Barley beta-glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H2O2-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O2-) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O2-, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure

Cognitive modulation of psychophysical, respiratory and autonomic responses to cold pressor test

In healthy subjects with high hypnotisability (highs) under hypnosis, subjectively effective suggestions for analgesia abolish the increases in blood pressure associated with cold pressor test (cpt) by reducing the peripheral vascular resistance. The aim of the present study was to investigate the effects of the suggestions of analgesia on the responses to cpt in healthy highs (n = 22) and in low hypnotisable participants (lows, n = 22) out of hypnosis. Cpt was administered without (CPT) and with suggestions for analgesia (CPT+AN). Psychophysical (pain intensity, pain threshold, cpt duration (time of immersion) and pain tolerance, defined as the difference between cpt duration and pain threshold), respiratory (amplitude and frequency) and autonomic variables (tonic skin conductance, mean RR interval (RR = 1/heart rate), blood pressure, skin blood flow) were studied. The suggestions for analgesia increased cpt duration and RR in both groups, but decreased pain intensity and enhanced pain threshold only in highs; in both groups they did not modulate systolic blood pressure, tonic skin conductance and skin blood flow; thus, increased parasympathetic activity appears responsible for the heart rate reduction induced by suggestions in both groups. In conclusion, our findings show that suggestions modulate pain experience differentially in highs and lows, and are partially effective also in lows. We hypothesize that the mechanisms responsible for the efficacy of suggestions in healthy lows may be involved also in their efficacy in chronic pain patients with low hypnotisability

Phenotype-specific association of the TGFBR3 locus with nonsyndromic cryptorchidism

PURPOSE: Based on a genome-wide association study of testicular dysgenesis syndrome showing a possible association with TGFBR3, we analyzed data from a larger, phenotypically restricted cryptorchidism population for potential replication of this signal. MATERIALS AND METHODS: We excluded samples based on strict quality control criteria, leaving 844 cases and 2,718 controls of European ancestry that were analyzed in 2 separate groups based on genotyping platform (ie Illumina® HumanHap550, version 1 or 3, or Human610-Quad, version 1 BeadChip in group 1 and Human OmniExpress 12, version 1 BeadChip platform in group 2). Analyses included genotype imputation at the TGFBR3 locus, association analysis of imputed data with correction for population substructure, subsequent meta-analysis of data for groups 1 and 2, and selective genotyping of independent cases (330) and controls (324) for replication. We also measured Tgfbr3 mRNA levels and performed TGFBR3/betaglycan immunostaining in rat fetal gubernaculum. RESULTS: We identified suggestive (p ≤ 1× 10(-4)) association of markers in/near TGFBR3, including rs9661103 (OR 1.40; 95% CI 1.20, 1.64; p = 2.71 × 10(-5)) and rs10782968 (OR 1.58; 95% CI 1.26, 1.98; p = 9.36 × 10(-5)) in groups 1 and 2, respectively. In subgroup analyses we observed strongest association of rs17576372 (OR 1.42; 95% CI 1.24, 1.60; p = 1.67 × 10(-4)) with proximal and rs11165059 (OR 1.32; 95% CI 1.15, 1.38; p = 9.42 × 10(-4)) with distal testis position, signals in strong linkage disequilibrium with rs9661103 and rs10782968, respectively. Association of the prior genome-wide association study signal (rs12082710) was marginal (OR 1.13; 95% CI 0.99, 1.28; p = 0.09 for group 1), and we were unable to replicate signals in our independent cohort. Tgfbr3/betaglycan was differentially expressed in wild-type and cryptorchid rat fetal gubernaculum. CONCLUSIONS: These data suggest complex or phenotype specific association of cryptorchidism with TGFBR3 and the gubernaculum as a potential target of TGFβ signaling

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9- sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient biallelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo

Prevalence of Salmonella enterica and Listeria monocytogenes contamination in foods of animal origin in Italy.

The present survey collected and analyzed the results of routine testing for Salmonella enterica and Listeria monocytogenes on foods of animal origin submitted for official controls in Italy during 2001 to 2002. Salmonella was detected in 2.2% of 71,643 food samples examined, and the isolation rates ranged from 9.9% for raw poultry meat to less than 0.1% for dairy products. Isolation rates were also high in raw pork (4.9%) and processed meats (5.3%), which often involved pork. Low rates were observed in seafood (0.5%) and in ready-to-eat foods, such as grocery products (0.7%) and ice creams (0.1%). Serotyping showed that approximately 50% of the isolates belonged to the serotypes most commonly isolated from humans in Italy, thus confirming that most cases of human salmonellosis have a foodborne origin. Levels of L. monocytogenes were higher than what is accepted by the current regulation in 2.4% of 42,300 food samples. The positivity rates ranged from 10.3% in raw pork to none in eggs and egg products. Contamination rates were higher in other meat products (between 2 and 5%) and fish (6.5%) than in cheeses (1.1%) and other dairy products (0.6%). Routine control activities on the microbial contamination of foods can generate data with statistical and epidemiological value. Such data can be used as a basis for estimating the exposure of consumers to foodborne pathogens, following the trends of contamination over time, and evaluating the effects of control measures on the contamination of food

Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/β-catenin signaling

Bcl9 and Pygopus (Pygo) are obligate Wnt/β-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, β-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the β-catenin–BCL9–Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective β-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects