Marixanthotalea marina gen. nov., sp. nov., a bacterium in the family Flavobacteriaceae isolated from seawater.

A Gram-stain-negative, yellow-pigmented, non-motile, rod-shaped, catalase-positive, strictly aerobic marine bacterium, designated XHP0103T, was isolated from seawater collected from the southern Yellow Sea, PR China (34° 45' 53″ N 119° 25' 30″ E). Strain XHP0103T grew optimally at 28 °C, pH 7.5 and in 1.0-3.0 % (w/v) sea salt. MK-6 was the major respiratory quinone. The major cellular fatty acids (>10%) were iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The polar lipid profile contained phosphatidylethanolamine, an unidentified aminolipid, an unidentified glycolipid and an unidentified lipid. Results of 16S rRNA gene sequence analysis indicated that strain XHP0103T displayed highest sequence similarity to Aestuariibaculum marinum IP7T (94.1 %). However, the phylogenetic trees based on 16S rRNA gene sequences suggested that strain XHP0103T clustered with Tamlana crocina HST1-43T (93.4 % sequence similarity) and Aestuariivivens insulae AH-MY3T (93.5 %). Genome sequencing revealed that strain XHP0103T comprised 3 134 388 bp with 2770 protein-coding genes, and the DNA G+C content was 35.5 %. The average nucleotide identity and digital DNA-DNA hybridization values between strain XHP0103T and T. crocina HST1-43T were 73.6 and 17.3 %, respectively. Based on phylogenetic, phenotypic, genomic and chemotaxonomic evidence, strain XHP0103T represents a novel genus in the family Flavobacteriaceae, for which the name Marixanthotalea marina gen. nov., sp. nov. is proposed. The type strain is XHP0103T (=MCCC 1K06060T=JCM 34682T).

TRPC channels blockade abolishes endotoxemic cardiac dysfunction by hampering intracellular inflammation and Ca2+ leakage.

Intracellular Ca2+ dysregulation is a key marker in septic cardiac dysfunction; however, regulation of the classic Ca2+ regulatory modules cannot successfully abolish this symptom. Here we show that the knockout of transient receptor potential canonical (TRPC) channel isoforms TRPC1 and TRPC6 can ameliorate LPS-challenged heart failure and prolong survival in mice. The LPS-triggered Ca2+ release from the endoplasmic reticulum both in cardiomyocytes and macrophages is significantly inhibited by Trpc1 or Trpc6 knockout. Meanwhile, TRPC's molecular partner - calmodulin - is uncoupled during Trpc1 or Trpc6 deficiency and binds to TLR4's Pococurante site and atypical isoleucine-glutamine-like motif to block the inflammation cascade. Blocking the C-terminal CaM/IP3R binding domain in TRPC with chemical inhibitor could obstruct the Ca2+ leak and TLR4-mediated inflammation burst, demonstrating a cardioprotective effect in endotoxemia and polymicrobial sepsis. Our findings provide insight into the pathogenesis of endotoxemic cardiac dysfunction and suggest a novel approach for its treatment.

TRPC1 promotes the genesis and progression of colorectal cancer via activating CaM-mediated PI3K/AKT signaling axis.

Transient receptor potential canonical (TRPC) channels are the most prominent nonselective cation channels involved in various diseases. However, the function, clinical significance, and molecular mechanism of TRPCs in colorectal cancer (CRC) progression remain unclear. In this study, we identified that TRPC1 was the major variant gene of the TRPC family in CRC patients. TRPC1 was upregulated in CRC tissues compared with adjacent normal tissues and high expression of TRPC1 was associated with more aggressive tumor progression and poor overall survival. TRPC1 knockdown inhibited cell proliferation, cell-cycle progression, invasion, and migration in vitro, as well as tumor growth in vivo; whereas TRPC1 overexpression promoted colorectal tumor growth and metastasis in vitro and in vivo. In addition, colorectal tumorigenesis was significantly attenuated in Trpc1-/- mice. Mechanistically, TRPC1 could enhance the interaction between calmodulin (CaM) and the PI3K p85 subunit by directly binding to CaM, which further activated the PI3K/AKT and its downstream signaling molecules implicated in cell cycle progression and epithelial-mesenchymal transition. Silencing of CaM attenuated the oncogenic effects of TRPC1. Taken together, these results provide evidence that TRPC1 plays a pivotal oncogenic role in colorectal tumorigenesis and tumor progression by activating CaM-mediated PI3K/AKT signaling axis. Targeting TRPC1 represents a novel and specific approach for CRC treatment.

