Secretory delivery of heterologous proteins in attenuated <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> for potential use in vaccine design

To synthesize and secrete heterologous proteins in an attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different antigen-delivery systems based on bacterial-originated secretion signal peptides (SPs) were designed and identified in this work. Four SPs were derived from hemolysin of Escherichia coli, RTX protein of V. cholerae, hemolysin of V. anguillarum, zinc-metalloprotease of V. anguillarum, respectively, and their abilities to support secretion of green fluorescent protein (GFP) in an attenuated V. anguillarum strain MVAV6203 were assayed. Immunodetection of GFP showed that the capability of the tested signal leaders to direct secretion of GFP varied greatly. Although all the four signal peptide-fused GFPs could be expressed correctly and trapped intracellularly in recombinant strains, only the EmpA signal peptide could confer efficient secretion to GFP. For the investigation of its potential application in live bacteria carrier vaccines, a heterologous protein EseB of Edwardsiella tarda was fused to the SP(empA) antigen-delivery system and introduced into the strain MVAV6203. Further analysis of EseB demonstrated that the constructed SP(empA) antigen-delivery system could be used to secrete foreign protein in attenuated V. anguillarum and be available for carrier vaccines development.     

Molecular epidemiology of scrub typhus in Taiwan during 2006–2016

Scrub typhus is the most common endemic vector-borne disease in Taiwan. We identified a total of 4,857 laboratory-confirmed cases during 2006–2016 with hyperendemic foci on offshore islands, including Penghu (778 cases, 16.0%) and Kinmen (716 cases, 14.7%), and eastern Taiwan, including Taitung (628 cases, 12.9%) and Hualien (508 cases, 10.5%). Scrub typhus cases occur year-round throughout Taiwan, with a summer peak in June and July. A total of 545 O. tsutsugamushi isolates were successfully obtained from patients infected in diverse geographic areas, including Taiwan and three offshore islands, and the complete open reading frame of the 56 kDa type-specific antigen gene (tsa56) sequence of these isolates was examined. High phylogenetic diversity was found in these isolates, which could be grouped into 36 distinct sequence types. Most isolates belonged to the Karp (49.9%; 272/545), followed by the TW-22 (17.8%; 97/454) and Kawasaki (14.7%; 80/545) genotypes. In conclusion, our data indicate the widespread presence of tsa56 genotypes closely related to Thailand and Korean strains and the presence of the unique endemic strains TW-12, TW-22, TW-29, and TW-36 in Taiwan.     

Genetic differentiation and phylogeography of rotifer Polyarthra dolichoptera and P. vulgaris populations between Southeastern China and eastern North America: High intercontinental differences

Genetic differentiations and phylogeographical patterns of small organisms may be shaped by spatial isolation, environmental gradients, and gene flow. However, knowledge about genetic differentiation of rotifers at the intercontinental scale is still limited. Polyarthra dolichoptera and P. vulgaris are cosmopolitan rotifers that are tolerant to environmental changes, offering an excellent model to address the research gap. Here, we investigated the populations in Southeastern China and eastern North America and evaluated the phylogeographical patterns from their geographical range sizes, geographic–genetic distance relationships and their responses to spatial‐environmental factors. Using the mitochondrial cytochrome c oxidase subunit I gene as the DNA marker, we analyzed a total of 170 individuals. Our results showed that some putative cryptic species, also known as entities were widely distributed, but most of them were limited to single areas. The divergence of P. dolichoptera and P. vulgaris indicated that gene flow between continents was limited while that within each continent was stronger. Oceanographic barriers do affect the phylogeographic pattern of rotifers in continental waters and serve to maintain genetic diversity in nature. The genetic distance of P. dolichoptera and P. vulgaris populations showed significant positive correlation with geographic distance. This might be due to the combined effects of habitat heterogeneity, long‐distance colonization, and oceanographic barriers. Furthermore, at the intercontinental scale, spatial distance had a stronger influence than environmental variables on the genetic differentiations of both populations. Wind‐ and animal‐mediated transport and even historical events of continental plate tectonics are potential factors for phylogeography of cosmopolitan rotifers.     

