The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP₂), we found that PIP₂ increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP₂ or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr³⁷⁰ in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.     

Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection

Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment.     

Identification of bovine NPC1 gene cSNPs and their effects on body size traits of Qinchuan cattle

NPC1 gene is an important gene closely related to the Niemann–Pick type C (NPC). Mutations in the NPC1 gene tend to cause Niemann–Pick type C, a lysosomal storage disorder. Previous studies have shown that NPC1 protein plays an important role in subcellular lipid transport, homeostasis, platelet function and formation, which are basic metabolic activities in the process of development. In this study, to explore the association between the NPC1 gene variation and body size traits in Qinchuan cattle, we detected four novel coding single nucleotide polymorphisms (cSNPs) in the bovine NPC1 gene, including one missense mutation (SNP1) and three synonymous mutations (SNP2, SNP3 and SNP4). Population genetic analyses of 518 individuals and association correlations between cSNPs and bovine body size traits were conducted in this research. A missense mutation at SNP1 locus was found to be significantly related to the heart girth, hip width and body weight (P < 0.01 or P < 0.05, 3.5-year-old). Two synonymous mutations at SNP2 and SNP3 loci also showed significant effects on hip width (P < 0.05, 3.5-year-old). One synonymous mutation at SNP4 locus showed significant effect on body weight (P < 0.05, 2.0-year-old). Combined haplotypes H₂H₆ and H₆H₆ showed significant effects on body size traits such as heart girth, hip width, and body weight (3.5-year-old, P < 0.01 or P < 0.05). This study provides evidence that the NPC1 gene might be involved in the regulation of bovine growth and body development, and may be considered as a candidate gene for marker assisted selection (MAS) in beef cattle breeding industry.     

Genetic diversity of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta) in Southeast Asia

The commercial importance of carrageenophytes Kappaphycus and Eucheuma is well known, with much interest in terms of cultivation, marketing, and research. Considering the many lucrative prospects, these red seaweeds were introduced into various parts of the world for farming, where merely a few were comprehensively documented. Despite being extensively cultivated throughout Southeast Asia, the genetic diversity of Kappaphycus and Eucheuma is poorly studied, where heavy reliance is placed on the use of local or commercial names for identifications. This study used the mitochondrial-encoded cox1 and cox2–3 spacer genetic markers to investigate the Kappaphycus and Eucheuma haplotypes, cultivated and wild, available throughout Southeast Asia. Concatenated cox1–cox2–3 spacer datasets were also analyzed. The near full-length cox1 gene is preferred at revealing the genetic diversity of Kappaphycus and Eucheuma, provided a larger reference database is available. Both molecular markers were capable of delineating common members of the genus Kappaphycus (i.e., Kappaphycus alvarezii, Kappaphycus striatus, and Kappaphycus cottonii) and Eucheuma denticulatum, and revealed interesting genotypes and new species which may be potential alternatives to the common cultivars as well as materials for research. The relative scarcity of Eucheuma species is discussed and future sites for sampling are recommended.     

Key to the fungus-feeder Phlaeothripinae species from China(Thysanoptera: Phlaeothripidae)

In China, 31 genera and 95 species of fungivorous Phlaeothripinae are recorded here, of which 7 species are newly recorded and illustrated. An illustrated identification key to the 94 species is also provided, together with the information of specimens examined, and distribution of each species.     

CRF-based models of protein surfaces improve protein-protein interaction site predictions

Background

The identification of protein-protein interaction sites is a computationally challenging task and important for understanding the biology of protein complexes. There is a rich literature in this field. A broad class of approaches assign to each candidate residue a real-valued score that measures how likely it is that the residue belongs to the interface. The prediction is obtained by thresholding this score.Some probabilistic models classify the residues on the basis of the posterior probabilities. In this paper, we introduce pairwise conditional random fields (pCRFs) in which edges are not restricted to the backbone as in the case of linear-chain CRFs utilized by Li et al. (2007). In fact, any 3D-neighborhood relation can be modeled. On grounds of a generalized Viterbi inference algorithm and a piecewise training process for pCRFs, we demonstrate how to utilize pCRFs to enhance a given residue-wise score-based protein-protein interface predictor on the surface of the protein under study. The features of the pCRF are solely based on the interface predictions scores of the predictor the performance of which shall be improved.

Results

We performed three sets of experiments with synthetic scores assigned to the surface residues of proteins taken from the data set PlaneDimers compiled by Zellner et al. (2011), from the list published by Keskin et al. (2004) and from the very recent data set due to Cukuroglu et al. (2014). That way we demonstrated that our pCRF-based enhancer is effective given the interface residue score distribution and the non-interface residue score are unimodal.Moreover, the pCRF-based enhancer is also successfully applicable, if the distributions are only unimodal over a certain sub-domain. The improvement is then restricted to that domain. Thus we were able to improve the prediction of the PresCont server devised by Zellner et al. (2011) on PlaneDimers.

Conclusions

Our results strongly suggest that pCRFs form a methodological framework to improve residue-wise score-based protein-protein interface predictors given the scores are appropriately distributed. A prototypical implementation of our method is accessible at http://ppicrf.informatik.uni-goettingen.de/index.html.     

Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis

Mesenchymal stem cell (MSC)-based therapy is a promising approach to treat various inflammatory disorders including multiple sclerosis. However, the fate of MSCs in the inflammatory microenvironment is largely unknown. Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal model of multiple sclerosis. We demonstrated that autophagy occurred in MSCs during their application for EAE treatment. Inflammatory cytokines, e.g., interferon gamma and tumor necrosis factor, induced autophagy in MSCs synergistically by inducing expression of BECN1/Beclin 1. Inhibition of autophagy by knockdown of Becn1 significantly improved the therapeutic effects of MSCs on EAE, which was mainly attributable to enhanced suppression upon activation and expansion of CD4⁺ T cells. Mechanistically, inhibition of autophagy increased reactive oxygen species generation and mitogen-activated protein kinase 1/3 activation in MSCs, which were essential for PTGS2 (prostaglandin-endoperoxide synthase 2 [prostaglandin G/H synthase and cyclooxygenase]) and downstream prostaglandin E2 expression to exert immunoregulatory function. Furthermore, pharmacological treatment of MSCs to inhibit autophagy increased their immunosuppressive effects on T cell-mediated EAE. Our findings indicate that inflammatory microenvironment-induced autophagy downregulates the immunosuppressive function of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel strategy to improve MSC-based immunotherapy.     

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

Food allergy is mediated by a combination of genetic and environmental risk factors, potentially mediated by epigenetic mechanisms. CD4+ T-cells are key drivers of the allergic response, and may therefore harbor epigenetic variation in association with the disease phenotype. Here we retrospectively examined genome-wide DNA methylation profiles (~450 000 CpGs) from CD4+ T-cells on a birth cohort of 12 children with IgE-mediated food allergy diagnosed at 12-months, and 12 non-allergic controls. DNA samples were available at two time points, birth and 12-months. Case:control comparisons of CD4+ methylation profiles identified 179 differentially methylated probes (DMP) at 12-months and 136 DMP at birth (FDR-adjusted P value < 0.05, delta β > 0.1). Approximately 30% of DMPs were coincident with previously annotated SNPs. A total of 96 allergy-associated non-SNP DMPs were present at birth when individuals were initially disease-free, potentially implicating these loci in the causal pathway. Pathway analysis of differentially methylated genes identified several MAP kinase signaling molecules. Mass spectrometry was used to validate 15 CpG sites at 3 candidate genes. Combined analysis of differential methylation with gene expression profiles revealed gene expression differences at some but not all allergy associated differentially methylated genes. Thus, dysregulation of DNA methylation at MAPK signaling-associated genes during early CD4+ T-cell development may contribute to suboptimal T-lymphocyte responses in early childhood associated with the development of food allergy.     

The Interaction Effects of Temperature and Humidity on Emergency Room Visits for Respiratory Diseases in Beijing,China

Few epidemiological studies have been reported as to whether there was any interactive effect between temperature and humidity on respiratory morbidity, especially in Asian countries. The present study used time-series analysis to explore the modification effects of humidity on the association between temperature and emergency room (ER) visits for respiratory, upper respiratory tract infection (URI), pneumonia, and bronchitis in Beijing between 2009 and 2011. Results showed that an obvious joint effect of temperature and humidity was revealed on ER visits for respiratory, URI, pneumonia, and bronchitis. Below temperature threshold, the temperature effect was stronger in low humidity level and presented a trend fall with humidity level increase. The effect estimates per 1 °C increase in temperature in low humidity level were ?2.88 % (95 % confidence interval (CI) ?3.08, ?2.67) for all respiratory, ?3.24 % (?3.59, ?2.88) for URI, ?1.48 % (?1.93, ?1.03) for pneumonia, and ?3.79 % (?4.37, ?3.21) for bronchitis ER visits, respectively. However, above temperature threshold, temperature effect was greater in high humidity level and trending upward with humidity level increasing. In high humidity level, a 1 °C increase in temperature, the effect estimates were 1.84 % (1.55, 2.13) for all respiratory, 1.76 % (1.41, 2.11) for URI, and 7.48 % (4.41, 10.65) for bronchitis ER visits. But, there was no statistically significant for pneumonia. This suggests that the modifying effects of the humidity should be considered when analyzing health impacts of temperature.     

p600 stabilizes microtubules to prevent the aggregation of CaMKIIα during photoconductive stimulation

The large microtubule-associated/Ca²⁺-signalling protein p600 (also known as UBR4) is required for hippocampal neuronal survival upon Ca²⁺ dyshomeostasis induced by glutamate treatment. During this process, p600 prevents aggregation of the Ca²⁺/calmodulin-dependent kinase IIα (CaMKIIα), a proxy of neuronal death, via direct binding to calmodulin in a microtubuleindependent manner. Using photoconductive stimulation coupled with live imaging of single neurons, we identified a distinct mechanism of prevention of CaMKIIα aggregation by p600. Upon direct depolarization, CaMKIIα translocates to microtubules. In the absence of p600, this translocation is interrupted in favour of a sustained self-aggregation that is prevented by the microtubule-stabilizing drug paclitaxel. Thus, during photoconductive stimulation, p600 prevents the aggregation of CaMKIIα by stabilizing microtubules. The effectiveness of this stabilization for preventing CaMKIIα aggregation during direct depolarization but not during glutamate treatment suggests a model wherein p600 has two modes of action depending on the source of cytosolic Ca²⁺.