Effects of Increased Von Willebrand Factor Levels on Primary Hemostasis in Thrombocytopenic Patients with Liver Cirrhosis

In patients with liver cirrhosis procoagulant and anticoagulant changes occur simultaneously. During primary hemostasis, platelets adhere to subendothelial structures, via von Willebrand factor (vWF). We aimed to investigate the influence of vWF on primary hemostasis in patients with liver cirrhosis. Therefore we assessed in-vitro bleeding time as marker of primary hemostasis in cirrhotic patients, measuring the Platelet Function Analyzer (PFA-100) closure times with collagen and epinephrine (Col-Epi, upper limit of normal ≤165 s) or collagen and ADP (Col-ADP, upper limit of normal ≤118 s). If Col-Epi and Col-ADP were prolonged, the PFA-100 was considered to be pathological. Effects of vWF on primary hemostasis in thrombocytopenic patients were analyzed and plasma vWF levels were modified by adding recombinant vWF or anti-vWF antibody. Of the 72 included cirrhotic patients, 32 (44.4%) showed a pathological result for the PFA-100. They had mean closure times (± SD) of 180±62 s with Col-Epi and 160±70 s with Col-ADP. Multivariate analysis revealed that hematocrit (P = 0.027) and vWF-antigen levels (P = 0.010) are the predictors of a pathological PFA-100 test in cirrhotic patients. In 21.4% of cirrhotic patients with platelet count ≥150/nL and hematocrit ≥27.0%, pathological PFA-100 results were found. In thrombocytopenic (<150/nL) patients with cirrhosis, normal PFA-100 results were associated with higher vWF-antigen levels (462.3±235.9% vs. 338.7±151.6%, P = 0.021). These results were confirmed by multivariate analysis in these patients as well as by adding recombinant vWF or polyclonal anti-vWF antibody that significantly shortened or prolonged closure times, respectively. In conclusion, primary hemostasis is impaired in cirrhotic patients. The effect of reduced platelet count in cirrhotic patients can at least be partly compensated by increased vWF levels. Recombinant vWF could be an alternative to platelet transfusions in the future.     

Effects of polarization in the presence and absence of biocides on biofilms in a simulated paper machine water

The antifouling potential of electric polarization combined and not combined with biocides was studied in nonsaline warm water with high organic content. Deinococcus geothermalis is a bacterium known for forming colored biofilms in paper machines and for its persistence against cleaning and chemical treatments. When D. geothermalis biofilms grown for 24 h in simulated paper machine water were exposed to cathodic or cathodically weighted pulsed polarization at least 60% (P < 0.05) of the biofilms were removed from stainless steel (AISI 316L). Biofilm removal by 25 ppm (effective substances 5-25 ppm) of oxidizing biocides (bromochloro-5,5-dimethylhydantoin, 2,2-dibromo-2-cyanoacetamide, peracetic acid) increased to 70% when combined with cathodically weighted pulsed polarization. Using a novel instrument that allows real-time detection of reactive oxygen species (ROS) we showed that the polarization program effective in antifouling generated ROS in a pulsed manner on the steel surface. We thus suggest that the observed added value of oxidative biocides combined with polarization depended on ROS. This suggestion was supported by the finding that a reductive biocide, methylene bisthiocyanate, counteracted the antifouling effect of polarization.     

Antibodies to a full-length VAR2CSA immunogen are broadly strain-transcendent but do not cross-inhibit different placental-type parasite isolates

The high molecular weight, multidomain VAR2CSA protein mediating adhesion of Plasmodium falciparum-infected erythrocytes in the placenta is the leading candidate for a pregnancy malaria vaccine. However, it has been difficult so far to generate strong and consistent adhesion blocking antibody responses against most single-domain VAR2CSA immunogens. Recent advances in expression of the full-length recombinant protein showed it binds with much greater specificity and affinity to chondroitin sulphate A (CSA) than individual VAR2CSA domains. This raises the possibility that a specific CSA binding pocket(s) is formed in the full length antigen and could be an important target for vaccine development. In this study, we compared the immunogenicity of a full-length VAR2CSA recombinant protein containing all six Duffy binding-like (DBL) domains to that of a three-domain construct (DBL4-6) in mice and rabbits. Animals immunized with either immunogen acquired antibodies reacting with several VAR2CSA individual domains by ELISA, but antibody responses against the highly conserved DBL4 domain were weaker in animals immunized with full-length DBL1-6 recombinant protein compared to DBL4-6 recombinant protein. Both immunogens induced cross-reactive antibodies to several heterologous CSA-binding parasite lines expressing different VAR2CSA orthologues. However, antibodies that inhibited adhesion of parasites to CSA were only elicited in rabbits immunized with full-length immunogen and inhibition was restricted to the homologous CSA-binding parasite. These findings demonstrate that partial and full-length VAR2CSA immunogens induce cross-reactive antibodies, but inhibitory antibody responses to full-length immunogen were highly allele-specific and variable between animal species.     