Estimation of Cross-Species Introgression Rates Using Genomic Data Despite Model Unidentifiability

Full-likelihood implementations of the multispecies coalescent with introgression (MSci) model treat genealogical fluctuations across the genome as a major source of information to infer the history of species divergence and gene flow using multilocus sequence data. However, MSci models are known to have unidentifiability issues, whereby different models or parameters make the same predictions about the data and cannot be distinguished by the data. Previous studies of unidentifiability have focused on heuristic methods based on gene trees and do not make an efficient use of the information in the data. Here we study the unidentifiability of MSci models under the full-likelihood methods. We characterize the unidentifiability of the bidirectional introgression (BDI) model, which assumes that gene flow occurs in both directions. We derive simple rules for arbitrary BDI models, which create unidentifiability of the label-switching type. In general, an MSci model with k BDI events has 2k unidentifiable modes or towers in the posterior, with each BDI event between sister species creating within-model parameter unidentifiability and each BDI event between nonsister species creating between-model unidentifiability. We develop novel algorithms for processing Markov chain Monte Carlo samples to remove label-switching problems and implement them in the bpp program. We analyze real and synthetic data to illustrate the utility of the BDI models and the new algorithms. We discuss the unidentifiability of heuristic methods and provide guidelines for the use of MSci models to infer gene flow using genomic data.     

Oligodendrocytes regulate formation of nodes of Ranvier via the recognition molecule OMgp

The molecular mechanisms underlying the involvement of oligodendrocytes in formation of the nodes of Ranvier (NORs) remain poorly understood. Here we show that oligodendrocyte-myelin glycoprotein (OMgp) aggregates specifically at NORs. Nodal location of OMgp does not occur along demyelinated axons of either Shiverer or proteolipid protein (PLP) transgenic mice. Over-expression of OMgp in OLN-93 cells facilitates process outgrowth. In transgenic mice in which expression of OMgp is down-regulated, myelin thickness declines, and lateral oligodendrocyte loops at the node-paranode junction are less compacted and even join together with the opposite loops, which leads to shortened nodal gaps. Notably, each of these structural abnormalities plus modest down-regulation of expression of Na(+) channel alpha subunit result in reduced conduction velocity in the spinal cords of the mutant mice. Thus, OMgp that is derived from glia has distinct roles in regulating nodal formation and function during CNS myelination.     

Mitofusin 2 Protects Hepatocyte Mitochondrial Function from Damage Induced by GCDCA

Mitochondrial impairment is hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Mitofusin 2 (Mfn2) regulates mitochondrial morphology and signaling and is involved in the development of numerous mitochondrial-related diseases; however, a functional role for Mfn2 in chronic liver cholestasis which is characterized by increased levels of toxic bile acids remain unknown. Therefore, the aims of this study were to evaluate the expression levels of Mfn2 in liver samples from patients with extrahepatic cholestasis and to investigate the role Mfn2 during bile acid induced injury in vitro. Endogenous Mfn2 expression decreased in patients with extrahepatic cholestasis. Glycochenodeoxycholic acid (GCDCA) is the main toxic component of bile acid in patients with extrahepatic cholestasis. In human normal hepatocyte cells (L02), Mfn2 plays an important role in GCDCA-induced mitochondrial damage and changes in mitochondrial morphology. In line with the mitochondrial dysfunction, the expression of Mfn2 decreased significantly under GCDCA treatment conditions. Moreover, the overexpression of Mfn2 effectively attenuated mitochondrial fragmentation and reversed the mitochondrial damage observed in GCDCA-treated L02 cells. Notably, a truncated Mfn2 mutant that lacked the normal C-terminal domain lost the capacity to induce mitochondrial fusion. Increasing the expression of truncated Mfn2 also had a protective effect against the hepatotoxicity of GCDCA. Taken together, these findings indicate that the loss of Mfn2 may play a crucial role the pathogenesis of the liver damage that is observed in patients with extrahepatic cholestasis. The findings also indicate that Mfn2 may directly regulate mitochondrial metabolism independently of its primary fusion function. Therapeutic approaches that target Mfn2 may have protective effects against hepatotoxic of bile acids during cholestasis.     

Chaperone-mediated refolding of recombinant prochymosin

It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable. Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present. The helping effect varies with the oxidation states of prochymosin. GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively. For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28%. Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI.