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Discoveries of bacterial communities in environments that previously have been described as sterile have in recent years radically challenged the view of these environments. In this study we aimed to use 16S rRNA sequencing to describe the composition and temporal stability of the bacterial microbiota in bovine milk from healthy udder quarters, an environment previously believed to be sterile. Sequencing of the 16S rRNA gene is a technique commonly used to describe bacterial composition and diversity in various environments. With the increased use of 16S rRNA gene sequencing, awareness of methodological pitfalls such as biases and contamination has increased although not in equal amount. Evaluation of the composition and temporal stability of the microbiota in 288 milk samples was largely hampered by background contamination, despite careful and aseptic sample processing. Sequencing of no template control samples, positive control samples, with defined levels of bacteria, and 288 milk samples with various levels of bacterial growth, revealed that the data was influenced by contaminating taxa, primarily Methylobacterium. We observed an increasing impact of contamination with decreasing microbial biomass where the contaminating taxa became dominant in samples with less than 104 bacterial cells per mL. By applying a contamination filtration on the sequence data, the amount of sequences was substantially reduced but only a minor impact on number of identified taxa and by culture known endogenous taxa was observed. This suggests that data filtration can be useful for identifying biologically relevant associations in milk microbiota data. [ABSTRACT FROM AUTHOR]

Aim: Regorafenib is an oral small-molecule multi kinase inhibitor. Recently, several clinical trials have revealed that regorafenib has an anti-tumor activity in gastric cancer. However, only part of patients benefit from regorafenib, and the mechanisms of regorafenib’s anti-tumor effect need further demonstrating. In this study, we would assess the potential anti-tumor effects and the underlying mechanisms of regorafenib in gastric cancer cells, and explore novel biomarkers for patients selecting of regorafenib. Methods: The anti-tumor effects of regorafenib on gastric cancer cells were analyzed via cell proliferation and invasion. The underlying mechanisms were demonstrated using molecular biology techniques. Results: We found that regorafenib inhibited cell proliferation and invasion at the concentration of 20μmol/L and in a dose dependent manner. The anti-tumor effects of regorafenib related to the decreased expression of CXCR4, and elevated expression and activation of CXCR4 could reverse the inhibition effect of regorafenib on gastric cancer cells. Further studies revealed that regorafenib reduced the transcriptional activity of Wnt/β-Catenin pathway and led to decreased expression of Wnt pathway target genes, while overexpression and activation of CXCR4 could attenuate the inhibition effect of regorafenib on Wnt/β-Catenin pathway. Conclusions: Our findings demonstrated that regorafenib effectively inhibited cell proliferation and invasion of gastric cancer cells via decreasing the expression of CXCR4 and further reducing the transcriptional activity of Wnt/β-Catenin pathway. [ABSTRACT FROM AUTHOR]

This work examined the mechanisms underlying auditory motion processing in the auditory cortex of awake monkeys using functional magnetic resonance imaging (fMRI). We tested to what extent auditory motion analysis can be explained by the linear combination of static spatial mechanisms, spectrotemporal processes, and their interaction. We found that the posterior auditory cortex, including A1 and the surrounding caudal belt and parabelt, is involved in auditory motion analysis. Static spatial and spectrotemporal processes were able to fully explain motion-induced activation in most parts of the auditory cortex, including A1, but not in circumscribed regions of the posterior belt and parabelt cortex. We show that in these regions motion-specific processes contribute to the activation, providing the first demonstration that auditory motion is not simply deduced from changes in static spatial location. These results demonstrate that parallel mechanisms for motion and static spatial analysis coexist within the auditory dorsal stream. [ABSTRACT FROM AUTHOR]

The present study was performed to evaluate the food safety of TT51-1, a new type of genetically modified rice that expresses the Cry1Ab/Ac protein (Bt toxin) and is highly resistant to most lepidopteran pests. Sixteen male and 16 female cynomolgus monkeys were randomly divided into four groups: conventional rice (non-genetically modified rice, non-GM rice), positive control, 17.5% genetically modified rice (GM rice) and 70% GM rice. Monkeys in the non-GM rice, positive control, and GM rice groups were fed on diets containing 70% non-GM rice, 17.5% GM rice or 70% GM rice, respectively, for 182 days, whereas animals in the positive group were intravenously injected with cyclophosphamide every other day for a total of four injections before the last treatment. Six months of treatment did not yield abnormal observations. Specifically, the following parameters did not significantly differ between the non-GM rice group and GM rice groups: body weight, food consumption, electrocardiogram, hematology, immuno-phenotyping of lymphocytes in the peripheral blood, mitogen-induced peripheral blood lymphocyte proliferation, splenocyte proliferation, KLH-T cell-dependent antibody response, organ weights and ratios, and histological appearance (p>0.05). Animals from the GM rice group differed from animals in the non-GM rice group (p<0.05) in several parameters: specifically, their body temperatures and serum alanine aminotransferase (ALT) levels were higher, whereas their levels of serum K+, Cl- and cytokines (IL-2, IL-4 and IL-5) were lower. Because dose- or time-dependent changes were not observed in this study and animals appeared histologically normal, the aforementioned differences were not considered to be adverse or related to the treatment with GM rice. In conclusion, a 6-month feeding study of TT51-1 did not show adverse immunotoxicological effects on cynomolgus monkeys. [ABSTRACT FROM AUTHOR]

