Browsing by Author "Jiang, Xi"

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    Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae
    Buren, Stefan; Pratt, Katelin; Jiang, Xi; Guo, Yisong; Jimenez-Vicente, Emilio; Echavarri-Erasun, Carlos; Dean, Dennis R.; Saaem, Ishtiaq; Gordon, D. Benjamin; Voigt, Christopher A.; Rubio, Luis M. (National Academy of Sciences, 2019-12-10)
    The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.
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    Development and evaluation of two subunit vaccine candidates containing antigens of hepatitis E virus, rotavirus, and astrovirus
    Xia, Ming; Wei, Chao; Wang, Leyi; Cao, Dianjun; Meng, Xiang-Jin; Jiang, Xi; Tan, Ming (Springer Nature, 2016-05-19)
    Hepatitis E virus (HEV), rotavirus (RV), and astrovirus (AstV) are important pathogens that transmit through a common fecal-oral route, causing hepatitis (HEV) and gastroenteritis (RV and AstV) respectively in humans. In this study, we developed and evaluated two subunit vaccine candidates that consisted of the same protruding or spike protein antigens of the three viruses in two formats, a fusion of the three antigens into one molecule (fused vaccine) vs. a mixture of the three free antigens together (mixed vaccine). Both vaccines were easily made via E. coli expression system. Mouse immunization experiments showed that the fused vaccine elicited significantly higher antibody responses against the three viral antigens than those induced by the mixed vaccine. In addition, the mouse post-immune antisera of the fused vaccine revealed significantly higher neutralizing titers against HEV infection in cell culture, as well as significantly higher 50% blocking titers (BT50) against RV VP8-HBGA receptor interactions than those of the post-immune antisera after immunization of the mixed vaccine. Thus, the fused vaccine is a promising trivalent vaccine candidate against HEV, RV, and AstV, which is worth for further development.
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    EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity
    Sun, Zhixiong; Xu, Xiguang; He, Jianlin; Murray, Alexander; Sun, Ming-an; Wei, Xiaoran; Wang, Xia; McCoig, Emmarose; Xie, Evan; Jiang, Xi; Li, Liwu; Zhu, Jinsong; Chen, Jianjun; Morozov, Alexei; Pickrell, Alicia M.; Theus, Michelle H.; Xie, Hehuang David (2019-08-29)
    Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.
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    Enterobacter cloacae inhibits human norovirus infectivity in gnotobiotic pigs
    Lei, Shaohua; Samuel, Helen; Twitchell, Erica; Bui, Tammy; Ramesh, Ashwin; Wen, Ke; Weiss, Mariah; Li, Guohua; Yang, Xingdong; Jiang, Xi; Yuan, Lijuan (2016-04-26)
    Human noroviruses (HuNoVs) are the leading cause of epidemic gastroenteritis worldwide. Study of HuNoV biology has been hampered by the lack of an efficient cell culture system. Recently, enteric commensal bacteria Enterobacter cloacae has been recognized as a helper in HuNoV infection of B cells in vitro. To test the influences of E. cloacae on HuNoV infectivity and to determine whether HuNoV infects B cells in vivo, we colonized gnotobiotic pigs with E. cloacae and inoculated pigs with 2.74 × 10(4) genome copies of HuNoV. Compared to control pigs, reduced HuNoV shedding was observed in E. cloacae colonized pigs, characterized by significantly shorter duration of shedding in post-inoculation day 10 subgroup and lower cumulative shedding and peak shedding in individual pigs. Colonization of E. cloacae also reduced HuNoV titers in intestinal tissues and in blood. In both control and E. cloacae colonized pigs, HuNoV infection of enterocytes was confirmed, however infection of B cells was not observed in ileum, and the entire lamina propria in sections of duodenum, jejunum, and ileum were HuNoV-negative. In summary, E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs, with or without E. cloacae colonization.
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    Evaluation of the 50% Infectious Dose of Human Norovirus Cin-2 in Gnotobiotic Pigs: A Comparison of Classical and Contemporary Methods for Endpoint Estimation
    Ramesh, Ashwin; Parreño, Viviana; Schmidt, Philip J.; Lei, Shaohua; Zhong, Weiming; Jiang, Xi; Emelko, Monica B.; Yuan, Lijuan (MDPI, 2020-08-28)
    Human noroviruses (HuNoVs) are the leading causative agents of epidemic and sporadic acute gastroenteritis that affect people of all ages worldwide. However, very few dose–response studies have been carried out to determine the median infectious dose of HuNoVs. In this study, we evaluated the median infectious dose (ID50) and diarrhea dose (DD50) of the GII.4/2003 variant of HuNoV (Cin-2) in the gnotobiotic pig model of HuNoV infection and disease. Using various mathematical approaches (Reed–Muench, Dragstedt–Behrens, Spearman–Karber, exponential, approximate beta-Poisson dose–response models, and area under the curve methods), we estimated the ID50 and DD50 to be between 2400–3400 RNA copies, and 21,000–38,000 RNA copies, respectively. Contemporary dose–response models offer greater flexibility and accuracy in estimating ID50. In contrast to classical methods of endpoint estimation, dose–response modelling allows seamless analyses of data that may include inconsistent dilution factors between doses or numbers of subjects per dose group, or small numbers of subjects. Although this investigation is consistent with state-of-the-art ID50 determinations and offers an advancement in clinical data analysis, it is important to underscore that such analyses remain confounded by pathogen aggregation. Regardless, challenging virus strain ID50 determination is crucial for identifying the true infectiousness of HuNoVs and for the accurate evaluation of protective efficacies in pre-clinical studies of therapeutics, vaccines and other prophylactics using this reliable animal model.
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    High Protective Efficacy of Probiotics and Rice Bran against Human Norovirus Infection and Diarrhea in Gnotobiotic Pigs
    Lei, Shaohua; Ramesh, Ashwin; Twitchell, Erica; Wen, Ke; Bui, Tammy; Weiss, Mariah; Yang, Xingdong; Kocher, Jacob; Li, Guohua; Giri-Rachman, Ernawati; Trang, Nguyen Van; Jiang, Xi; Ryan, Elizabeth P.; Yuan, Lijuan (2016)
    Probiotics have been recognized as vaccine adjuvants and therapeutic agents to treat acute gastroenteritis in children. We previously showed that rice bran (RB) reduced human rotavirus diarrhea in gnotobiotic pigs. Human noroviruses (HuNoVs) are the major pathogens causing non-bacterial acute gastroenteritis worldwide. In this study, Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle 1917 (EcN) were first screened for their ability to bind HuNoV P particles and virions derived from clinical samples containing HuNoV genotype GII.3 and GII.4, then the effects of LGG+EcN and RB on HuNoV infection and diarrhea were investigated using the gnotobiotic pig model. While LGG+EcN colonization inhibited HuNoV shedding, probiotic cocktail regimens in which RB feeding started 7 days prior to or 1 day after viral inoculation in the LGG+EcN colonized gnotobiotic pigs exhibited high protection against HuNoV diarrhea and shedding, characterized by significantly reduced incidence (89 versus 20%) and shorter mean duration of diarrhea (2.2 versus 0.2 days), as well as shorter mean duration of virus shedding (3.2 versus 1.0 days). In both probiotic cocktail groups, the diarrhea reduction rates were 78% compared with the control group, and diarrhea severity was reduced as demonstrated by the significantly lower cumulative fecal scores. The high protective efficacy of the probiotic cocktail regimens was attributed to stimulation of IFN-γ(+) T cell responses, increased production of intestinal IgA and IgG, and maintenance of healthy intestinal morphology (manifested as longer villi compared with the control group). Therefore, probiotic cocktail regimens containing LGG+EcN and RB may represent highly efficacious strategies to prevent and treat HuNoV gastroenteritis, and potentially other human enteric pathogens.
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    Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency
    Lei, Shaohua; Ryu, Junghyun; Wen, Ke; Twitchell, Erica; Bui, Tammy; Ramesh, Ashwin; Weiss, Mariah; Li, Guohua; Samuel, Helen; Clark-Deener, Sherrie; Jiang, Xi; Lee, Kiho; Yuan, Lijuan (Nature Publishing Group, 2016-04-27)
    Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.
