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Clonorchis sinensis was a food-borne zoonotic parasite in the worldwide and also an important risk factor of hepatic fibrosis. Excretory/secretion products of C. sinensis (CsESPs) are involved in parasite-host interactions and contribute to the development of hepatic damage. The aim of the present study was to investigate whether CsESPs and CsTP (adult protein) could induce autophagy of hepatic stellate cells (HSCs) and further activate HSCs so as to participate in the pathogenesis of hepatic fibrosis. The human hepatic stellate cell line LX-2 was stimulated by CsESPs and CsTP. CsESPs showed the effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay while CsTP failed. Autophagosomes and autolysosomes were observed after the transmission mRFP-EGFP-LC3 plasmid into the LX-2 cells. CsESPs had more powerful to induce the accumulation of autophagosomes and autolysosomes to enhance autophagic flux compared with CsTP. Western-blotting analysis confirmed that the ratio of LC3-II/I in LX-2 cells was up-regulated after CsESPs treatment for 6 h, which further proved that CsESPs could induce autophagy in LX-2 cells. Meanwhile, q-PCR results showed that the mRNA levels of collagen I, collagen III and α-SMA decreased in LX-2 cells after treatment with autophagy inhibitor chloroquine, whereas they increased when combination with CsESPs. These results suggested that CsESPs-induced autophagy might be involved in the activation of HSCs, and consequently participate in the pathogenesis of hepatic fibrosis caused by C. sinensis infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here, we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid, and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable, and advantageous additional model to be used alongside existing models to advance tuberculosis research.

The Gram-negative bacterium

How mammalian neuronal identity is progressively acquired and reinforced during development is not understood. We have previously shown that loss of RP58 (ZNF238 or ZBTB18), a BTB/POZ-zinc finger-containing transcription factor, in the mouse brain leads to microcephaly, corpus callosum agenesis, and cerebellum hypoplasia and that it is required for normal neuronal differentiation. The transcriptional programs regulated by RP58 during this process are not known. Here, we report for the first time that in embryonic mouse neocortical neurons a complex set of genes normally expressed in other cell types, such as those from mesoderm derivatives, must be actively repressed in vivo and that RP58 is a critical regulator of these repressed transcriptional programs. Importantly, gene set enrichment analysis (GSEA) analyses of these transcriptional programs indicate that repressed genes include distinct sets of genes significantly associated with glioma progression and/or pluripotency. We also demonstrate that reintroducing RP58 in glioma stem cells leads not only to aspects of neuronal differentiation but also to loss of stem cell characteristics, including loss of stem cell markers and decrease in stem cell self-renewal capacities. Thus, RP58 acts as an in vivo master guardian of the neuronal identity transcriptome, and its function may be required to prevent brain disease development, including glioma progression.

Triple-negative breast cancer (TNBC) cannot be treated with current hormonal therapies and has a higher risk of relapse than other breast cancers. To identify potential therapeutic targets for TNBC, we conducted microRNA sequencing (RNA-Seq) in human TNBC specimens and tumor-matched controls. We found that growth differentiation factor-10 (GDF10), a member of the TGF-β superfamily, was downregulated in tumor samples. Further analysis of GDF10 expression in a larger set of clinical TNBC samples using qPCR confirmed its downregulation and association with parameters of disease severity. Using human-derived TNBC cell lines, we carried out GDF10 under- and overexpression experiments, which showed that GDF10 loss promoted cell proliferation and invasion. By contrast, overexpression of GDF10 inhibited proliferation, invasion, and epithelial mesenchymal transition (EMT) via upregulation of Smad7 and E-Cadherin, downregulation of p-Smad2 and N-Cadherin, and reduction of nuclear Smad4 expression. In addition, overexpression of GDF10 reduced tumor burden and induced apoptosis in a TNBC xenograft mouse model. These findings indicate that GDF10 acts as a tumor suppressor in mammary epithelial cells that limits proliferation and suppresses EMT. Efforts aimed at restoring GDF10 expression may thus bring a long-sought therapeutic alternative in the treatment of patients with TNBC.