Pseudoxanthomonas bacteria that drive deposit formation of wood extractives can be flocculated by cationic polyelectrolytes

Runnability problems caused by suspended bacteria in water using industries, have, in contrast to biofilms, received little attention. We describe here that Pseudoxanthomonas taiwanensis, a wide-spread and abundant bacterium in paper machine water circuits, aggregated dispersions of wood extractives ("pitch") and resin acid, under conditions prevailing in machine water circuits (10⁹ cfu ml⁻¹, pH 8, 45°C). The aggregates were large enough (up to 50 μm) so that they could be expected to clog wires and felts and to reduce dewatering of the fiber web. The Pseudoxanthomonas bacteria were negatively charged over a pH range of 3.2–10. Cationic polyelectrolytes of the types used as retention aids or fixatives to flocculate "anionic trash" in paper machines were effective in flocculating the Pseudoxanthomonas bacteria. The polyelectrolyte most effective for this purpose was of high molecular weight (7–8 × 10⁶ g mol⁻¹) and low charge density (1 meq g⁻¹), whereas polyelectrolytes that effectively zeroed the electrophoretic mobility (i.e., neutralized the negative charge) of the bacterium were less effective in flocculating the bacteria. Based on the results, we concluded that the polyelectrolytes functioning by bridging mechanism, rather than by neutralization of the negative charge, may be useful as tools for reducing harmful deposits resulting from interaction of bacteria with wood extractives in warm water industry.     

In vitro studies on intestinal calcium and phosphate transport in horses

Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca^2 +) and inorganic phosphate (P_i) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca^2 + and P_i transport are limited. The aim of the present study was to characterize the intestinal Ca^2 + and P_i transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca^2 + transport at both mRNA and protein level. Among the different intestinal segments, the duodenum showed significant and highest active Ca^2 + absorption. The findings from RT-PCR and Western blot analysis suggest that the epithelial Ca^2 + channel TRPV6, the cytosolic calcium binding protein calbindin-D_9K and the plasma membrane calcium ATPase PMCA may be involved in active transcellular Ca^2 + transport. Regarding the P_i transport, the results indicate significant active P_i secretion in the jejunum, but the contributing mechanisms remain unclear. A significant inhibiting effect of ouabain as an antagonist of the basolateral Na⁺/K⁺-ATPase on the serosal-to-mucosal P_i transport suggests a pivotal role of Na⁺ in jejunal P_i transport in the horse.     

Current geographical ranges of Malagasy dung beetles are not delimited by large rivers

Aim We investigated whether the largest river (Mangoro) on the east coast of Madagascar acts as a barrier to dispersal in dung beetles by comparing species composition and genetic differentiation of the most common species on the two banks of the river. Moreover, by analysing the current geographical ranges of all wet forest dung beetle species, possible long‐term effects of the largest rivers on the distribution of species were assessed. Location Madagascar. Methods Dung beetles were sampled with baited pitfall traps at a downstream and an upstream locality on the two banks of the Mangoro River. The most common species, Nanos binotatus (Canthonini), was sequenced for cytochrome c oxidase subunit I (COI; 804 bp) to characterize within‐population diversity and between‐population genetic differentiation. For the analysis of species geographical range boundaries in relation to the position of the largest rivers on the east coast, a database including all the records for 158 wet forest species was used. The congruence of species range boundaries with the positions of the rivers was tested with a randomization test. Results All common species were found on both sides of the Mangoro River. In Nanos binotatus, haplotype and nucleotide diversities ranged from 0.25 to 0.85 and 0.001 to 0.01, respectively. Population differentiation was high and significant in all comparisons (P < 0.01; average F_ST = 0.61). The differentiation was not significantly higher across than along the river, as would be expected by the riverine barrier hypothesis. There was no indication that the range boundaries of wet forest dung beetle species would generally coincide with the largest rivers in eastern Madagascar. Main conclusions The results provide little support for the riverine barrier hypothesis as an explanation for the current range boundaries of dung beetles in eastern Madagascar. However, extensive deforestation of the coastal regions in eastern Madagascar may have caused a great shrinkage of the ranges of many forest‐dwelling species. Thus the present‐day distributions may not reflect accurately the patterns of the past geographical ranges of the species.     

Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet

Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability.     

Extracytoplasmic processes impaired by inactivation of trxA (thioredoxin gene) in Bacillus subtilis

The trxA gene is regarded as essential in Bacillus subtilis, but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is expected if the TrxA protein is important for the activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phosphosulfate reductase. We demonstrate here that a TrxA deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD deficiency, indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.