Novel H7N2 and H5N6 Avian Influenza A Viruses in Sentinel Chickens: A Sentinel Chicken Surveillance Study.

In 2014, a sentinel chicken surveillance for avian influenza viruses was conducted in aquatic bird habitat near Wuxi City, Jiangsu Province, China. Two H7N2, one H5N6, and two H9N2 viruses were isolated. Sequence analysis revealed that the H7N2 virus is a novel reassortant of H7N9 and H9N2 viruses and H5N6 virus is a reassortant of H5N1 clade 2.3.4 and H6N6 viruses. Substitutions V186 and L226 (H3 numbering) in the hemagglutinin (HA) gene protein was found in two H7N2 viruses but not in the H5N6 virus. Two A138 and A160 mutations were identified in the HA gene protein of all three viruses but a P128 mutation was only observed in the H5N6 virus. A deletion of 3 and 11 amino acids in the neuraminidase stalk region was found in two H7N2 and H5N6 viruses, respectively. Moreover, a mutation of N31 in M2 protein was observed in both two H7N2 viruses. High similarity of these isolated viruses to viruses previously identified among poultry and humans, suggests that peridomestic aquatic birds may play a role in sustaining novel virus transmission. Therefore, continued surveillance is needed to monitor these avian influenza viruses in wild bird and domestic poultry that may pose a threat to poultry and human health.

Pandemic (H1N1) 2009 Influenza Virus Infection in A Survivor Who Has Recovered from Severe H7N9 Virus Infection, China.

We firstly report a patient who presented with severe complications after infection with influenza A(H1N1) pdm2009, more than 1 year after recovery from severe H7N9 virus infections. The population of patients who recovered from severe H7N9 infections might be at a higher risk to suffer severe complications after seasonal influenza infections, and they should be included in the high-risk populations recommended to receive seasonal influenza vaccination.

Characterization of a novel reassortant influenza A virus (H2N2) from a domestic duck in Eastern China.

While H2N2 viruses have been sporadically isolated from wild and domestic birds, H2N2 viruses have not been detected among human populations since 1968. Should H2N2 viruses adapt to domestic poultry they may pose a risk of infection to people, as most anyone born after 1968 would likely be susceptible to their infection. We report the isolation of a novel influenza A virus (H2N2) cultured in 2013 from a healthy domestic duck at a live poultry market in Wuxi City, China. Sequence data revealed that the novel H2N2 virus was similar to Eurasian avian lineage avian influenza viruses, the virus had been circulating for ≥ two years among poultry, had an increase in α2,6 binding affinity, and was not highly pathogenic. Approximately 9% of 100 healthy chickens sampled from the same area had elevated antibodies against the H2 antigen. Fortunately, there was sparse serological evidence that the virus was infecting poultry workers or had adapted to infect other mammals. These findings suggest that a novel H2N2 virus has been circulating among domestic poultry in Wuxi City, China and has some has increased human receptor affinity. It seems wise to conduct better surveillance for novel influenza viruses at Chinese live bird markets.

[A survey of levels of antibodies against influenza viruses in the population of Wuxi City].

OBJECTIVE: To investigate levels of antibodies against type A and type C influenza viruses and those against the 2009 H1N1 influenza A virus (before and after the 2009 H1N1 pandemic) among residents in Wuxi. To compare levels of antibodies against the 2009 H1N1 influenza virus (one year after the pandemic) in the unvaccinated population with those in the population who received vaccine. METHODS: Serum samples were collected from subjects (aged 1-60 years) during September 2008 to May 2009, and during September 2010 to January 2011. Also collected were serum samples from adults who had received vaccines for pandemic (H1N1) 2009 for one year. Antibody response to influenza viruses was measured using hemagglutination inhibition (HI) assay. Seropositivity rate, seroprotection rate and geometric mean titer (GMT) were compared for each age group during different periods. RESULTS: Before the outbreak of the 2009 H1N1 pandemic, seropositivity rate, seroprotection rate and GMT among the study subjects in were 2.86% (4/140), 0.71% (1/140) and 5.23, respectively. One year after the outbreak, seropositivity rate, seroprotection rate and GMT among the study subjects were 66.33%, 37.76% and 19.17, respectively. Among them, adult subjects showed 50.00% seropositivity rate, 19.44% seroprotection rate and 13.09 GMT, while adult subjects who had received vaccine for one year showed 61.36% seropositivity rate, 22.73% seroprotection rate and 14.14 GMT. No significant difference was observed between these two populations (P > 0.05 for all three indexes). Furthermore, before the outbreak of the 2009 H1N1 pandemic, levels of antibodies against seasonal influenza viruses among the study subjects were as follows: for H1N1 virus, seropositivity rate, seroprotection rate and GMT were 55.00%, 35.00% and 16.90, respectively; for H3N2 virus, seropositivity rate, seroprotection rate and GMT were 86.40%, 84.30% and 58.56, respectively. CONCLUSION: One year after the 2009 H1N1 influenza A virus had spread to Wuxi, the population levels of antibodies against this virus have approached those against seasonal influenza viruses, as reflected by seropositivity rates, seroproection rates and GMT. Moreover, considerable levels of antibodies against seasonal influenza viruses were observed in populations, indicating no seasonal influenza outbreak would occur recently.

Immunoproteomic analysis of human serological antibody responses to vaccination with whole-cell pertussis vaccine (WCV).

BACKGROUND: Pertussis (whooping cough) caused by Bordetella pertussis (B.p), continues to be a serious public health threat. Vaccination is the most economical and effective strategy for preventing and controlling pertussis. However, few systematic investigations of actual human immune responses to pertussis vaccines have been performed. Therefore, we utilized a combination of two-dimensional electrophoresis (2-DE), immunoblotting, and mass spectrometry to reveal the entire antigenic proteome of whole-cell pertussis vaccine (WCV) targeted by the human immune system as a first step toward evaluating the repertoire of human humoral immune responses against WCV. METHODOLOGY/PRINCIPAL FINDINGS: Immunoproteomic profiling of total membrane enriched proteins and extracellular proteins of Chinese WCV strain 58003 identified a total of 30 immunoreactive proteins. Seven are known pertussis antigens including Pertactin, Serum resistance protein, chaperonin GroEL and two OMP porins. Sixteen have been documented to be immunogenic in other pathogens but not in B.p, and the immunogenicity of the last seven proteins was found for the first time. Furthermore, by comparison of the human and murine immunoproteomes of B.p, with the exception of four human immunoreactive proteins that were also reactive with mouse immune sera, a unique group of antigens including more than 20 novel immunoreactive proteins that uniquely reacted with human immune serum was confirmed. CONCLUSIONS/SIGNIFICANCE: This study is the first time that the repertoire of human serum antibody responses against WCV was comprehensively investigated, and a small number of previously unidentified antigens of WCV were also found by means of the classic immunoproteomic strategy. Further research on these newly identified predominant antigens of B.p exclusively against humans will not only remarkably accelerate the development of diagnostic biomarkers and subunit vaccines but also provide detailed insight into human immunity mechanisms against WCV. In particular, this work highlights the heterogeneity of the B.p immunoreactivity patterns of the mouse model and the human host.

