Microscopic Mechanisms of Strain Hardening in Glassy Polymers

The mechanisms underlying the increase in stress for large mechanical strains of a polymer glass, quantified by the strain-hardening modulus, are still poorly understood. In the present paper we aim to elucidate this matter and present new mechanisms. Molecular-dynamics simulations of two polymers with very different strain-hardening moduli (polycarbonate and polystyrene) have been carried out. Nonaffine displacements occur because of steric hindrances and connectivity constraints. We argue that it is not necessary to introduce the concept of entanglements to understand strain hardening, but that hardening is rather coupled with the increase in the rate of nonaffine particle displacements. This rate increases faster for polycarbonate, which has the higher strain-hardening modulus. Also more nonaffine chain stretching is present for polycarbonate. It is shown that the inner distances of such a nonaffinely deformed chain can be well described by the inner distances of the worm-like chain, but with an effective stiffness length (equal to the Kuhn length for an infinite worm-like chain) that increases during deformation. It originates from the finite extensibility of the chain. In this way the increase in nonaffine particle displacement can be understood as resulting from an increase in the effective stiffness length of the perturbed chain during deformation, so that at larger strains a higher rate of plastic events in terms of nonaffine displacement is necessary, causing in turn the observed strain hardening in polymer glasses.

Localization of eIF4A-III in the nucleolus and splicing speckles is an indicator of plant stress

The mechanisms of long-term adaptation to low oxygen environment are quite well studied, but little is known about the sensing of oxygen shortage, the signal transduction and the short-term effects of hypoxia in plant cells. We have found that an RNA helicase eIF4A-III, a putative component of the Exon Junction Complex, rapidly changes its pattern of localisation in the plant nucleus under hypoxic conditions. In normal cell growth conditions GFP- eIF4A-III was mainly nucleoplasmic, but in hypoxia stress conditions it moved to the nucleolus and splicing speckles. This transition occurred within 15-20 min in Arabidopsis culture cells and seedling root cells, but took more than 2 h in tobacco BY-2 culture cells. Inhibition of respiration, transcription or phosphorylation in cells and ethanol treatment had similar effects to hypoxia. The most likely consequence is that a certain mRNA population will remain bound to the eIF4A-III and other mRNA processing proteins, rather than being transported from the nucleus to the cytoplasm, and thus its translation will be suspended.

Instability of surface-temperature filaments in strain and shear

The effects of uniform straining and shearing on the stability of a surface quasi-geostrophic temperature filament are investigated. Straining is shown to stabilize perturbations for wide filaments but only for a finite time until the filament thins to a critical width, after which some perturbations can grow. No filament can be stabilized in practice, since there are perturbations that can grow large for any strain rate. The optimally growing perturbations, defined as solutions that reach a certain threshold amplitude first, are found numerically for a wide range of parameter values. The radii of the vortices formed through nonlinear roll-up are found to be proportional to θ/s, where θ is the temperature anomaly of the filament and s the strain rate, and are not dependent on the initial size of the filament. Shearing is shown to reduce the normal-mode growth rates, but it cannot stabilize them completely when there are temperature discontinuities in the basic state; smooth filaments can be stabilized completely by shearing and a simple scaling argument provides the shear rate required. Copyright © 2010 Royal Meteorological Society

Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein

CSRP3 or muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with alpha-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myo filaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli. (C) 2009 Elsevier Inc. All rights reserved.

Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed. (c) 2008 Elsevier Inc. All rights reserved.

Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway

We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-beta production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-beta stimulation. Through the generation of recombinant viruses, we were able to show that this is not due to a loss of the ability of the NS1 genes to suppress IFN-beta induction; indeed, the NS1 genes behaved similarly with respect to their abilities to block dsRNA signaling. Interestingly, replication of A/Sydney/5/97 virus was less Susceptible to pre-treatment with IFN-alpha than the other viruses. In contrast to the universal effect on dsRNA signaling, we noted differences in the effect of NS1 proteins on expression of interferon stimulated genes and also genes induced by a distinct pathway. The majority of NS1 proteins blocked expression From both IFN-dependent and TNF-dependent promoters by an apparent post-transcriptional mechanism. The NS1 gene of A/PR/8/34 NS1 did not confer these blocks. We noted striking differences in the Cellular localization of different influenza A virus NS1 proteins during infection, which might explain differences in biological activity. (C) 2005 Elsevier Inc. All rights reserved.