Animal models have long been used in tuberculosis research to understand disease pathogenesis and to evaluate novel vaccine candidates and anti-mycobacterial drugs. However, all have limitations and there is no single animal model which mimics all the aspects of mycobacterial pathogenesis seen in humans. Importantly mice, the most commonly used model, do not normally form granulomas, the hallmark of tuberculosis infection. Thus there is an urgent need for the development of new alternative in vivo models. The insect larvae, Galleria mellonella has been increasingly used as a successful, simple, widely available and cost-effective model to study microbial infections. Here we report for the first time that G. mellonella can be used as an infection model for members of the Mycobacterium tuberculosis complex. We demonstrate a dose-response for G. mellonella survival infected with different inocula of bioluminescent Mycobacterium bovis BCG lux, and demonstrate suppression of mycobacterial luminesence over 14 days. Histopathology staining and transmission electron microscopy of infected G. mellonella phagocytic haemocytes show internalization and aggregation of M. bovis BCG lux in granuloma-like structures, and increasing accumulation of lipid bodies within M. bovis BCG lux over time, characteristic of latent tuberculosis infection. Our results demonstrate that G. mellonella can act as a surrogate host to study the pathogenesis of mycobacterial infection and shed light on host-mycobacteria interactions, including latent tuberculosis infection.

Nasopharyngeal carcinoma (NPC) is a kind of malignancy generated from the nasopharyngeal epithelium. Recently, long noncoding RNA (lncRNA) has been shown to be involved in the regulation of many signaling pathways and is closely associated with carcinogenesis and tumor progression. However, the precise role of lncRNA Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in NPC is not well understood. Here, we find that OIP5-AS1 is overexpressed in NPC patient specimens and NPC cell lines. Further investigations reveal that knockdown of OIP5-AS1 significantly inhibits the proliferation, migration, and invasion and accelerates the apoptosis of NPC cells in vitro. Consistent with these findings, NPC progression is significantly slowed in mice when OIP5-AS1 is knocked down. Interestingly, there is a functional link between OIP5-AS1 and microRNA-203 (miR-203), a tumor suppressor, in NPC cells. In conclusion, our data demonstrate that OIP5-AS1 plays an important role in the development and progression of NPC by targeting miR-203 and therefore provide a promising target for the treatment of NPC.

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1, BB/G018553/1, BB/S002103/1, and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. Funding for LL provided by the ‘National Natural Science Foundation of China’ (No.31520103917). MTGH and DH were supported by the Wellcome Trust (grant number 098051). We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain. Publisher PDF

Wound exudate holds great clinical and research potential in wound repair via paracrine signaling. In essence, exudate is modified serum that contains a high concentration of exosomes. The aim of this study was to investigate the role of serum-derived exosomes in scald wound healing of NIH mice skin and to explore the underlying mechanisms. Hence, we constructed a deep second-degree scald model in NIH mice, testing the benefits of exosomes in the scald wound healing. The scratch wound assay, apoptosis assay and MTT assay were conducted to assess the effects of serum-derived exosomes on migration, apoptosis and proliferation of HaCaT cells and fibroblasts. Our results showed that serum-derived exosomes injected subcutaneously entered cells and effectively accelerated wound healing processes in mice. Additionally, serum-derived exosomes optimized functions of cells related to skin injury repair by stimulating fibroblast proliferation, promoting HaCaT cell migration, and suppressing apoptosis of HaCaT cells induced by heat stress. Further study revealed that serum-derived exosomes enhanced phosphorylation of the serine-threonine kinase Akt in scalded skin tissue. These results suggest a potential clinical use of serum-derived exosomes for treating skin scald.

The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen Actinobacillus pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 h time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes.

The larvae of the insect Galleria mellonella, have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points (4, 48, 168 h). Reverse transcriptase quantitative PCR confirmed induction of five genes (gloverin, cecropin, IMPI, hemolin, and Hdd11), which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/β-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella, mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (β-actin). Cecropins (antimicrobial peptides), which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.

Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialized animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.

Based on the co-crystal structures of LXRβ and its agonists (spiro [pyrrolidine-3,3'-oxindole] derivatives) discovered by us previously, we designed and synthesized a compound library to explore the agonistic activities. The library was screened with luciferase reporter assays, interestingly, it resulted in the discovery of 10 LXR inverse agonists besides 5 LXR agonists. To clarify the mechanism of the actions, we conducted molecular dynamics (MD) simulations on the LXR and inverse agonists complexes, and revealed that H3, H11 and H12 configurations are the key to turn on agonism or inverse agonism status for LXR. Binding tightly with H3, pushing H11 out and destabilizing H12 could form a bigger hydrophobic groove to accommodate NCOR1 to turn on LXR inverse agonism. The inverse agonist 10rr was further studied, and found as a lipogenesis inhibitor through down-regulating LXR target genes SREBP-1c, ACC, FAS and SCD-1, and demonstrated lipid-lowering effects in 3T3-L1 cells, HepG2 cells and mice with Triton WR-1339-induced hyperlipidemia. Therefore, we have proved that LXR inverse agonists can be promising agents for hyperlipidemia treatment.

Alpha-helical transmembrane protein (αTMP) is one of the two major categories of transmembrane protein (TMP). They are abundant existing in eukaryotic cells and involved in many biological processes. The special physicochemical properties, the structures of αTMP are hard to be experimentally solved, but αTMP's sequential segments are important to determine their conformations, so that TM-specific alignment is necessary to benefit their structure prediction. We used segment information extracted from topology structure and evolutionary information as features to implement a αTMP Segment Alignment method (TMSA). The method was trained using one non-redundant dataset and tested using another non-redundant dataset. Comparing the results to a general alignment method HHalign, TMSA achieved higher alignment accuracy, and easier to recognize the fold of αTMPs.

BACKGROUND Ischemia-reperfusion injury of whole brain involves a complicated pathophysiology mechanism. Dexmedetomidine (Dex) has been shown to have neuro protective functions. This study observed the effect of Dex on serum S100B and cerebral intracellular adhesion molecule-1 (ICAM-1) in a rat model of cerebral ischemia-reperfusion. MATERIAL AND METHODS Healthy Sprague Dawley (SD) rats (males, 7 weeks old) were randomly divided into sham, model, and Dex groups (n=20 each). A cerebral ischemia-reperfusion model was prepared by clipping of the bilateral common carotid artery combined with hypotension. Dex (9 μg/kg) was infused intravenously immediately after reperfusion in the Dex group, while the other two groups received an equal volume of saline. Neural defect score (NDS) was measured at 6 hours, 24 hours, and 72 hours after surgery, with pathological observation of brain tissues. ELISA was then used to test serum S100B protein level. Malondialdehyde (MDA) and superoxide dismutase (SOD) were assayed by spectrometry. Nuclear factor-kappa B (NF-kB) and ICAM-1 levels were determined by real-time (RT)-PCR. RESULTS Model rats had significant injury in the hippocampal CA1 region as shown by elevated NDS, S100B, and MDA levels, higher NF-κB and ICAM-1 mRNA expression, and lower SOD levels (p

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world's population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Furanasperterpenes A (1) and B (2) with a novel 6/6/6/6/5 pentacyclic skeleton and a new 11-acetoxy-terretonin E (3), were isolated from the marine-derived Aspergillus terreus GZU-31-1. Their structures were elucidated based on spectroscopic methods, and the absolute configurations were determined by X-ray diffraction and electronic circular dichroism (ECD) calculations. A possible biogenetic pathway was proposed. These compounds were evaluated for their lipid-lowering effects in 3T3-L1 adipocytes. Furanasperterpene A (1) showed the equivalent activity in reducing TG levels to positive control (berberine) at the concentration of 5 μM.

Cathepsin L-like protease is an important member of the papain-like cysteine protease and plays numerous indispensable roles in the biology of parasitic organisms. In a previous study, we identified a gene encoding a cathepsin L-like protease of Clonorchis sinensis (CsCPL) that was detected in the cercaria, metacercaria, and adult worm stages by immunolocalization, suggesting that this cysteine protease may be important and involved in the development of C. sinensis. In this study, the mature domain of CsCPL (CsCPL-m) was cloned and expressed in the form of inclusion bodies in Escherichia coli. After refolding, the recombinant CsCPL-m displayed optimal protease activity towards Z-Phe-Arg-AMC substrates but not towards Z-Arg-Arg-AMC, and the activity of the protease was inhibited completely by the cysteine protease-specific inhibitors E-64 and IAA, which further demonstrated that CsCPL belongs to the cathepsin L-like cysteine protease family. Recombinant CsCPL-m exhibited considerable activity at temperatures ranging from 28 to 42 °C, with the highest activity observed at 42 °C. Furthermore, recombinant CsCPL-m exhibited activity across a broad range of pH values (pH 4.0–8.0), with an optimal pH of 5.5. The Km and Vmax of the recombinant CsCPL-m towards Z-Phe-Arg-AMC were determined to be 5.71 × 10−6 M and 0.6 μM/min, respectively, at 37 °C and pH 5.5. The recombinant CsCPL-m could degrade BSA and gelatine, but could not degrade human hemoglobin and human immunoglobulin G. These results implied that CsCPL might participate in the catabolism of host proteins for nutrition during the parasitic life cycle of C. sinensis; thus, CsCPL could be used as a potential vaccine antigen and drug target against C. sinensis infection.

Haemophilus parasuis is the aetiological agent of Glasser's disease, which is responsible for cases of fibrinous polyserositis, polyarthritis and meningitis. No vaccine is known that provides cross-protection against all serovars. The identification of novel immunoprotective antigens would undoubtedly contribute to the development of efficient subunit vaccines. In the present study, an immunoproteomic approach was used to analyze secreted proteins of H. parasuis and six proteins with high immunogenicity were identified. Five of them were successfully expressed, and their immunogenicity and protective efficacy were assessed in a mouse challenge model. All five proteins elicited strong humoral antibody and cellular immune responses in mice. They all effectively reduced the growth of H. parasuis in mouse organs and conferred different levels of protection (40–80%) against challenge. IgG subtype analysis revealed that the five proteins induce a bias toward a Th1-type immune response, and a significant increase was observed in the cytokine levels of IL-2, IFN-γ and Th2-specific IL-4 in the culture supernatants of splenocytes isolated from immunized mice. The results suggest that both Th1 and Th2 responses are involved in mediating protection. These data suggest that the five proteins could be potential subunit vaccine candidates for use to prevent H. parasuis infection. Biological significance Haemophilus parasuis can cause huge financial loss in the swine industry worldwide. There are still no vaccines which can provide cross-protection against all serovars. To address this need, we applied an immunoproteomic approach involving 2-DE, MALDI-TOF/TOF MS and Western-blot to identify the secreted proteins which may be able to provide immunoprotection to this disease. We identified six immunogenic proteins, and the immunogenicity and protective efficacy were validated. This result provides a foundation for developing novel subunit vaccines against Haemophilus parasuis.

Liver X Receptor (LXR) is a potential drug target for atherosclerosis. One of the major challenges in taking LXR modulators to the clinic is steatosis. It was reported that sterol LXR agonists selectively activate LXR in the intestine and macrophage cells rather than in the liver. We hypothesize that sterol LXR agonists may selectively inhibit atherosclerosis without causing hepatic lipogenesis. Thus, based on LXR structure, 12 sterol compounds were designed and tested in a dual-luciferase reporter gene experiment. It was confirmed that compounds 4 and 6 were LXR agonists. Further experiments demonstrated that compounds 4 and 6 inhibit the formation of macrophage foam cells without inducing triglyceride accumulation in either hepatocytes or adipocytes. In vivo studies demonstrated that compound 4 promotes reverse cholesterol transport without inducing hepatic lipogenesis. Thus, we report that these compounds with sterol scaffolds can be promising leads for the treatment of atherosclerosis without inducing steatosis.

Natural immunity againstNeisseria meningitidisis thought to develop following nasopharyngeal colonization with this bacterium or other microbes expressing cross-reactive antigens.Neisseria lactamicais a commensal of the upper respiratory tract which is often carried by infants and young children; epidemiological evidence indicates that colonization with this bacterium can elicit serum bactericidal activity (SBA) againstNeisseria meningitidis, the most validated correlate of protective immunity. Here we demonstrate experimentally that immunization of mice with liveN. lactamicaprotects animals against lethal meningococcal challenge and that some, but not all, strains ofN. lactamicaelicit detectable SBA in immunized animals regardless of the serogroup ofN. meningitidis. While it is unlikely that immunization with liveN. lactamicawill be implemented as a vaccine against meningococcal disease, understanding the basis for the induction of cross-protective immunity and SBA should be valuable in the design of subunit vaccines for the prevention of this important human infection.

Cysteine proteases (CPs) were associated with the pathogenicity and excystment of Clonorchis sinensis. Most of them were potential antigens for the immunodiagnosis of clonorchiasis. More researches on CPs will let us know more about their functions, and further employ them for the development of more efficient diagnostic reagent and prevention strategies. In the current study, a full-length sequence encoding cathepsin L from C. sinensis (CsCL41.5) was identified from our adult cDNA library. Bioinformatic analysis showed that CsCL41.5 included typical motifs of cathepsin L (ERFNIN and GNFD motifs) and conserved amino acid positions which constituted the active center of the enzyme. The identity of its amino acid sequence with the cathepsin L of Schistosoma japonicum was 49.6 %. Recombinant CsCL41.5 (rCsCL41.5) was highly expressed in the form of inclusion body in Escherichia coli, and soluble rCsCL41.5 was obtained after purification and renaturation. Western blotting analysis indicated that CsCL41.5 is an excretory-secretory antigen of C. sinensis adult. Immunolocalization demonstrated that CsCL41.5 is distributed in the intestine and eggs in the uterus of adult worm, tegument of metacercaria, oral suck, and tail of cercaria. ELISA assays showed that IgG4 was the predominant IgG isotype responding to rCsCL41.5 in sera from clonorchiasis patients. The sensitivity and specificity of specific IgG4 detection with rCsCL41.5 was 62.5 % (15/24) and 81.7 % (49/60), respectively. It was concluded that there were differences in biological function, efficiency of serodiagnosis, and characterization of immune reactivity between CsCL41.5 and other CPs of C. sinensis, combining with previous studies.

Clonorchiasis is a common zoonosis in southern and northeastern parts of China, especially in Guangdong, Guangxi and Jilin province. Anti-Clonorchis sinensis antibody detection by enzyme-linked immunosorbent assay (ELISA) has been used for epidemiological surveys of clonorchiasis for its convenience and celerity, but it is still a meaningful work to screen ideal diagnostic antigen or antibody subtype for improvement of diagnostic sensitivity and specificity and for judgement of curative effect. In the present study, recombinant CsCatL-propeptide (rCsCatL-propeptide) was highly expressed in form of inclusion body in Escherichia coli. Soluble rCsCatL-propeptide with high purity were obtained after purification in denatured condition by using His Bind Purification kit, and then renatured. The major antibody subtypes responding to rCsCatL-propeptide in sera from clonorchiasis patients were IgG1 and IgG4, but the level of IgG4 was more predominant (P 0.05). The sensitivity of specific IgG4 detection (91.7%) was statistically significantly higher than that of IgG1 (25.0%) with rCsCatL-propeptide (P 0.01). The specificities of IgG1 and IgG4 detection with rCsCatL-propeptide were 83.3% and 88.5%, respectively, and the difference between them was not statistically significant (P 0.05). Cross-reactions took place when we detected IgG1 of sera from patients infected with Schistosoma japonicum, Paragonimus westermani, hookworm, Trichuris trichiura and Ascaris lumbricoides with rCsCatL-propeptide, while cross-reactions only took place in sera from patients infected with S. japonicum and P. westermani when we detected specific IgG4. The positive rate of IgG4 detection in sera from clonorchiasis patients with1,000, 1,000-4,999, 5,000-9,999, and ≥10,000 eggs per gram faeces (EPG) were 76.9%, 89.3%, 95.6%, and 100.0%, respectively. The positive rates of serodiagnosis correlated well with the EPG (r = 0.93). Overall, rCsCatL-propeptide is a valuable candidate for specific IgG4 detection in sera from clonorchiasis patients by the method of ELISA for its few cross-reaction and acceptable sensitivity. In addition, specific IgG4 detection can be used to valuate infected degree and therapeutic effect of clonorchiasis patients.

OBJECTIVE Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia. RESEARCH DESIGN AND METHODS Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose. RESULTS The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91phox or p47phox, or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function. CONCLUSIONS Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes.

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis(formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortium. M. T. G. H. was supported by the Wellcome Trust (grant number 098051). The MIC work was funded from the former AHVLA’s Research and Development Internal Investment Fund (grant number RD0030c). Objectives: The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Methods: Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. Results: A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC > 32 mg/L) and sulfisoxazole (MIC ≥ 256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. Conclusions: This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. Publisher PDF

The association of enteroviruses with myocardial disease has been investigated extensively by molecular biological techniques to detect viral RNA, but remains controversial. This retrospective study investigated the involvement of enterovirus in myocarditis or dilated cardiomyopathy (DCM) by detection of viral antigens in myocardial samples from a new patient series using an optimized immunohistochemical technique. Formalin-fixed, paraffin-embedded biopsy, autopsy or explanted myocardial tissue samples were obtained from 136 subjects. These comprised histologically proven cases of acute fatal myocarditis (n=10), DCM (n=89, including 10 patients with healing/borderline myocarditis) and a comparison group of samples from 37 unused donor hearts and cases with other conditions. A monoclonal antibody 5-D8/1 directed against a conserved, non-conformational epitope in capsid protein VP1 was employed for broad detection of different enterovirus serotypes. Investigations were performed blindly. Histological sections from 7 of 10 fatal myocarditis cases, 47 of 89 patients (52.8%) with DCM were positive for the viral capsid protein VP1 by immunohistochemical staining. Consecutive sections of positive samples were negative when the antibody was omitted or replaced with subclass- and concentration-matched normal mouse IgG. In contrast, only 3 of 37 samples (8.1%) in the comparison group were positive (Yates corrected chi(2)=19.99, P0.001: odds ratio =12.68). VP1 staining was distributed in individual or grouped myofibers and localized in the cytoplasm of myocytes. In some cases, VP1 was detected in only a few myofibers within an entire section. These results provide further evidence of enterovirus involvement in a high proportion of DCM cases and demonstrate that VP1 is present in disease stages from acute myocarditis, healing myocarditis to end-stage DCM requiring cardiac transplantation, indicating translation of viral protein during persistent enterovirus infection.

Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 10(4) to 10(7) c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.

Background A recently-licensed 10-valent pneumococcal conjugate vaccine (PHiD-CV; Synflorix, GSK) uses Protein D from Haemophilus influenzae as a carrier protein. PHiD-CV therefore has the potential to provide additional protection against nontypeable H. influenzae (NTHi). NTHi frequently causes respiratory tract infections and is associated with significant morbidity and mortality worldwide and there is currently no vaccine. Methods We developed mouse models of NTHi infection and influenza/NTHi superinfection. Mice were immunized with PHiD-CV, heat-killed NTHi, or a 13-valent pneumococcal conjugate vaccine that did not contain Protein D (PCV13; Prevenar, Pfizer) and then infected intranasally with NTHi. Results Infection with NTHi resulted in weight loss, inflammation and airway neutrophilia. In a superinfection model, prior infection with pandemic H1N1 influenza virus (strain A/England/195/2009) augmented NTHi infection severity, even with a lower bacterial challenge dose. Immunization with PHiD-CV produced high levels of antibodies that were specific against Protein D, but not heat-killed NTHi. Immunization with PHiD-CV led to a slight reduction in bacterial load, but no change in disease outcome. Conclusions PHiD-CV induced high levels of Protein D-specific antibodies, but did not augment pulmonary clearance of NTHi. We found no evidence to suggest that PHiD-CV will offer added benefit by preventing NTHi lung infection.

Objective: The mechanisms by which Coxsackie B viruses cause myocarditis or dilated cardiomyopathy are not well understood. This study examined changes in the expression of cardiac genes resulting from Coxsackievirus B3 (CVB3) infection of mice. Methods: Mice (five per group) were experimentally infected with CVB3 or mock-infected with diluent. Altered expression of genes was initially identified by cDNA array, and confirmed by semiquantitative RT-PCR, western blot and immunohistochemistry. Results: Forty-two up-regulated or down-regulated genes were observed in cDNA arrays carrying 588 known mouse genes. Among these, one down-regulated gene, Bag-1, known to be involved in inhibition of apoptosis and modulation of chaperone activity, was investigated further. Semiquantitative RT-PCR showed that Bag-1 expression was down-regulated by up to 30% in virus-infected mouse heart on day 7 compared to the mock-infected. Cell fractionation and western blot analysis confirmed that Bag-1 isoform p32 was predominant in the cytoplasm of mouse myocardium and down-regulated at 4 days or 7 days after CVB3 infection. In contrast, Bag-1 isoform p50 appeared to increase in the nuclear fraction of mouse heart at 7 days after infection. Down regulated expression and distribution of Bag-1 protein or evidence of apoptosis in the infected mouse heart was demonstrated by immunostaining or histochemistry (TUNEL assay), respectively. Conclusion: CVB3 infection induced differential expression of Bag-1 in cytoplasmic and nuclear fractions of mouse heart and apoptosis. This may be important in the pathogenesis of enterovirus heart muscle disease.

Increased expression of inducible nitric oxide synthase (iNOS) has been found in inflammatory myocardial disease and increased production of nitric oxide (NO) has both an inhibitory effect on virus replication and a cytotoxic effect on host cells. To investigate the relationship between severity of enteroviral myocarditis and iNOS expression, a characterised murine model was infected with either cardiovirulent or an attenuated Coxsackievirus B3 and myocardial samples were collected on Day 7. The ability of these viruses to induce NOS expression was compared by measurement of iNOS enzyme activity and localisation of iNOS protein or peroxynitrite, a product of excessive NO production. In accordance with previous reports, high expression of iNOS was detected in mice infected with the cardiovirulent virus. The iNOS protein was located mainly in infiltrating macrophages in and around foci of necrotic myofibres where viral genomic RNA was detected. In contrast, the level of iNOS expression was significantly lower in mice infected with the attenuated virus. This correlates with fewer and smaller myocarditic lesions and less infiltrating cells in the heart. iNOS was not detected in mock-infected mice by the above assays. These findings suggest that one mechanism of attenuation may be associated with the reduced ability of the variant to induce NOS expression in the heart. This also confirms a cytotoxic role for NO in the pathogenesis of Coxsackievirus B3-induced myocarditis.

Aims Cardiomyocyte apoptosis contributes to cardiac complications of diabetes. The aim of this study was to investigate the role of calpain in cardiomyocyte apoptosis induced by hyperglycaemia. Methods and results In cultured adult rat ventricular cardiomyocytes, high glucose (33 mM) increased calpain activity and induced apoptosis, concomitant with the impairment of Na+/K+ ATPase activity. These effects of high glucose on cardiomyocytes were abolished by various pharmacological calpain inhibitors, knockdown of calpain-1 but not calpain-2 using siRNA, or over-expression of calpastatin, a specific endogenous calpain inhibitor. The effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor of Na+/K+ ATPase. Furthermore, blocking gp91 phox -NADPH oxidase activation, L-type calcium channels, or ryanodine receptors prevented calpain activation and apoptosis in high glucose-stimulated cardiomyocytes. In a mouse model of streptozotocin-induced diabetes, administration of different calpain inhibitors blocked calpain activation, increased the Na+/K+ ATPase activity, and decreased apoptosis in the heart. Conclusion Calpain-1 activation induces apoptosis through down-regulation of the Na+/K+ ATPase activity in high glucose-stimulated cardiomyocytes and in vivo hyperglycaemic hearts. High glucose-induced calpain-1 activation is mediated through the NADPH oxidase-dependent pathway and associated with activation of L-type calcium channels and ryanodine receptors. Our data suggest that calpain activation may be important in the development of diabetic cardiomyopathy and thus may represent a potential therapeutic target for diabetic heart diseases.

Cathepsin L of parasite plays multiple roles in growth, food uptake, and invasion into host and pathogenesis, which makes it a valuable target for diagnosis, vaccine, and drug. In this study, we identified a cDNA encoding cathepsin L homolog (CsCPL) from the library of Clonorchis sinensis adult by bioinformatics analysis. Sequence encoding proenzyme of CsCPL (removal of signal peptide, CsproCPL) was highly expressed in form of inclusion body in Escherichia coli, and soluble rCsproCPL (about 1 mg/ml) in high purity were obtained after denaturation, purification, and renaturation. Western blot analysis indicated that CsCPL is a component of excretory-secretory products of adult, in mature form of protease. Reverse transcription polymerase chain reaction showed that CsCPL is also expressed in metacercaria and cercaria stage. Immunolocalization demonstrated that CsCPL is deposited at adult intestine, or tegument, and tegumentary cell of metacercaria and cercaria (especially at dorsal tegument of cercaria), indicating different secretory routine roles in adult and larva. The characteristics of CsCPL suggested that it may involve in invasion of cercaria into fish and development to metacercaria, excystment of metacercaria, and protein digestion of adult, which may render it a candidate antigen for fish vaccine and serodiagnosis of human clonorchiasis.

We addressed the hypothesis that meningococcal secreted proteins (MSPs) can elicit protective immunity against meningococcal disease. Endotoxin-depleted MSP preparations were used to immunise a group of 15 six-week-old BALB/c mice (25 μg MSPs/dose mixed with Freund's complete adjuvant) on days 0, 14 and 21. Mice were challenged 2 weeks later with 107 colony forming units of live Neisseria meningitidis strain MC58 (serogroup B, ET-5). Negative and positive control groups of 15 mice each were injected with adjuvant only, or a live attenuated strain of MC58, respectively. Seven out of 15 mice (47%) from the negative control group died after 72 h of challenge, whereas none of test or positive control group died. Protection afforded by the anti-MSP immune response can be at least partly attributed to complement-mediated bacterial lysis, detectable in vitro using the serum of immunised mice. Murine anti-MC58 MSP sera were bactericidal against homologous and five unrelated ET-5 serogroup B strains. However, failed to kill strains from other hypervirulent clonal lineages belonging to the same or different serogroups, despite the presence of cross-reactive antibodies detectable by immunoblotting. Similar sera raised against MSPs from an isolate belonging to the ET-37 electropherotype lineage were bactericidal against all tested isolates of this lineage and, in addition, against some but not all isolates belonging to the ET-5 lineage. FACS analysis of intact bacteria treated with anti-MSPs confirmed surface-binding of antibodies.

Human clonorchiasis, caused by the infection of Clonorchis sinensis, is endemic in China. In this study, a full-length cDNA sequence encoding C. sinensis tegumental protein 22.3 kDa (CsTP22.3) was identified from adult cDNA library. Recombinant CsTP22.3 was expressed and purified from Escherichia coli BL21. CsTP22.3 was immunolocalized at the tegument of adult C. sinensis by using anti-recombinant CsTP22.3 sera. ELISA was performed using rCsTP22.3 protein to investigate the IgG response to CsTP22.3. Sera from clonorchiasis patients had clear IgG response to CsTP22.3. We used the CotC, a major component of the Bacillus subtilis spore coat, as a fusion partner for the expression of C. sinensis TP22.3 on the spore coat. Western blotting and immunofluorescence analyses were used to identify TP22.3 surface expression on spores. Recombinant spores displaying the TP22.3 antigen were used for oral immunization and were shown to generate mucosal response in rats. TP22.3-specific secretory IgA in faeces reached significant levels 2 weeks after oral dosing. In addition, the recombinant spores induced a significant level of protection (44.7%, p0.05) in rats challenged with C. sinensis metacercariae. This report shows that C. sinensis TP22.3 expressed on B. subtilis spores was immunogenic and oral administration of TP22.3 spore can provide protection against C. sinensis.

A cDNA clone encoding a novel lysophospholipase with a predicted molecular weight of 25.2 kDa was isolated from a Clonorchis sinensis adult cDNA library. The enzyme activity of the recombinant protein expressed in Escherichia coli was determined using phosphatidylcholine and lysophosphatidylcholine as substrates. Western blotting analysis indicated that it belonged to excretory/secretory proteins of the adults. The sensitivity and specificity of the recombinant antigen for serodiagnosis were evaluated with immunoglobulin enzyme-linked immunosorbent assay using serum samples from 20 patients with clonorchiasis and 20 patients with schistosomiasis. The sensitivity (75%) and specificity (80%) of the recombinant protein were comparable to those of crude extracts, at 65 and 82.5%, respectively. The sensitivity of the recombinant protein was 77% using 100 serum samples of clonorchiasis patients with various parasite burden. The results suggested that the recombinant lysophospholipase protein was not a satisfactory candidate for diagnosis of clonorchiasis, although it might be an excretory/secretory protein.

From a Clonorchis sinensis adult cDNA plasmid library, a cDNA clone encoding a novel lysophosphatidic acid phosphatase (LPAP) homologue was isolated. The predicted molecular weight of putative protein was 48.8 kDa and the deduced amino acid sequence had 45%, 32%, and 29% identity with LPAP of Schistosoma japonicum, Danio rerio, and Homo sapiens, respectively. Prediction of signal peptide and Western blot analysis indicated that the CsLPAP homologue was an excretory–secretory antigen (ES antigen) of C. sinensis. Immunostaining revealed that the CsLPAP was markedly localized in the intestinal cecum, seminal receptacle and eggs of the adult worm. The recombinant CsLPAP showed slightly higher sensitivity (82.14%) and specificity (85.86%) than the crude worm antigen by enzyme-linked immunosorbent assay (ELISA), a result which suggested that the recombinant antigen might be valuable in the serodiagnosis of human clonorchiasis.

Some effective antiparasitic drugs for chemotherapy schistosomiasis, such as praziquantel and artemether, are proved to act on lactate dehydrogenase (LDH) in vitro, but detailed molecular action mechanisms of these have not yet been elucidated. To this end, the sequence encoding LDH of Schistosoma japonicum (SjLDH) was amplified from the cDNA library of the adult parasite. Sequence analysis revealed an open reading frame (ORF) of 999 bp, encoding 332 amino acids with a molecular weight of 36.120 kDa. It has three transmembrane regions and may be located in the cytoplasm membrane. The encoding ORF was inserted into pET-28a vector and expressed in Escherichia coli (E. coli) induced by IPTG. A 36-kDa protein with 6×His-tag was purified by metal affinity column and identified by SDS-PAGE and Western blot, which had high LDH activity of 379 U/mg. Characterization such as Michaelis constant (K m) and maximum velocity (V max), optimum pH, and temperature of the protein were assayed. It provides a model for drug screening on SjLDH.

Meningococcal disease is a life-threatening infection caused by Neisseria meningitidis . Currently, there are no vaccines to prevent infection with serogroup B N. meningitidis strains, the leading cause of meningococcal meningitis in Europe and North America. Here we describe the construction and characterization of two attenuated serogroup B N. meningitidis strains, YH102 (MC58Δ sia Δ rfaF ) and YH103 (MC58Δ sia Δ metH ). Both strains are markedly attenuated in their capacity to cause bacteremia in rodent models and have a reduced ability to survive in a human whole-blood assay. Immunization of adult mice with these strains leads to the development of bactericidal antibodies and confers sterilizing protection against challenge with homologous live bacteria. Furthermore, we show that the strains confer protection against infection by other serogroups. Use of the attenuated strains in animals with gene knockouts or after depletion of immunological effectors could be used to define the basis of protection, and human volunteer studies could be undertaken to examine the immune response following exposure to this important human pathogen.