Molecular cloning and characterization of a prenyltransferase from the cotton aphid, Aphis gossypii.

Prenyltransferases play a key role in isoprenoid biosynthesis. Here, a cDNA encoding a prenyltransferase was isolated from the cotton aphid, Aphis gossypii, which consists of 1354 nucleotides and encodes a protein of 394 amino acids (AgIPPS). Subsequent sequencing of AgIPPS genomic DNA resulted in one 3138-bp sequence. Southern blotting analysis indicated that only a single IPPS gene exists in the cotton aphid. Real-time quantitative PCR showed that AgIPPS transcripts were mainly present at the corpora allatum, but small quantity could be detected in tissues other than the corpora allatum. Transcript abundance changed in an alternative manner at different life stages. High expression was observed in embryos, second and forth instar nymphs and adults, but only low level was detected in the first and third instars. Functional expression, activity assay and product analysis revealed that the mature form of AgIPPS (AgIPPS-S) could efficiently convert labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to both geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). These data suggested that, unlike the green peach aphid and the pea aphid, the cotton aphid appears to contain only a single IPPS with dual FPP/GPP synthase activity.

Comparative proteomic analysis of Campylobacter jejuni cultured at 37C and 42C.

Campylobacter jejuni has the potential to thrive at 37C (e.g., in the human intestinal tract) and 42C (e.g., in the poultry intestinal tract). We aimed to determine the protein expression profiles of C. jejuni cultured at 37C and 42C in vitro by two-dimensional electrophoresis (2DE). The differentially expressed spots/proteins between C. jejuni cultured at 37C and 42C were defined when their expression differed by twofold. The differently expressed spots detected from C. jejuni cultured both on agar and in broth at 37C and 42C were subjected to protein identification by MALDI-TOF/TOF. Overall, 15 and 20 differentially expressed proteins were defined for C. jejuni cultured at the two temperatures on agar and in broth, respectively. All of the identified differentially expressed proteins could be clustered as proteins involving the metabolism, regulator system, periplasmic proteins and the major antigens of C. jejuni. In conclusion, there are subsets of proteins that are optimally expressed at 37C, which may contribute to the host adaptation and/or the pathogenicity in the human intestinal tract.

Toxoplasma gondii: proteomic analysis of antigenicity of soluble tachyzoite antigen.

The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals. The tachyzoite of T. gondii is the main life-cycle stage that is responsible for toxoplasmosis. Study of the antigenicity of soluble tachyzoite antigen (STAg) is important for discovery of protective antigens which will aid in the detection and prevention of toxoplasmosis. At present, no complete proteome map of T. gondii STAg is established, although a large-scale whole proteomic analysis of tachyzoites is underway. In this study, 1227 protein spots of T. gondii soluble tachyzoite antigen (STAg) were fractionated by 2-dimensional electrophoresis (2-DE) at pH range 3-10. By mass spectrometry (MS) analysis, among the separated 1227 protein spots, 426 were identified by searching the Swissport and NCBI nr databases. Two hundred and thirty of these identified spots (230/426, 54%) were demonstrated to be T. gondii protein by MS. Of the 21 Toxoplasma protein spots identified by Western blot with rabbit anti-T. gondii serum, 16 had immunoregulatory functions and five had immune defense functions. Due to multiple spots for a single protein, these 16 spots represented 11 proteins: a putative protein disulfide isomerase (PDI), heat shock protein 60 (Hsp60), a pyruvate kinase (PK), a putative glutamate dehydrogenase (GDH), a coronin, a heat shock protein 70 (Hsp70), a protein kinase C receptor 1 (RACK1), a malate dehydrogenase (MDH), a major surface antigen 1 (SAG1), an uridine phosphorylase (UPase) and a peroxiredoxin (Prx). Among the identified 11 proteins, except that the antigenicity and immunogenicity of the SAG1 has been reported and antigenicity of Hsp70 has been disputed, the remaining antigenic proteins were first identified in this study. In conclusion, we obtained nine novel types of immunogenic proteins that might be potential candidates of vaccine development for toxoplasmosis, which we will confirm in later studies.

[Etiology monitoring of influenza virus and variation in hemagglutinin genes of H13 subtype in Wuxi area].

OBJECTIVE: To monitor the seasonal distribution of influenza types and subtypes in Wuxi area during 2005-2008, and to investigate the variation in hemagglutinin (HA) genes of A/H3N2 strains in 2008. METHODS: Nose-throat swab specimens were collected in Wuxi area from flu-like patients from outpatient departments of hospitals as well as from clustering flu-like outbreak patients from workspace, followed by MDCK cell inoculation. Types and subtypes of positive influenza isolates were identified using standard antiserum. We then sequenced the HA genes for H3 subtype influenza viruses isolated from 2008 specimens to investigate the variation in HA genes. RESULTS: During 2005 and September 2008, 435 strains of influenza viruses were isolated from flu-like patients in Wuxi Area, among which 164 isolates are of A/H1N1 subtype, 80 isolates are of A/H3N2 subtype, and 191 isolates are of B type. These types/subtypes have significant seasonal distributions. Sequences of HA genes for H3 subtype show that the 9 strains isolated in Wuxi area are similar to those of strains isolated in Shanghai within the same period. Many of the sequences belong to the same branch of the phylogenetic tree, and are similar to sequences of vaccine strains in WHO 2008-2009 repositories. CONCLUSION: A/H1N1, A/H3N2 and B still attribute to most of the sporadic and local outbreaks of influenza infection in Wuxi area in recent years. HA genes of A/H3N2 strains isolated in Wuxi area are similar to those of strains isolated in Shanghai in the same period, and also similar to those of vaccine strains recommended by WHO for 2008-2009.

[Intranasal immunization with mucosal complex vaccine protects mice against Toxoplasma gondii].

OBJECTIVE: To study the mucosal and systemic immune response after intranasal immunization with mucosal complex vaccine for Toxoplasma gondii, and to observe the protective effect on mice. METHODS: The mucosal complex vaccine was made of soluble tachyzoite antigen (STAg) and cholera toxin (CT), which were mixed and dissolved in PBS (1 ml PBS containing 1 mg STAg and 50 microg CT). Fifty-two BALB/c mice were randomly divided into two groups: immunized group and control. Mice were intranasally immunized with 20 microl mucosal complex vaccine (20 microg STAg and l microg CT) per mouse twice at an interval of two weeks, while the control mice were given PBS solution instead. Six mice of each group were killed by dislocation of cervical vertebra on day 14 after the last immunization. The specific IgG antibodies in serum and IgA in feces were detected by ELISA. Lymphocytes in spleen, Peyer's patches (PP) and intestinal intraepithelial lymphocyte (IEL) were isolated and counted. Percentage of CD4+ and CD8+ T cells was determined by immunocytochemistry. Other mice were challenged intragastrically each with 4 x 10(4) tachyzoites of RH strain Toxoplasma gondii on day 14 after the last immunization. Their health condition was observed and the number of tachyzoites in liver and brain was determined microscopically on the 30 th day after challenge. RESULTS: IgG antibodies in serum and IgA antibodies in feces of immunized mice were higher than the control (P < 0.05). Lymphocytes in spleen, PP and IEL significantly increased after immunization (P < 0.01). The CD4+ and CD8+ T cells were both higher than that of the control (P < 0.05) in spleen and PP. The number of CD8+ T cells in IEL increased significantly (P < 0.01), and the ratio of CD4+ and CD8+ T cells was reversed with significance (P < 0.05). On the day 30 after challenge, the survival rate of immunized mice was higher than that of control (P < 0.05), while the tachyzoite load in liver and brain was significantly smaller respectively. CONCLUSION: Intranasal inoculation with mucosal complex vaccine effectively induces the mucosal and systemic immune response, and protects mice against Toxoplasma gondii.