Genome Sequence of the Fish Pathogen Yersinia ruckeri SC09 Provides Insights into Niche Adaptation and Pathogenic Mechanism.

Yersinia ruckeri is the etiologic agent of enteric red mouth disease (ERM), a severe fish disease prevailing in worldwide aquaculture industries. Here we report for the first time the complete genome of Y. ruckeri (Yersinia ruckeri) SC09, a highly virulent strain isolated from Ictalurus punctatus with severe septicemia. SC09 possesses a single chromosome of 3,923,491 base pairs, which contains 3651 predicted protein coding sequences (CDS), 19 rRNA genes, and 79 tRNA genes. Among the CDS, we have identified a Ysa locus containing genes encoding all the components of a type III secretion system (T3SS). Comparative analysis suggest that SC09-Ysa share extensive similarity in sequence, gene content, and gene arrangement with Salmonella enterica pathogenicity island 1 (SPI1) and chromosome-encoded T3SS from Yersinia enterocolitica biotype 1B. Furthermore, phylogenetic analysis shown that SC09-Ysa and SPI1-T3SS belong on the same branch of the phylogenetic tree. These results suggest that SC09-Ysa and SPI1-T3SS appear to mediate biological function to adapt to specific hosts with a similar niche, and both of them are likely to facilitate the development of an intracellular niche. In addition, our analysis also indicated that a substantial part of the SC09 genome might contribute to adaption in the intestinal microenvironment, including a number of proteins associated with aerobic or anaerobic respiration, signal transduction, and various stress reactions. Genomic analysis of the bacterium offered insights into the pathogenic mechanism associated with intracellular infection and intestinal survivability, which constitutes an important first step in understanding the pathogenesis of Y. ruckeri.

Clostridium beijerinckii mutant with high inhibitor tolerance obtained by low-energy ion implantation.

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l(-1) of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g(-1). When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l(-1) of ABE with a yield of 0.34 g g(-1), including 2.2 g l(-1) acetone, 6.8 g l(-1) butanol, and 0.5 g l(-1) ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.

Chaperone Copolymer Assisted G-Quadruplex-Based Signal Amplification Assay for Highly Sensitive Detection of VEGF.

Vascular endothelial growth factor (VEGF) is a critical biomarker in the angiogenesis of several cancers. Nowadays, novel approaches to rapid, sensitive, and reliable VEGF detection are urgently required for early cancer diagnosis. Cationic comb-type copolymer, poly(L-lysine)-graft-dextran (PLL-g-Dex) accelerates DNA hybridization and chain exchange reaction while stabilizing the DNA assembly structure. In this work, we examined the chaperone activity of PLL-g-Dex to assist G-quadruplex-based fluorescent DNA biosensors for sensitive detection of VEGF. This convenient and effective strategy is based on chitosan hydrogel, c-myc, Thioflavin T (ThT), VEGF aptamer, and its partially complementary strand. The results show that chaperone copolymer PLL-g-Dex significantly promotes the accumulation of G-quadruplex and assembles into G-wires, allowing an effective signal amplification. Using this method, the detection limit of VEGF was as low as 23 pM, better than many previous works on aptamer-based VEGF detection. This chaperone copolymer-assisted signal amplification strategy has potential applications in the highly sensitive detection of target proteins, even including viruses.

Molecular Characterization, Phylogenetic, Expression, and Protective Immunity Analysis of OmpF, a Promising Candidate Immunogen Against Yersinia ruckeri Infection in Channel Catfish.

Outer membrane porins, as the major components of Gram-negative bacterial membrane proteins, have been proven to be involved in interactions with the host immune system and potent protective antigen candidates against bacterial infection in fish. Outer membrane porin F (OmpF) is one of the major porins of Yersinia ruckeri (Y. ruckeri), the causative agent of enteric red mouth disease of salmonid and non-salmonid fish. In the present study, the molecular characterization and phylogenetic analysis of OmpF gene was studied, heterogenous expression, immunogenicity and protective immunity of OmpF were systemically evaluated as a subunit vaccine for channel catfish against Y. ruckeri infection. The results showed that OmpF gene was highly conserved among 15 known Yersinia species based on the analysis of conserved motifs, sequences alignment and phylogenetic tree, and was subjected to negative/purifying selection with global dN/dS ratios value of 0.649 throughout the evolution. Besides, OmpF was also identified to have immunogenicity by western blotting and was verified to be located on the surface of Y. ruckeri using cell surface staining and indirect immunofluorescence assays. Moreover, recombinant OmpF (rtOmpF) as a subunit vaccine was injected with commercial adjuvant ISA763, significantly enhanced the immune response by increasing serum antibody levels, lysozyme activity, complement C3 activity, total protein content, SOD activity, immune-related genes expression in the head kidney and spleen, and survival percent of channel catfish against Y. ruckeri infection. Thus, our present results not only enriched the information of molecular characterization and phylogenetics of OmpF, but also demonstrated that OmpF holds promise to be used as a potential antigen against Y. ruckeri infection in fish.

