A fucosylated chondroitin sulfate from echinoderm modulates in vitro fibroblast growth factor 2-dependent angiogenesis

Fucosylated chondroitin sulfate (FucCS), a glycosaminoglycan obtained from sea cucumber, has the same structure as mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. This new polysaccharide has a more favorable effect than heparin on vascular cell growth. It inhibits smooth muscle cell proliferation as heparin, and it has a potent enhancing effect on endothelial cell proliferation and migration in the presence of heparin-binding growth factors. We now extend our studies to the effect of this glycosaminoglycan on endothelial cells to an in vitro angiogenesis model on Matrigel. FucCS, in the presence of fibroblast growth factor-2 (FGF-2), strongly increases the capacity of endothelial cells to form vascular tubes on Matrigel with a well-organized capillary-like network and typical closed structures. Comparison between the activity of native and chemically modified chondroitin sulfate from sea cucumber reveals that the sulfated fucose branches are the structural motif for the proangiogenic activity. Heparin does not induce angiogenesis in this experimental model. We also have evidence for the proposition that endothelial cell proliferation is not the sole event involved in the in vitro FGF-2-induced angiogenesis. It implies a variety of other modifications of the endothelial cells and of their interaction with the extracellular matrix, such as integrin expression and actin cytoskeleton reorganization. Finally, the proangiogenic effect of FucCS, concomitant with its capacity to prevent venous and arterial thrombosis, in animal models makes this new glycosaminoglycan a promising molecule with possible beneficial effects in pathological conditions affecting blood vessels such as the neovascularization of ischemic areas.     

Carbapenem Non-Susceptible Enterobacteriaceae in Quebec,Canada: Results of a Laboratory Surveillance Program (2010–2012)

The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) represent a major public health concern because these bacteria are usually extensively resistant to most antibiotics. In order to evaluate their dissemination in Quebec, a surveillance program was introduced in 2010. We report the molecular and epidemiological profiles of CPE isolates collected. Between August 2010 and December 2012, a total of 742 non-duplicate isolates non-susceptible to carbapenems were analysed. AmpC β-lactamase and metallo-β-lactamase production were detected by Etest and carbapenemase production by the modified Hodge test (MHT). Antibiotic susceptibility profiles were determined using broth microdilution or Etest. Clonality of Klebsiella pneumoniae carbapenemase (KPC) strains was analyzed by pulsed-field gel electrophoresis (PFGE). The presence of genes encoding carbapenemases as well as other β-lactamases was detected using PCR. Of the 742 isolates tested, 169 (22.8%) were CPE. Of these 169 isolates, 151 (89.3%) harboured a bla _KPC gene while the remaining isolates carried bla _SME (n = 9), bla _OXA-48 (n = 5), bla _NDM (n = 3), and bla _NMC (n = 1) genes. Among the 93 KPC strains presenting with a unique pattern (unique PFGE pattern and/or unique antibiotics susceptibility profile), 99% were resistant to ertapenem, 95% to imipenem, 87% to meropenem, 97% to aztreonam, 31% to colistin and 2% to tigecycline. In 19 patients, 2 to 5 KPC strains from different species or with a different PFGE pattern were isolated. CPE strains were present in the province of Quebec with the majority of strains harbouring KPC. Alternately, SME, OXA-48 and NMC containing strains were rarely found.     

Cardioversion and Risk of Adverse Events with Dabigatran versus Warfarin—A Nationwide Cohort Study

Aim

Cardioversion can rapidly and effectively restore sinus rhythm in patients with persistent atrial fibrillation. Since 2011 dabigatran has been available as an alternative to warfarin to prevent thromboembolic events in patients with non-valvular atrial fibrillation undergoing cardioversion. We studied time to cardioversion, risk of adverse events, and risk of readmission with atrial fibrillation after cardioversion according to anticoagulation therapy.

Methods and Results

Through the nationwide Danish registries we included 1,230 oral anticoagulation naïve patients with first time non-valvular atrial fibrillation and first time cardioversion from 2011 to 2012; 37% in the dabigatran group (n = 456), and 63% in the warfarin group (n = 774). Median time to cardioversion was 4.0 (interquartile range [IQR] 2.9 to 6.5) and 6.9 (IQR 3.9 to 12.1) weeks in the dabigatran and warfarin groups respectively, and the adjusted odds ratio of cardioversion within the first 4 weeks was 2.3 (95% confidence interval [CI] 1.7 to 3.1) in favor of dabigatran. The cumulative incidence of composite endpoint of stroke, bleeding or death were 2.0% and 1.0% at 30 weeks in the warfarin and dabigatran groups respectively, with an adjusted hazard ratio of 1.33 (95% CI 0.33 to 5.42). Cumulative incidence of readmission with atrial fibrillation after 30 weeks were 9% and 11% in the warfarin and dabigatran groups, respectively, and an adjusted hazard ratio of 0.66 (95% CI 0.41 to 1.08).

Conclusion

Anticoagulation treatment with dabigatran allows shorter time to cardioversion for atrial fibrillation than warfarin, and appears to be an effective and safe alternative treatment strategy to warfarin.     

Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis.

In order to study some of its enzymatic properties, phosphatidylinositol synthase 1 (AtPIS1) from the plant Arabidopsis thaliana was expressed in Escherichia coli, a host naturally devoid of phosphatidylinositol (PtdIns). In the context of the bacterial membrane and in addition to de novo synthesis, the plant enzyme is capable of catalysing the exchange of the inositol polar head for another inositol. Our data clearly show that the CDP-diacylglycerol-independent exchange reaction can occur using endogenous PtdIns molecular species or PtdIns molecular species from soybean added exogenously. Exchange has been observed in the absence of cytidine monophosphate (CMP), but is greatly enhanced in the presence of 4 microm CMP. Our data also show that AtPIS1 catalyses the removal of the polar head in the presence of much higher concentrations of CMP, in a manner that suggests a reverse of synthesis. All of the PtdIns metabolizing activities require free manganese ions. EDTA, in the presence of low Mn2+ concentrations, also has an enhancing effect.     

Crop plants with improved culture and quality traits for food,feed and other uses

The large French research project GENIUS (2012–2019, https://www6.inra.genius-project_eng/) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.

     

Study in vitro of the impact of endophytic bacteria isolated from Centella asiatica on the disease incidence caused by the hemibiotrophic fungus Colletotrichum higginsianum

Thirty-one endophytic bacteria isolated from healthy leaves of Centella asiatica were screened in vitro for their ability to reduce the growth rate and disease incidence of Colletotrichum higginsianum, a causal agent of anthracnose. Isolates of Cohnella sp., Paenibacillus sp. and Pantoea sp. significantly stimulated the growth rate of C. higginsianum MUCL 44942, while isolates of Achromobacter sp., Acinetobacter sp., Microbacterium sp., Klebsiella sp. and Pseudomonas putida had no influence on this plant pathogen. By contrast, Bacillus subtilis BCA31 and Pseudomonas fluorescens BCA08 caused a marked inhibition of C. higginsianum MUCL 44942 growth by 46 and 82 %, respectively. Cell-free culture filtrates of B. subtilis BCA31 and P. fluorescens BCA08 were found to contain antifungal compounds against C. higginsianum MUCL 44942. Inoculation assays on in vitro-cultured plants of C. asiatica showed that foliar application of B. subtilis BCA31, three days before inoculation with C. higginsianum MUCL 44942, significantly reduced incidence and severity of the disease. The role of endophytic bacteria in maintaining the apparent inactivity of C. higginsianum MUCL 44942 in C. asiatica grown in the wild is discussed.     

