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1.
Article in English | MEDLINE | ID: mdl-38345957

ABSTRACT

In this paper, we introduce Neural-ABC, a novel parametric model based on neural implicit functions that can represent clothed human bodies with disentangled latent spaces for identity, clothing, shape, and pose. Traditional mesh-based representations struggle to represent articulated bodies with clothes due to the diversity of human body shapes and clothing styles, as well as the complexity of poses. Our proposed model provides a unified framework for parametric modeling, which can represent the identity, clothing, shape and pose of the clothed human body. Our proposed approach utilizes the power of neural implicit functions as the underlying representation and integrates well-designed structures to meet the necessary requirements. Specifically, we represent the underlying body as a signed distance function and clothing as an unsigned distance function, and they can be uniformly represented as unsigned distance fields. Different types of clothing do not require predefined topological structures or classifications, and can follow changes in the underlying body to fit the body. Additionally, we construct poses using a controllable articulated structure. The model is trained on both open and newly constructed datasets, and our decoupling strategy is carefully designed to ensure optimal performance. Our model excels at disentangling clothing and identity in different shape and poses while preserving the style of the clothing. We demonstrate that Neural-ABC fits new observations of different types of clothing. Compared to other state-of-the-art parametric models, Neural-ABC demonstrates powerful advantages in the reconstruction of clothed human bodies, as evidenced by fitting raw scans, depth maps and images. We show that the attributes of the fitted results can be further edited by adjusting their identities, clothing, shape and pose codes. The dataset and trained parametric model will be available at https://ustc3dv.github.io/NeuralABC/.

2.
Front Public Health ; 11: 1189969, 2023.
Article in English | MEDLINE | ID: mdl-37427288

ABSTRACT

Backgrounds: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a global threat since 2020. The emergence of the Omicron variant in 2021, which replaced Delta as the dominant variant of concern, has had a significant adverse impact on the global economy and public health. During this period, Zhejiang Province implemented dynamic zeroing and focused on preventing imported cases. This study aimed to gain clear insight into the characteristics of imported COVID-19 cases in Zhejiang Province. Methods: We conducted a systematic molecular epidemiological analysis of 146 imported cases between July 2021 and November 2022 in Zhejiang Province. Virus samples with cycle threshold (Ct) value less than 32 were performed next generation sequencing. Basing the whole genome sequence obtained after quality control and assembly of reads, the whole genome variation map and phylogenetic tree were constructed and further analyzed. Results: Our study identified critical months and populations for surveillance, profiled the variation of various lineages, determined the evolutionary relationships among various lineages of SARS-CoV-2, and compared the results in Zhejiang with those obtained worldwide during this period. Conclusion: The continuous molecular epidemiological surveillance of imported cases of COVID-19 in Zhejiang Province during 2021 to 2022 is consistent with the global epidemic trend.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/epidemiology , Phylogeny , China/epidemiology
3.
Front Microbiol ; 13: 964823, 2022.
Article in English | MEDLINE | ID: mdl-35928146

ABSTRACT

Bacillus cereus is a major food-borne bacterial pathogen in the world, which can cause diarrhea and emetic syndrome. This study aimed to reveal the quantitative prevalence of B. cereus in ready-to-eat (RTE) rice products in Eastern China and to gain essential information on the characteristics of B. cereus isolates. A total of 91 out of the 1071 samples were positive for B. cereus. The contamination level of B. cereus in 0.5 % of RTE rice product samples outnumbered 103 CFU/g. The number of B. cereus attained 105-106 CFU/g in one sample. The distribution patterns of virulence genes in B. cereus isolates were identified. 84.6% of the B. cereus isolates had at least one enterotoxin or emetic toxin gene. The predominant pattern was XXV. 9.9% of isolates belonged to it and possessed one enterotoxin gene entFM. The occurrence rate of hblACD and nheABC was 36.3% and 47.3%, respectively. Antimicrobial susceptibility tests revealed a high resistance rate toward penicillin, and 23.1% of the isolates were multi-drug resistant. B. cereus isolates were genotyped by using ERIC-PCR. 89 genotypes were determined. The Hunter Gaston Discriminatory Index (HGDI) attained 0.9995. Relationships analysis revealed that Group A B. cereus isolates tended to carry hblA, hblC, hblD, nheA, nheB, and show resistance to penicillin/trimethoprim/sulfamethoxazole. This study was useful for updating the knowledge of the contamination status of B. cereus in RTE rice products in China.