Design and Characterization of a Multifunctional pH-Triggered Peptide C8 for Selective Anticancer Activity.

Most drug delivery systems have been developed for efficient delivery to tumor sites via targeting and on-demand strategies, but the carriers rarely execute synergistic therapeutic actions. In this work, C8, a cationic, pH-triggered anticancer peptide, is developed by incorporating histidine-mediated pH-sensitivity, amphipathic helix, and amino acid pairing self-assembly design. We designed C8 to function as a pH-responsive nanostructure whose cytotoxicity can be switched on and off by its self-assembly: Noncytotoxic ß-sheet fibers at high pH with neutral histidines, and positively charged monomers with membrane lytic activity at low pH. The selective activity of C8, tested for three different cancer cell lines and two noncancerous cell lines, is shown. Based on liposome leakage assays and multiscale computer simulations, its physical mechanisms of pore-forming action and selectivity are proposed, which originate from differences in the lipid composition of the cellular membrane and changes in hydrogen bonding. C8 is then investigated for its potential as a drug carrier. C8 forms a nanocomplex with ellipticine, a nonselective model anticancer drug. It selectively targets cancer cells in a pH-responsive manner, demonstrating enhanced efficacy and selectivity. This study provides a novel powerful strategy for the design and development of multifunctional self-assembling peptides for therapeutic and drug delivery applications.

Enhanced cyclic adenosine monophosphate production by Arthrobacter A302 through rational redistribution of metabolic flux.

Cyclic adenosine monophosphate (cAMP) was synthesized through the purine salvage synthesis pathway by Arthrobacter A302. Results showed that hypoxanthine was the best of the precursors, and the cAMP concentration reached 4.06 g/L. For inhibition of the glycolytic pathway, sodium fluoride was found the optimal effector, which was further studied on cAMP production. With the addition of 0.4 g/L of sodium fluoride, the maximal cAMP concentration reached 11.04 g/L, and the concentrations of lactic acid, alpha-ketoglutarate and citric acid were decreased by 77%, 86% and 76%, respectively. Meanwhile, the specific activities of glyceraldehyde 3-phosphate dehydrogenase, phosphofructokinase and pyruvate kinase were decreased by 66%, 61%, and 46%, respectively. By contrast the activity of 6-phosphoglucose dehydrogenase was increased by 100%, which demonstrated the redistribution of metabolic flux. This is the first study to reveal the regulatory mechanisms of different effectors on cAMP production among the EMP pathway, HMP pathway and TCA cycle.

Characterization and biological evaluation of paclitaxel-loaded poly(L-lactic acid) microparticles prepared by supercritical CO2.

In this study, paclitaxel loaded poly( L-lactic acid) (PTX-PLLA) microparticles were prepared using solution enhanced dispersion by supercritical CO2(SEDS) technique. This supercritical antisolvent technique offers the advantage of negligible organic solvent residua in the drug loaded microparticles. Scanning electron microscopy (SEM) showed that microparticles exhibited rather spherical shape and small particle size with narrow particle size distribution. X-ray diffraction (XRD) and differential scanning calorimeter (DSC) indicated that PTX was amorphously dispersed in the PLLA matrix. The drug loading and encapsulation efficiency of PTX-PLLA microparticles were 14.33% and 62.68%, respectively. In vitro cytotoxicity evaluation of PTX-PLLA microparticles against nonsmall-cell lung cancer A549 and ovarian cancer SKOV3 cell lines indicated that PTX-PLLA had superior antiproliferation activity against the A549 and SKOV3 cell lines, compared with free PTX formulations. The cellular internalization of fluorescent microparticles was evidenced by fluorescence microscope and further confirmed by transmission electron microscopy (TEM). This was attributed to the efficient intracellular accumulation of PTX via cell phagocytosis and sustained release of PTX from PLLA matrix. The anticancer activity of PTX-PLLA was associated with PTX-induced cell apoptosis such as nuclear aberrations, condensation of chromatin and swelling damage in mitochondria. The cell apoptosis index detected by flow cytometry was higher in PTX-PLLA group than in free PTX. The PTX-PLLA formulation, which was obtained through micronization of PTX and encapsulation of micronized PTX into PLLA simultaneously in the SEDS process, significantly potentiated the anticancer activity of PTX.