Simultaneous saccharification and fermentation of Kanlow switchgrass by thermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading, temperature and higher solid loadings

Switchgrass (Panicum virgatum) was subjected to hydrothermolysis pretreatment and then used to study the effect of enzyme loading and temperature in a simultaneous saccharification and fermentation (SSF) with the thermotolerant yeast strain Kluyveromyces marxianus IMB3 at 8% solid loading. Various loadings of Accellerase 1500 between 0.1 and 1.1 mL g(-1) glucan were tested in SSF at 45 °C (activity of enzyme was 82.2 FPU mL(-1)). The optimum enzyme loading was 0.7 mL g(-1) glucan based on the six different enzyme loadings tested. SSFs were performed at 37, 41 and 45 °C with an enzyme loading of 0.7 mL g(-1) glucan. The highest ethanol concentration of 22.5 g L(-1) was obtained after 168 h with SSF at 45 °C, which was equivalent to 86% yield. Four different batch and fed-batch strategies were evaluated using a total solid loading of 12% (dry basis). About 32 g L(-1) ethanol was produced with the four strategies, which was equivalent to 82% yield.     

Asthenozoospermia in mice with targeted deletion of the sperm mitochondrion-associated cysteine-rich protein (Smcp) gene

The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp(-/-) female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp(-/-) mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.     

S100A13-lipid interactions-role in the non-classical release of the acidic fibroblast growth factor

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca2+-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca2+ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.     

Polymorphism associated with the Schistosoma mansoni tetraspanin-2 gene

A vaccine against schistosomiasis would contribute significantly to reducing the 3-70 million disability-adjusted life years lost annually to the disease. Towards this end, inoculation with the large extracellular loop (EC-2) of Schistosoma mansoni tetraspanin-2 protein (Sm-TSP-2) has proved effective in reducing worm and egg burdens in S. mansoni-infected mice. The EC-2 loop of Schistosoma japonicum TSP-2, however, has been found to be highly polymorphic, perhaps diminishing the likelihood that this antigen can be used for vaccination against this species. Here, we examine polymorphism of the EC-2 of Sm-TSP-2 in genetically unique worms derived from six individuals from Kisumu, Kenya.     

The effects of a constant sprint-to-rest ratio and recovery mode on repeated sprint performance

It is unclear if a constant sprint-to-rest ratio allows full performance recovery between repeated sprints over different distances. This is important for the development of sprint-training programs. Additionally, there is conflicting evidence on whether active recovery enhances sprint performance. Three repeated sprint protocols were used (22 × 15, 13 × 30, and 8 × 50 m), with each having an active and passive recovery. Each trial was conducted with an initial sprint-to-rest ratio of 1:10. Repeated sprints were analyzed by comparing the first sprint to the last sprint. For the 15-m trials, there were no significant main effects for recovery or time and no significant interaction. For the 30-m trials, there was no main effect for recovery, but a main effect for time (F[1,10] = 15.995, p = 0.003; mean difference = 0.20 seconds, 95% confidence interval [CI] = 0.09-0.31 seconds, d = 1.4 [large effect]). There was no interaction of recovery and time in the 30-m trials. For the 50-m trials, there was no main effect for recovery, but a main effect for time (F[1,10] = 34.225, p = 0.0002; mean difference = 0.39 seconds, 95% CI = 0.24-0.55 seconds, d = 1.3 [large effect]). There was no interaction of recovery and time in the 50-m trials. The results demonstrate that a 1:10 sprint-to-rest ratio allows full performance recovery between 15-m sprints, but not between sprints of 30 or 50 m, and that recovery mode did not influence repeated sprint performance.     

Toll-like Receptor 4 Is Expressed on Intestinal Stem Cells and Regulates Their Proliferation and Apoptosis via the p53 Up-regulated Modulator of Apoptosis

Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.     

Significance of serum trace element status in patients with rheumatic heart disease: a prospective study

It is known that certain trace elements can affect various heart diseases. In this study, we aimed to evaluate the changes in concentrations of certain serum trace elements in patients with chronic rheumatic heart disease (RHD). Serum analysis of selenium (Se), zinc (Zn), and copper (Cu) trace elements was assayed by atomic absorption spectrophotometry. RHD patients had significantly lower serum concentrations of Se and Zn than control subjects (p < 0.05 and p < 0.001, respectively). However, the serum Cu concentration was significantly higher in RHD patients than in controls (1.93 +/- 0.59 microg/L vs 1.06 +/- 0.29 microg/L; p < 0.001). Similarly, the Cu/Zn ratio in RHD patients was higher than in control subjects (4.70 +/- 0.92 vs 1.68 +/- 0.45; p < 0.001). Additionally, no significant correlation was found among these trace element concentrations and the functional capacity classes (p > 0.05). RHD patients had decreased serum Se and Zn element concentrations and increased serum Cu element concentration. We suggest that Se and Zn deficiency might be contributory factors in the development of rheumatic heart disease, and a high Cu concentration and a high Cu/Zn ratio might reflect an ongoing inflammatory process in this disease.