A validated model of passive skeletal muscle to predict force and intramuscular pressure

The passive properties of skeletal muscle are often overlooked in muscle studies, yet they play a key role in tissue function in vivo. Studies analyzing and modeling muscle passive properties, while not uncommon, have never investigated the role of fluid content within the tissue. Additionally, intramuscular pressure (IMP) has been shown to correlate with muscle force in vivo and could be used to predict muscle force in the clinic. In this study, a novel model of skeletal muscle was developed and validated to predict both muscle stress and IMP under passive conditions for the New Zealand White Rabbit tibialis anterior. This model is the first to include fluid content within the tissue and uses whole muscle geometry. A nonlinear optimization scheme was highly effective at fitting model stress output to experimental stress data (normalized mean square error or NMSE fit value of 0.993) and validation showed very good agreement to experimental data (NMSE fit values of 0.955 and 0.860 for IMP and stress, respectively). While future work to include muscle activation would broaden the physiological application of this model, the passive implementation could be used to guide surgeries where passive muscle is stretched.     

Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.     

Purification of the Chlorella HUP1 hexose—proton symporter to homogeneity and its reconstitution in vitro

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose—proton sym- porter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of d -glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-β-d -glucoside in the presence of Escherichia colil -α-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H⁺-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate d -glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of d -glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.     

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine. An effective, mild procedure based on N alpha-fluorenylmethyloxycarbonyl (Fmoc) protection and side-chain anchoring to a tris(alkoxy)benzylamide (PAL) handle

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N alpha-Fmoc-C alpha-tert.-butyl aspartate or glutamate via the free omega-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF3COOH--CH2Cl2, 90 min at 25 degrees, releases the desired peptides in greater than 95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.     

Linkage disequilibrium studies in multiple endocrine neoplasia type 1 (MEN1)

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary. The MEN1 gene has been localised to a 2-Mb region of chromosome 11q13 by meiotic mapping studies in MEN1 families. Such studies may have a limited resolution of approximately 1 cM (i.e. 1 Mb) and we have therefore investigated 96 MEN1 families (40 British, 17 French, 12 Finnish, 7 Swedish, 7 Dutch, 7 North American, 2 Australian, 1 New Zealand, 1 German, 1 Spanish and 1 Danish) for linkage disequilibrium, in order to facilitate a finer mapping resolution. We have utilised five microsatellite DNA sequence polymorphisms from the candidate region and have accurately determined their allele sizes, which ranged from 161 bp to 272 bp. The heterozygosity and number of alleles (given in brackets), respectively, at the loci were: D11S1883 (76%, 11), D11S457 (55%, 5), PYGM (94%, 18), D11S1783 (10%, 4) and D11S449 (87%, 16). Allelic association was assessed by Chi-square 2 ×n contingency tables, by Fisher exact 2 ×n contingency tables and by a likelihood-based approach. The results of haplotype analysis revealed 91 different affected haplotypes in the 96 families, an identical affected haplotype being observed in no more than two families. These results indicate the absence of an ancestral affected haplotype. Significant linkage disequilibrium (P < 0.005) could be established amongst the microsatellite loci but not between the loci and MEN1 in either the total population or in any of the geographical sub-populations. The absence of linkage disequilibrium between MEN1 and the polymorphic loci is probably the result of the occurrence of multiple different disease-causing mutations in MEN1. Received: 1 April 1997 / Accepted: 25 June 1997     

How reliable are the Powell–Wetherall plot method and the maximum-length approach? Implications for length-based studies of growth and mortality

Length-based methods are the cornerstone of many population studies and stock assessments. This study tested two widely used methods: the Powell–Wetherall (P–W) plot and the L_max approach (i.e., estimating L_∞ directly from L_max). In most simulations, P–W estimates of the ratio total mortality/growth (Z/K ratio) were biased beyond acceptable limits (bias?>?30%). Bias in Z/K showed a complex behavior, without possible corrections. Estimates of asymptotic length (L_∞) were less biased than Z/K, but were very sensitive to intra-cohort variability in growth and to changes in the occurrence of large individuals in the sample. Exclusion of the largest size classes during the regression procedure or weighing by abundance does not solve these issues. Perfect linearization of the data and extremely narrow confidence intervals for Z/K will lead users to erroneous overconfidence in outputs. Clearly, the P–W method is not suitable for the assessment of Z/K ratios of natural populations. Estimation of L_∞ may be tentatively possible under very specific conditions, with necessary external verifications. Also, this study demonstrates that there is no way to estimate L_∞ directly from L_max, since there is no particular relationship to expect a priori between L_∞ and L_max. Errors in estimating L_∞ directly affect the estimate of the growth constant K and all other subsequent calculations in population studies, stock assessments and ecosystem models. New approaches are urgently needed for length-based studies of body growth (e.g., unconstrained curve fit with subsequent bootstrapping), that consider the inherent uncertainty regarding the underlying data and processes.     

Capturing pair-wise epistatic effects associated with three agronomic traits in barley

Genetic association mapping has been widely applied to determine genetic markers favorably associated with a trait of interest and provide information for marker-assisted selection. Many association mapping studies commonly focus on main effects due to intolerable computing intensity. This study aims to select several sets of DNA markers with potential epistasis to maximize genetic variations of some key agronomic traits in barley. By doing so, we integrated a MDR (multifactor dimensionality reduction) method with a forward variable selection approach. This integrated approach was used to determine single nucleotide polymorphism pairs with epistasis effects associated with three agronomic traits: heading date, plant height, and grain yield in barley from the barley Coordinated Agricultural Project. Our results showed that four, seven, and five SNP pairs accounted for 51.06, 45.66 and 40.42% for heading date, plant height, and grain yield, respectively with epistasis being considered, while corresponding contributions to these three traits were 45.32, 31.39, 31.31%, respectively without epistasis being included. The results suggested that epistasis model was more effective than non-epistasis model in this study and can be more preferred for other applications.