of extrovert men; the lowest parameters were recorded in the second trimester. Annual changes in neurodynamic parameters in women were smaller than in men.     

Non-standard O(2) consumption-temperature curves during rest and isometric exercise in human skeletal muscle

The present work was aimed at measuring intramuscular oxygen consumption (O(2)) as a function of temperature (T), in human forearm, during rest and aerobic isometric exercise (4% of the maximal voluntary contraction, MVC). Based upon results from in vitro experiments performed on isolated mitochondria of animal species, it was hypothesised that, during isometric exercise, the O(2)-T curve should display a maximum for some 'optimal' T. Intramuscular T and measurements were performed using a combined deep body temperature/near infrared probe during muscle cooling. At rest, O(2) increased non-linearly and monotonically as a function of T (n=8). O(2) increased approximately 2 times when going from 26 to 36 degrees C. A log(O(2))-T plot or a log(O(2))-1/T did not linearise the data. During isometric contraction, O(2) values at 26.8+/-0.6, 28.6+/-0.9, 31.9+/-0.9 and 35.9+/-0.9 degrees C were 3.04+/-1.26, 7.60+/-1.64, 4.43+/-1.95, and 6.64+/-1.37 micromol 100 g(-1) min(-1), respectively (n=6). The O(2) value at 28.6 degrees C was significantly higher (P<0.05) than that at 26.8 and 31.9 degrees C. The 'sudden' O(2) change at 28.6 degrees C is compatible with the phenomenon observed at the mitochondrial level.     

Statistical distributions of test statistics used for quantitative trait association mapping in structured populations

Background

Spurious associations between single nucleotide polymorphisms and phenotypes are a major issue in genome-wide association studies and have led to underestimation of type 1 error rate and overestimation of the number of quantitative trait loci found. Many authors have investigated the influence of population structure on the robustness of methods by simulation. This paper is aimed at developing further the algebraic formalization of power and type 1 error rate for some of the classical statistical methods used: simple regression, two approximate methods of mixed models involving the effect of a single nucleotide polymorphism (SNP) and a random polygenic effect (GRAMMAR and FASTA) and the transmission/disequilibrium test for quantitative traits and nuclear families. Analytical formulae were derived using matrix algebra for the first and second moments of the statistical tests, assuming a true mixed model with a polygenic effect and SNP effects.

Results

The expectation and variance of the test statistics and their marginal expectations and variances according to the distribution of genotypes and estimators of variance components are given as a function of the relationship matrix and of the heritability of the polygenic effect. These formulae were used to compute type 1 error rate and power for any kind of relationship matrix between phenotyped and genotyped individuals for any level of heritability. For the regression method, type 1 error rate increased with the variability of relationships and with heritability, but decreased with the GRAMMAR method and was not affected with the FASTA and quantitative transmission/disequilibrium test methods.

Conclusions

The formulae can be easily used to provide the correct threshold of type 1 error rate and to calculate the power when designing experiments or data collection protocols. The results concerning the efficacy of each method agree with simulation results in the literature but were generalized in this work. The power of the GRAMMAR method was equal to the power of the FASTA method at the same type 1 error rate. The power of the quantitative transmission/disequilibrium test was low. In conclusion, the FASTA method, which is very close to the full mixed model, is recommended in association mapping studies.     

An intronic signal for alternative splicing in the human genome

An important level at which the expression of programmed cell death (PCD) genes is regulated is alternative splicing. Our previous work identified an intronic splicing regulatory element in caspase-2 (casp-2) gene. This 100-nucleotide intronic element, In100, consists of an upstream region containing a decoy 3' splice site and a downstream region containing binding sites for splicing repressor PTB. Based on the signal of In100 element in casp-2, we have detected the In100-like sequences as a family of sequence elements associated with alternative splicing in the human genome by using computational and experimental approaches. A survey of human genome reveals the presence of more than four thousand In100-like elements in 2757 genes. These In100-like elements tend to locate more frequent in intronic regions than exonic regions. EST analyses indicate that the presence of In100-like elements correlates with the skipping of their immediate upstream exons, with 526 genes showing exon skipping in such a manner. In addition, In100-like elements are found in several human caspase genes near exons encoding the caspase active domain. RT-PCR experiments show that these caspase genes indeed undergo alternative splicing in a pattern predicted to affect their functional activity. Together, these results suggest that the In100-like elements represent a family of intronic signals for alternative splicing in the human genome.     