In some fish species, high or low temperature can switch the sex determination mechanisms and induce fish sex reversal when the gonads are undifferentiated. During this high or low temperature-induced sex reversal, the expressions of many genes are altered. However, genome-wide DNA methylation changes in fish gonads after high or low temperature treatment are unclear. Herein, we compared the global DNA methylation changes in the gonads from control females (CF), control males (CM), high temperature-treated females (TF), and high temperature-induced males (IM) from the F8 family of Nile tilapia (Oreochromis niloticus) using methylated DNA immunoprecipitation sequencing. The DNA methylation level in CF was higher than that in CM for various chromosomes. Both females and males showed an increase in methylation levels on various chromosomes after high-temperature induction. We identified 64,438 (CF/CM), 63,437 (TF/IM), 98,675 (TF/CF), 235,270 (IM/CM) and 119,958 (IM/CF) differentially methylated regions (DMRs) in Nile tilapia gonads, representing approximately 0.70% (CF/CM), 0.69% (TF/IM), 1.07% (TF/CF), 2.56% (IM/CM), and 1.30% (IM/CF)of the length of the genome. A total of 89 and 65 genes that exhibited DMRs in their gene bodies and promoters were mapped to the Nile tilapia genome. Furthermore, more than half of the genes with DMRs in the gene body in CF/CM were also included in the IM/CM, TF/CF, TF/IM, and IM/CF groups. Additionally, many important pathways, including neuroactive ligand-receptor interaction, extracellular matrix-receptor interaction, and biosynthesis of unsaturated fatty acids were identified. This study provided an important foundation to investigate the molecular mechanism of high temperature-induced sex reversal in fish species. [ABSTRACT FROM AUTHOR]

Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by abnormal remodeling of small, peripheral pulmonary arteries. Germline mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene are a major risk factor for developing PAH. At present, the correlation between the BMPR2 mutation and the patient's prognosis remains controversial despite several investigations. In this study, we explored the functional effects of four BMPR2 mutations to dissect the functional significance of the BMPR2 gene defect. Cellular immunofluorescence assay of four mutants (Tyr67Cys, Thr268fs, Ser863Asn, and Gln433X) revealed that the BMPR2 protein containing Thr268fs, Ser863Asn, or Gln433X exhibited abnormal subcellular localization. The BrdU incorporation and TUNEL assay suggested that any of the BMPR2 mutations Thr268fs, Ser863Asn, or Gln433X could improve endothelial cell apoptosis and decrease cell proliferation. All of the four mutants could inhibit nitric oxide (NO) synthesis in HLMVE cells, and ET-1 levels increased in the cells transfected with mutant Ser863Asn. Our results will improve the understanding of the genotype-phenotype correlations and mechanisms associated with BMPR2 mutations. [ABSTRACT FROM AUTHOR]

Streptococcus iniae is a Gram-positive bacterium and a severe pathogen to a wide range of economically important fish species. In addition, S. iniae is also a zoonotic pathogen and can cause serious infections in humans. In this study, we identified from a pathogenic S. iniae strain a putative bacteriocin, Sil, and examined its biological activity. Sil is composed of 101 amino acid residues and shares 35.6% overall sequence identity with the lactococcin 972 of Lactococcus lactis. Immunoblot analysis showed that Sil was secreted by S. iniae into the extracellular milieu. Purified recombinant Sil (rSil) exhibited a dose-dependent inhibitory effect on the growth of Bacillus subtilis but had no impact on the growths of other 16 Gram-positive bacteria and 10 Gram-negative bacteria representing 23 different bacterial species. Treatment of rSil by heating at 50°C abolished the activity of rSil. rSil bound to the surface of B. subtilis but induced no killing of the target cells. Cellular study revealed that rSil interacted with turbot (Scophthalmus maximus) head kidney monocytes and inhibited the innate immune response of the cells, which led to enhanced cellular infection of S. iniae. Antibody blocking of the extracellular Sil produced by S. iniae significantly attenuated the infectivity of S. iniae. Consistent with these in vitro observations, in vivo study showed that administration of turbot with rSil prior to S. iniae infection significantly increased bacterial dissemination and colonization in fish tissues. Taken together, these results indicate that Sil is a novel virulence-associated bacteriostatic and an immunoregulator that promotes S. iniae infection by impairing the immune defense of host fish. [ABSTRACT FROM AUTHOR]

Background: Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine. Methods: Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA). Results: KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively. Conclusion: KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndrome (MDS) is a group of progressive,clonal, neoplastic bone marrow disorders characterized by hematopoietic stem cell dysregulation and abnormalities in the immune system. Mesenchymal stem cells (MSC) appear to modulate the immune system at the very first step of the immune response through the inhibition of dendritic cells (DCs) differentiation and maturation. However, it is still unclear whether the effects of MSC on the development of DCs will be altered with disease state. In addition, it is not clear whether there are differences in the effects between low-risk and high-risk MDS-MSC on DCs development. In this study, our data confirm that MDS-MSC mediate a potent inhibition of DCs differentiation. Additionaly, MDS-MSC greatly alter DCs functions, including endocytosis, IL-12 secretion, their ability to inhibit T cell proliferation. Moreover, our results show that there are major differences in DCs development and function between low-risk and high-risk MDS-MSC. Compared to high-risk MDS-MSC, low-risk MDS-MSC is characterized by a poor ability to inhibit DCs differentiation and maturation; and correspondingly, less dysfunctional DC endocytosis, mildly decreased IL-12 secretion, and a reduction in DC-mediated inhibition of T cell proliferation. Finally, our results demonstrate that MDS-MSC derived TGF-β1 is largely responsible for the inhitory effects. These results elucidate the different immunoregulatory role of MSC in low-risk and high-risk MDS on DCs development, which may be important for understanding the pathogenesis of MDS and the development of novel immune therapies for the treatment of MDS. [ABSTRACT FROM AUTHOR]