4.
Foodborne Pathog Dis ; 19(8): 550-557, 2022 08.
Article in English | MEDLINE | ID: mdl-35787152

ABSTRACT

Vibrio parahaemolyticus is an important foodborne pathogen with diverse serotypes. In May 2021, we investigated a gastroenteritis outbreak that occurred in China, caused by V. parahaemolyticus O10:K4 infection. Based on the epidemiological curve, this outbreak was identified as a homologous exposure event. A case-control study demonstrated that emperor crab with mashed garlic (odds ratio [OR] = 4.60, p = 0.030; 95% confidence interval [95% CI]: 1.11-19.14), goose liver geoduck (OR = 4.50, p = 0.029; 95% CI: 1.12-18.13), shrimp (OR = 4.89, p = 0.021; 95% CI: 1.22-19.65), and sea cucumber (OR = 7.36, p = 0.005; 95% CI: 1.68-32.26) were the potential sources of the food poisoning. V. parahaemolyticus isolates from 18 laboratory-confirmed cases were all serotyped O10:K4, and determined to be sequence type ST3 via multilocus sequence typing. Pulsed field gel electrophoresis and whole-genome sequencing analysis revealed the identical pattern and 0-2 single nucleotide variation among these isolates. tdh was positive in all isolates, while trh and Orf8 were absent. Seven essential base positions in toxRS for pandemic clone identification were identical between the O10:K4 and O3:K6 pandemic clones. Phylogenetic analysis with 45 additional genomes of 13 different serotypes showed the closest genetic relationship between O10:K4 and O1: KUT. O10:K4 was thought to evolve from the O3:K6 pandemic clone. The new serovariant of O3:K6 poses a challenge for the prevention and control of V. parahaemolyticus disease outbreaks, or even epidemics, in the future.


Subject(s)
Gastroenteritis , Vibrio Infections , Vibrio parahaemolyticus , Case-Control Studies , China/epidemiology , Disease Outbreaks , Gastroenteritis/epidemiology , Humans , Phylogeny , Serogroup , Serotyping , Vibrio Infections/epidemiology , Vibrio parahaemolyticus/genetics
5.
Mol Biol Rep ; 48(11): 7575-7585, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34655017

ABSTRACT

BACKGROUND: Protein degradation systems play crucial roles in all the kingdoms of life. Their natural function is to eliminate proteins that are improperly synthesized, damaged, aggregated, or short-lived, ensuring the timely and accurate regulation of the response to abrupt environmental changes. Thus, proteolysis plays an important role in protein homeostasis, quality control, and the control of regulatory processes, such as adaptation and cell development. Except for the lysosome, ATPases Associated with various cellular Activities (AAA+) ATPase-protease complex is another major protein degradation system in the cell. METHODS AND RESULTS: The AAA+ ATPase-protease complex is a giant energy-dependent protease complex found in almost all kinds of cells, including bacteria, archaea and eukarya. Based on sequence analysis of ClpQ (HslV) and 20S proteasome beta subunits, it was found that bacterial ClpQ possess multiple same highly conserved motifs with 20S proteasome beta subunits of archaea and eukaryote. In this review, we also discussed the structure and functional mechanism, protein degradation signals and pathogenic role of proteasome / Clp protease complex in prokaryotes. CONCLUSION: Bacterial protein degradation systems play important roles in stress tolerance, protein quality control, DNA protection, transcription and pathogenicity of bacteria. But our current knowledge of the bacterial protease system is incomplete, and further research into the Clp protease complex and associated protein degradation signals will extend our understanding of the metabolism, physiology, reproduction, and pathogenicity of bacteria.