Assessment of boundary conditions for CFD simulation in human carotid artery

Computational fluid dynamics (CFD) is an increasingly used method for investigation of hemodynamic parameters and their alterations under pathological conditions, which are important indicators for diagnosis of cardiovascular disease. In hemodynamic simulation models, the employment of appropriate boundary conditions (BCs) determines the computational accuracy of the CFD simulation in comparison with pressure and velocity measurements. In this study, we have first assessed the influence of inlet boundary conditions on hemodynamic CFD simulations. We selected two typical patients suspected of carotid artery disease, with mild stenosis and severe stenosis. Both patients underwent digital subtraction angiography (DSA), magnetic resonance angiography, and the invasive pressure guide wire measured pressure profile. We have performed computational experiments to (1) study the hemodynamic simulation outcomes of distributions of wall shear stress, pressure, pressure gradient and (2) determine the differences in hemodynamic performances caused by inlet BCs derived from DSA and Womersley analytical solution. Our study has found that the difference is related to the severity of the stenosis; the greater the stenosis, the more the difference ensues. Further, in our study, the two typical subjects with invasively measured pressure profile and thirty subjects with ultrasound Doppler velocimeter (UDV) measurement served as the criteria to evaluate the hemodynamic outcomes of wall shear stress, pressure, pressure gradient and velocity due to different outlet BCs based on the Windkessel model, structured-tree model, and fully developed flow model. According to the pressure profiles, the fully developed model appeared to have more fluctuations compared with the other two models. The Windkessel model had more singularities before convergence. The three outlet BCs models also showed good correlation with the UDV measurement, while the Windkessel model appeared to be slightly better (\( R^{2} = 0.942 \)). The structured-tree model was seen to have the best performance in terms of available computational cost and accuracy. The results of our numerical simulation and the good correlation with the computed pressure and velocity with their measurements have highlighted the effectiveness of CFD simulation in patient-specific human carotid artery with suspected stenosis.     

Strategies for characterizing highly polymorphic markers in human gene mapping.

Before new markers are thoroughly characterized, they are usually screened for high polymorphism on the basis of a small panel of individuals. Four commonly used screening strategies are compared in terms of their power to correctly classify a marker as having heterozygosity of 70% or higher. A small number of typed individuals (10, say) are shown to provide good discrimination power between low- and high-heterozygosity markers when the markers have a small number of alleles. Characterizing markers in more detail requires larger sample sizes (e.g., at least 80-100 individuals) if there is to be a high probability of detecting most or all alleles. For linkage analyses involving highly polymorphic markers, the practice of arbitrarily assuming equal gene frequencies can cause serious trouble. In the presence of untyped individuals, when gene frequencies are unequal but are assumed to be equal in the analysis, recombination-fraction estimates tend to be badly biased, leading to strong false-positive evidence for linkage.     

A neuromusculoskeletal tracking method for estimating individual muscle forces in human movement

A neuromusculoskeletal tracking (NMT) method was developed to estimate muscle forces from observed motion data. The NMT method combines skeletal motion tracking and optimal neuromuscular tracking to produce forward simulations of human movement quickly and accurately. The skeletal motion tracker calculates the joint torques needed to actuate a skeletal model and track observed segment angles and ground forces in a forward simulation of the motor task. The optimal neuromuscular tracker resolves the muscle redundancy problem dynamically and finds the muscle excitations (and muscle forces) needed to produce the joint torques calculated by the skeletal motion tracker. To evaluate the accuracy of the NMT method, kinematics and ground forces obtained from an optimal control (parameter optimization) solution for maximum-height jumping were contaminated with both random and systematic noise. These data served as input observations to the NMT method as well as an inverse dynamics analysis. The NMT solution was compared to the input observations, the original optimal solution, and a simulation driven by the inverse dynamics torques. The results show that, in contrast to inverse dynamics, the NMT method is able to produce an accurate forward simulation consistent with the optimal control solution. The NMT method also requires 3 orders-of-magnitude less CPU time than parameter optimization. The speed and accuracy of the NMT method make it a promising new tool for estimating muscle forces using experimentally obtained kinematics and ground force data.     

Variation in the correction factor used for converting counts of individual Guillemots Uria aalge into breeding pairs

The correction factor k (breeding pairs/total count of birds) was measured for two nesting groups of Guillemots Uria aalge over 6–7 years. The value varied between different subgroups but there were no significant differences between groups or between years. These results, and those from other studies, suggest that a value of 0–67 is applicable to a range of colonies. Values of k for Razorbills Aha tarda were more variable. A plea is made for the presentation of original counts rather than 'corrected' figures in published work.     