Thiamine is synthesized by a salvage pathway in Rhizobium leguminosarum bv. viciae strain 3841

In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only "pin" colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(beta-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

Effect of time of day and rabbit strain on patterns of aortic wall permeability

Lipid deposits occur more frequently downstream of branch points than upstream in immature rabbit and human aortas but the opposite pattern is seen in mature vessels. These distributions correlate spatially with age-related patterns of aortic permeability, observed in rabbits, and may be determined by them. The mature but not the immature pattern of permeability is dependent on endogenous nitric oxide synthesis. Although the transport patterns have hitherto seemed robust, recent studies have given the upstream pattern in some mature rabbits but the downstream pattern in others. Here we show that transport in mature rabbits is significantly skewed to the downstream pattern in the afternoon compared with the morning (P < 0.05), and switches from a downstream to an upstream pattern at around 21 months in rabbits of the Murex strain, but at twice this age in Highgate rabbits (P < 0.001). The effect of time of day was not explained by changes in nitric oxide production, assessed from plasma levels of nitrate and nitrate, nor did it correlate with conduit artery tone, assessed from the shape of the peripheral pulse wave. The effect of strain could not be explained by variation in nitric oxide production nor by differences in wall structure. The effects of time of day and rabbit strain on permeability patterns explain recent discrepancies, provide a useful tool for investigating underlying mechanisms and may have implications for human disease.

Immunohistochemical localization of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in bovine oocytes during in vitro maturation and fertilization

The aim of this study was to evaluate the distribution of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in immature oocytes and in matured oocytes before and after IVF. Denuded oocytes were submitted to a whole-mount immunofluorescence procedure. Specimens were imaged and fluorescent intensities quantified by scanning laser confocal microscopy. Immunoreactivity for inhibin alpha subunit (both alpha(C) and pro-alpha. regions), abundant in the ooplasm of immature oocytes, decreased after maturation (a 68% and 88% decrease, respectively; P < 0.001), but increased after IVF by 2- and 5.7-fold, respectively (P < 0.01). Intense staining for PA was detected in immature oocytes (predominantly in the outer ooplasm and zona pellucida) but after maturation and fertilization it was localized mainly in the zona pellucida, perivitelline space and oolemma. Immunoreactivity for RA in the ooplasm decreased by 58% after maturation (P < 0.001) but increased again by 75% after fertilization (P < 0.01). Immunoreactivity for beta(B) was localized mainly in the zona pellucida and did not change after maturation. However, immurloreactivity for beta(B) was not detected in the zona pellucida after fertilization, but remained unchanged in unfertilized oocytes. Immunoreactivity for follistatin was detected in the ooplasm and zona pellucida of immature oocytes but decreased progressively in the ooplasm after maturation (a 63% decrease; P < 0.001) and did not change after IVF. Examination of partially denuded cumulus-oocyte complexes confirmed abundant expression of alpha(C), pro-alpha, beta(A) and follistatin immunoreactivity in cumulus cells, whereas beta(B) subunit staining was weak or absent in cumulus cells, but intense in the zona pellucida. In conclusion, the present study shows that qualitative and quantitative changes in the distribution of inhibin/activin subunits and follistatin accompany oocyte maturation and fertilization. The possibility, indicated by these observations, that activin A and activin B may play distinct roles in bovine oocyte maturation and fertilization warrants further study.

Damage rate is a predictor of fatigue life and creep strain rate in fatigue of tensile human cortical bone samples

We present results on the growth of damage in 29 fatigue tests of human femoral cortical bone from four individuals, aged 53–79. In these tests we examine the interdependency of stress, cycles to failure, rate of creep strain, and rate of modulus loss. The behavior of creep rates has been reported recently for the same donors as an effect of stress and cycles (Cotton, J. R., Zioupos, P., Winwood, K., and Taylor, M., 2003, "Analysis of Creep Strain During Tensile Fatigue of Cortical Bone," J. Biomech. 36, pp. 943–949). In the present paper we first examine how the evolution of damage (drop in modulus per cycle) is associated with the stress level or the "normalized stress" level (stress divided by specimen modulus), and results show the rate of modulus loss fits better as a function of normalized stress. However, we find here that even better correlations can be established between either the cycles to failure or creep rates versus rates of damage than any of these three measures versus normalized stress. The data indicate that damage rates can be excellent predictors of fatigue life and creep strain rates in tensile fatigue of human cortical bone for use in practical problems and computer simulations.

Analysis of creep strain during tensile fatigue of cortical bone

During fatigue tests of cortical bone specimens, at the unload portion of the cycle (zero stress) non-zero strains occur and progressively accumulate as the test progresses. This non-zero strain is hypothesised to be mostly, if not entirely, describable as creep. This work examines the rate of accumulation of this strain and quantifies its stress dependency. A published relationship determined from creep tests of cortical bone (Journal of Biomechanics 21 (1988) 623) is combined with knowledge of the stress history during fatigue testing to derive an expression for the amount of creep strain in fatigue tests. Fatigue tests on 31 bone samples from four individuals showed strong correlations between creep strain rate and both stress and “normalised stress” (σ/E) during tensile fatigue testing (0–T). Combined results were good (r2=0.78) and differences between the various individuals, in particular, vanished when effects were examined against normalised stress values. Constants of the regression showed equivalence to constants derived in creep tests. The universality of the results, with respect to four different individuals of both sexes, shows great promise for use in computational models of fatigue in bone structures.

Synthesis of prebiotic galactooligosaccharides using whole cells of a novel strain, Bifidobacterium bifidum NCIMB 41171

A novel strain of Bifidobacterium bifidum NCIMB 41171, isolated from a faecal sample from a healthy human volunteer and able to express beta-galactosidase activity, was used in synthesis reactions for the production of galactooligosaccharide from lactose. The beta-galactosidase activity of whole bifidobacterial cells showed an optimum activity at pH 6.8-7.0 and 40 degrees C. The transgalactosylation activity of the B. bifidum cells from 50% (w/w) lactose resulted in a galactooligosaccharide mixture (20% w/w) comprising (w/w): 25% disaccharides, 35% trisaccharides, 25% tetrasaccharides and 15% pentasaccharides. Using different initial lactose concentrations, the conversion rate to galactooligosaccharides was maximum (35%) when 55% (w/w) lactose was used. In fermentation experiments, B. bifidum showed an increased preference towards the produced galactooligosaccharide mixture, displaying higher growth rate and short-chain fatty acid production when compared with commercially available oligosaccharides.

Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-based substrates

The production of volatile compounds by the probiotic strain, Lactobacillus plantarum NCIMB 8826, in cereal-based media (oat, wheat. barley and malt) was investigated. Sixty compounds, including fatty acids and their esters, amides, alcohols, aldehydes, aromatic hydrocarbons, furans, ketones, peroxides and pyrans, were identified. L. plantarum significantly changed the aroma profile of the four cereal broths. and each substrate showed a specific volatiles profile. Oat and barley media were the substrates more influenced by the fermentation process. The most abundant volatiles detected in oat, wheat, barley and malt were oleic acid, linoleic acid. acetic acid, and 5-hydroxymethylfurfural, respectively. Analysis of these products confirmed the heterofermentative pathway of L plantarum. Maillard compounds were not detected during sterilisation and fermentation. This study is the first to report the volatile composition of probiotic drinks produce with non-supplemented cereal-based media and the results obtained could contribute to the development of new non-dairy probiotic formulations. (C) 2009 Elsevier Ltd. All rights reserved.