Nucleotide Variability and Translation Efficiency of the 5′ Untranslated Region of Hepatitis A Virus: Update from Clinical Isolates Associated with Mild and Severe Hepatitis

Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% ± 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.Hepatitis A virus (HAV) is a nonenveloped RNA virus of the Picornaviridae family. The viral genome consists of an approximately 7,500-nucleotide (nt)-long, positive-stranded RNA divided in three parts: a 5′ untranslated region (5′ UTR), a single open reading frame that encodes both structural and nonstructural proteins, and a 3′ UTR with a short poly(A) tail. By sequencing of the VP1-2A junction and the VP1 gene, 3 genotypes (I, II, and III) divided into A and B subtypes have been described in humans (7, 27). HAV is the main cause of acute viral hepatitis worldwide. The majority of cases follow a benign course, but some may be present with fulminant forms, characterized by acute liver failure (factor V levels of <50% and encephalopathy). HAV-induced liver disease appears to result primarily from immunologic mechanisms, chiefly on the basis of in vitro studies. Most HAV strains have no detectable cytopathic effect in cell culture and no apparent effect on cell growth or metabolism (16), and HAV-infected cells are lysed by cytotoxic T cells isolated from the liver of acutely infected patients (30, 31). Clinical studies have suggested that host factors such as age and underlying liver disease were involved in the severity of liver diseases (32, 33) and that the host immune response also played a role in the fulminant forms of hepatitis A, as evidenced by markedly low viral loads (26).Nevertheless, the existence of viral determinants of hepatitis A severity is suggested by both experimental and clinical studies. Indeed, mutations within the VP1-2A and 2C genes have been shown to enhance virulence in tamarins (9). It has also been suggested that 5′ UTR mutations associated with viral adaptation to cell culture were also responsible for viral attenuation in vivo (15). The 5′ UTR of HAV is about 735 nucleotides long and is considered the most conserved region of the genome. The 5′ UTR is involved in genome replication and translation initiation. Folding predictions and biochemical probing showed that this region forms a highly ordered secondary structure containing a pyrimidine-rich tract (PRT) and an internal ribosomal entry site (IRES) with 10 to 12 AUG triplets upstream of the initiator codon (18). The IRES allows the initiation of the cap-independent translation of the viral genome. Most knowledge of HAV IRES activity is derived from studies of the HM-175 reference strain and its cell culture-adapted variants (4, 5, 36). These experiments have shown that HAV presents the lowest IRES-dependent translation initiation activity among picornaviruses both in reticulocyte lysates and in a variety of cell lines, including the human hepatoma cell line HepG2 (type III IRES) (3, 6). These features have been attributed to a lower affinity of the HAV 5′ UTR for translation factors (6). The hypothesis that the slow growth of HAV in cell culture could be related to this inefficient translation is supported by the emergence of 5′ UTR mutations in cell culture-adapted variants with enhanced viral replication (8). The finding that these mutations were associated with viral attenuation in vivo supports the hypothesis of viral determinants of virulence in the 5′ UTR (15). Among the few clinical studies which have addressed this question, Fujiwara et al., by comparing full-length HAV genomes obtained from Japanese patients with benign or fulminant hepatitis, found less nucleotide variation in the 5′ UTRs from patients with fulminant hepatitis (12, 13) and suggested that two IRES mutations (G324A and C372G/T) might influence the course of HAV infection (14).The aim of the present study was to further examine the genetic variability of 5′ UTR sequences from field isolates, to assess the potential impact of nucleotide variations on IRES activity by using validated techniques, and to search for a relationship with disease severity by comparing isolates obtained from patients with benign or fulminant forms of hepatitis A.     

NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water

Bacillus sp. 3B6, bacterium isolated from cloud water, was incubated on sucrose for exopolysaccharide production. Dialysis of the obtained mixture (MWCO 500) afforded dialyzate (DIM) and retentate (RIM). Both were separated by size exclusion chromatography. RIM afforded eight fractions: levan exopolysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types with different degrees of polymerization (dp 2–7) and monosaccharides fructose:glucose = 9:1. Levan was composed of two components with molecular mass ∼3500 and ∼100 kDa in the ratio 2.3:1. Disaccharide fraction contained difructose anhydride DFA IV. 1-Kestose, 6-kestose, and neokestose were identified as trisaccharides in the ratio 2:1:3. Fractions with dp 4–7 were mixtures of FOSs of levan (2,6-βFruf) and inulin (1,2-βFruf) type. DIM separation afforded two dominant fractions: monosaccharides with fructose: glucose ratio 1:3; disaccharide fraction contained sucrose only. DIM trisaccharide fraction contained 1-kestose, 6-kestose, and neokestose in the ratio1.5:1:2, penta and hexasaccharide fractions contained FOSs of levan type (2,6-βFruf) containing α-glucose. In the pentasaccharide fraction also the presence of a homopentasaccharide composed of 2,6-linked βFruf units only was identified. Nystose, inulin (1,2-βFruf) type, was identified as DIM tetrasaccharide. Identification of levan 2,6-βFruf and inulin 1,2-βFruf type oligosaccharides in the incubation medium suggests both levansucrase and inulosucrase enzymes activity in Bacillus sp. 3B6.     

The oxidation of adrenaline and noradrenaline by the two forms of monoamine oxidase from human and rat brain

The selective monoamine oxidase inhibitors clorgyline and (−)-deprenyl were used to study the distribution of monoamine oxidase-A and -B (MAO-A, MAO-B) activities towards (−)-noradrenaline and (+),(−)-adrenaline in homogenates from seven different regions of human brain. The activities towards 5-hydroxytryptamine and 2-phenethylamine, which are essentially specific substrates for the A- and B-forms, respectively, under the conditions used in this work, were also determined. Noradreanline and adrenaline were substrates for both forms of the enzyme in all regions studied. The total MAO activity was found to be highest in the hypothalamus and lowest in the cerebellar cortex. Use of the selective MAO inhibitors clorgyline and (−)-deprenyl also showed adrenaline and noradrenaline to be substrates for both forms of the enzyme in rat brain. In human cerebral cortex and rat brain the two forms were found to have similar K_m-values and maximum velocities towards adrenaline. These values for the two forms were also found to be similar in human cerebral cortex when noradrenaline was used as the substrate. In contrast MAO-A showed a significantly lower K_m and a higher maximum velocity towards noradrenaline in rat brain. These results suggest that the rat may not provide a close model of the human for studies on the effects of MAO inhibitors on brain noradrenaline metabolism.     

Contribution of a time-dependent and hyperpolarization-activated chloride conductance to currents of resting and hypotonically shocked rat hepatocytes

Hepatocellular Cl- flux is integral to maintaining cell volume and electroneutrality in the face of the many transport and metabolic activities that describe the multifaceted functions of these cells. Although a significant volume-regulated Cl- current (VRAC) has been well described in hepatocytes, the Cl- channels underlying the large resting anion conductance have not been identified. We used a combination of electrophysiological and molecular approaches to describe potential candidates for this conductance. Anion currents in rat hepatocytes and WIF-B and HEK293T cells were measured under patch electrode-voltage clamp. With K+-free salts of Cl- comprising the major ions externally and internally, hyperpolarizing steps between -40 and -140 mV activated a time-dependent inward current in hepatocytes. Steady-state activation was half-maximal at -63 mV and 28-38% of maximum at -30 to -45 mV, previously reported hepatocellular resting potentials. Gating was dependent on cytosolic Cl-, shifting close to 58 mV/10-fold change in Cl- concentration. Time-dependent inward Cl- currents and a ClC-2-specific RT-PCR product were also observed in WIF-B cells but not HEK293T cells. All cell types exhibited typical VRAC in response to dialysis with hypertonic solutions. DIDS (0.1 mM) inhibited the hepatocellular VRAC but not the inward time-dependent current. Antibodies against the COOH terminus of ClC-2 reacted with a protein between 90 and 100 kDa in liver plasma membranes. The results demonstrate that rat hepatocytes express a time-dependent inward Cl- channel that could provide a significant depolarizing influence in the hepatocyte.