Methods for protecting silica sorbents used in high-performance liquid chromatography from strongly adsorbed impurities during purification of human recombinant insulin

One of the main stages of human recombinant insulin (HRI) production is the hormone purification by means of reversed phase high-performance liquid chromatography (RP HPLC). The optimization of this stage determines the increase of the total manufacturing yield. Therefore, the cost of the sorbent used in HPLC influences the cost of the manufacturing product, i.e. HRI substance. However, resolution between HRI and its admixtures decreases with time. The reason for this is the sorbent contamination with strongly adsorbed impurities (SAI) which are accumulated during elution. In the following research several methods for sorbent protection are studied. The opinion that SAI are mainly high-molecular weight compounds was examined using gel filtration. Different sorbent types were tested for the use in guard column. The results obtained were applied and improved at preparative level.     

Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection.

The organizer at the midbrain-hindbrain boundary (MHB organizer) has been proposed to induce and polarize the midbrain during development. We investigate the requirement for the MHB organizer in acerebellar mutants, which lack a MHB and cerebellum, but retain a tectum, and are mutant for fgf8, a candidate inducer and polarizer. We examine the retinotectal projection in the mutants to assay polarity in the tectum. In mutant tecta, retinal ganglion cell (RGC) axons form overlapping termination fields, especially in the ventral tectum, and along both the anterior-posterior and dorsal-ventral axis of the tectum, consistent with a MHB requirement in generating midbrain polarity. However, polarity is not completely lost in the mutant tecta, in spite of the absence of the MHB. Moreover, graded expression of the ephrin family ligand Ephrin-A5b is eliminated, whereas Ephrin-A2 and Ephrin-A5a expression is leveled in acerebellar mutant tecta, showing that ephrins are differentially affected by the absence of the MHB. Some RGC axons overshoot beyond the mutant tectum, suggesting that the MHB also serves a barrier function for axonal growth. By transplanting whole eye primordia, we show that mapping defects and overshooting largely, but not exclusively, depend on tectal, but not retinal genotype, and thus demonstrate an independent function for Fgf8 in retinal development. The MHB organizer, possibly via Fgf8 itself, is thus required for midbrain polarisation and for restricting axonal growth, but other cell populations may also influence midbrain polarity.     

A comparison of two commercially available ELISA methods for the quantification of human plasma heat shock protein 70 during rest and exercise stress

This study compared resting and exercise heat/hypoxic stress-induced levels of plasma extracellular heat shock protein 70 (eHSP70) in humans using two commercially available enzyme-linked immunosorbent assay (ELIS)A kits. EDTA plasma samples were collected from 21 males during two separate investigations. Participants in part A completed a 60-min treadmill run in the heat (HOT70; 33.0 ± 0.1 °C, 28.7 ± 0.8 %, n = 6) at 70 % V̇O_2max. Participants in part B completed 60 min of cycling exercise at 50 % V̇O_2max in either hot (HOT50; 40.5 °C, 25.4 relative humidity (RH)%, n = 7) or hypoxic (HYP50; fraction of inspired oxygen (F_IO₂) = 0.14, 21 °C, 35 % RH, n = 8) conditions. Samples were collected prior to and immediately upon termination of exercise and analysed for eHSP70 using EKS-715 high-sensitivity HSP70 ELISA and new ENZ-KIT-101 Amp’d™ HSP70 high-sensitivity ELISA. ENZ-KIT was superior in detecting resting eHSP70 (1.54 ± 3.27 ng·mL⁻¹; range 0.08 to 14.01 ng·mL⁻¹), with concentrations obtained from 100 % of samples compared to 19 % with EKS-715 assay. The ENZ-KIT requires optimisation prior to running samples in order to ensure participants fall within the standard curve, a step not required with EKS-715. Using ENZ-KIT, a 1:4 dilution allowed for quantification of resting HSP70 in 26/32 samples, with a 1:8 (n = 3) and 1:16 (n = 3) dilution required to determine the remaining samples. After exercise, eHSP70 was detected in 6/21 and 21/21 samples using EKS-715 and ENZ-KIT, respectively. eHSP70 was increased from rest after HOT70 (p < 0.05), but not HOT50 (p > 0.05) or HYP50 (p > 0.05) when analysed using ENZ-KIT. It is recommended that future studies requiring the precise determination of resting plasma eHSP70 use the ENZ-KIT (i.e. HSP70 Amp’d® ELISA) instead of the EKS-715 assay, despite additional assay development time and cost required.     

A photographic microfluorimetric method for determining the DNA content in individual human chromosomes

A method for determination of DNA contents in individual human chromosomes has been elaborated based of the two step analysis: 1) identification of chromosomes by Q-banding; 2) photographic microfluorimetry of chromosomes after the Feulgen staining (a fluorescent variant using a Schiff-type reagent of Auramine-SO2). The DNA content in 24 human chromosomes was calculated in absolute values (fg). The data obtained are compared with the evidence published elsewhere and provided by different cytophotometric methods.