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    Parenterally Administered P24-VP8* Nanoparticle Vaccine Conferred Strong Protection against Rotavirus Diarrhea and Virus Shedding in Gnotobiotic Pigs
    Ramesh, Ashwin; Mao, Jiangdi; Lei, Shaohua; Twitchell, Erica; Shiraz, Ashton K.; Jiang, Xi; Tan, Ming; Yuan, Lijuan (MDPI, 2019-11-06)
    Current live rotavirus vaccines are costly with increased risk of intussusception due to vaccine replication in the gut of vaccinated children. New vaccines with improved safety and cost-effectiveness are needed. In this study, we assessed the immunogenicity and protective efficacy of a novel P24-VP8* nanoparticle vaccine using the gnotobiotic (Gn) pig model of human rotavirus infection and disease. Three doses of P24-VP8* (200 μg/dose) intramuscular vaccine with Al(OH)3 adjuvant (600 μg) conferred significant protection against infection and diarrhea after challenge with virulent Wa strain rotavirus. This was indicated by the significant reduction in the mean duration of diarrhea, virus shedding in feces, and significantly lower fecal cumulative consistency scores in post-challenge day (PCD) 1–7 among vaccinated pigs compared to the mock immunized controls. The P24-VP8* vaccine was highly immunogenic in Gn pigs. It induced strong VP8*-specific serum IgG and Wa-specific virus-neutralizing antibody responses from post-inoculation day 21 to PCD 7, but did not induce serum or intestinal IgA antibody responses or a strong effector T cell response, which are consistent with the immunization route, the adjuvant used, and the nature of the non-replicating vaccine. The findings are highly translatable and thus will facilitate clinical trials of the P24-VP8* nanoparticle vaccine.
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    Simvastatin Reduces Protection and Intestinal T Cell Responses Induced by a Norovirus P Particle Vaccine in Gnotobiotic Pigs
    Kocher, Jacob; Castellucci, Tammy Bui; Wen, Ke; Li, Guohua; Yang, Xingdong; Lei, Shaohua; Jiang, Xi; Yuan, Lijuan (MDPI, 2021-07-01)
    Noroviruses (NoVs) are a leading cause of acute gastroenteritis worldwide. P particles are a potential vaccine candidate against NoV. Simvastatin is a cholesterol-reducing drug that is known to increase NoV infectivity. In this study, we examined simvastatin’s effects on P particle-induced protective efficacy and T-cell immunogenicity using the gnotobiotic pig model of human NoV infection and diarrhea. Pigs were intranasally inoculated with three doses (100 µg/dose) of GII.4/VA387-derived P particles together with monophosphoryl lipid A and chitosan adjuvants. Simvastatin-fed pigs received 8 mg/day orally for 11 days prior to challenge. A subset of pigs was orally challenged with 10 ID50 of a NoV GII.4/2006b variant at post-inoculation day (PID) 28 and monitored for 7 days post-challenge. Intestinal and systemic T cell responses were determined pre- and postchallenge. Simvastatin abolished the P particle’s protection and significantly increased diarrhea severity after NoV infection. Simvastatin decreased proliferation of virus-specific and non-specific CD8 T cells in duodenum and virus-specific CD4 and CD8 T cells in spleen and significantly reduced numbers of intestinal mononuclear cells in vaccinated pigs. Furthermore, simvastatin significantly decreased numbers of duodenal CD4+IFN-γ+, CD8+IFN-γ+ and regulatory T cells and total duodenal activated CD4+ and CD8+ T cells in vaccinated pigs pre-challenge at PID 28. Following challenge, simvastatin prevented the IFN-γ+ T cell response in spleen of vaccinated pigs. These results indicate that simvastatin abolished P particle vaccine-induced partial protection through, at least in part, impairing T cell immunity. The findings have specific implications for the development of preventive and therapeutic strategies against NoV gastroenteritis, especially for the elderly population who takes statin-type drugs.