Subject(s)
Bacteria/metabolism , Bacteria/pathogenicity , Bacterial Proteins/metabolism , Microbial Viability , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Bacteria/genetics , Bacterial Proteins/genetics , Proteasome Endopeptidase Complex/genetics
6.
Foodborne Pathog Dis ; 18(2): 139-146, 2021 02.
Article in English | MEDLINE | ID: mdl-33493422

ABSTRACT

Objectives: The previous researches revealed that Vibrio parahaemolyticus has been detected in freshwater fish samples. However, the molecular characteristics of V. parahaemolyticus isolated from freshwater fish, including pathogenic and pandemic strains, are still unknown. This study aims to characterize and identify molecular properties of the bacterium. In addition, it identifies the source of V. parahaemolyticus from freshwater fish samples in Zhejiang Province, China. Methods: Four hundred and twenty-one freshwater fish samples (from fishing farms, retail markets, and restaurants) and 212 seafood samples (from retail markets) were collected in 10 cities of Zhejiang Province. V. parahaemolyticus strains were isolated from these samples and comparatively analyzed by multilocus sequence typing, serotyping, antimicrobial susceptibility test, and polymerase chain reaction, targeting common toxin genes (tdh, trh) and markers for pandemic strains (orf8, toxRS/new). Results: Sixty-eight V. parahaemolyticus strains were isolated from the 421 freshwater fish samples, and 89 V. parahaemolyticus isolates were identified out of 212 seafood samples. The detection rate of V. parahaemolyticus was significantly different (p < 0.05) between the fishing farms, the retail markets, and the restaurants. The isolates from freshwater fish samples were divided into eight O serotypes with three O3:K6 isolates, which contain three pandemic complexes (tdh+, orf8+, toxRS/new+). A total of 53 different sequence types (STs) were identified among the 68 isolates, including 28 novel STs. Antimicrobial susceptibility results indicated that 76.5% of the strains were resistant to ampicillin. A third (3/9) of the isolates from fishing farm sources shared the same STs with their counterparts from retail markets. Compared with the isolates from the seafood samples collected in the same sampling sites, 13.2% (9/68) freshwater fish isolates overlapped with seafood isolates. Conclusions: Our study showed that V. parahaemolyticus population in freshwater fish is genetically diverse. The V. parahaemolyticus contaminates might have come from both fishing farm sources and cross-contamination from seafood in the closed area at the markets. Freshwater fish may work as a reservoir of pathogenic and pandemic V. parahaemolyticus isolates, indicating potential public health and food safety risks associated with the consumption of freshwater fish.


Subject(s)
Fishes/microbiology , Food Microbiology/statistics & numerical data , Seafood/microbiology , Vibrio Infections/veterinary , Vibrio parahaemolyticus/isolation & purification , Animals , China/epidemiology , Fresh Water/microbiology , Microbial Sensitivity Tests , Multilocus Sequence Typing , Polymerase Chain Reaction , Prevalence , Serogroup , Vibrio Infections/epidemiology , Vibrio Infections/microbiology
7.
mSystems ; 6(1)2021 Jan 12.
Article in English | MEDLINE | ID: mdl-33436512

ABSTRACT

Salmonella enterica serotype Kentucky is frequently associated with high-level fluoroquinolone resistance and has gained epidemiological importance globally. A retrospective screening was performed to understand the national prevalence of ciprofloxacin-resistant S Kentucky in China. S. enterica strains (n = 15,405) were collected within the frame of two national surveillance networks between 2013 and 2017. Thirty-three S. Kentucky strains were detected in 5 of 10 provinces, and 27 were assigned to sequence type 198 (ST198). The 27 isolates were multidrug resistant, with high-level resistance to ciprofloxacin, and 21 isolates were further resistant to extended-spectrum cephalosporins (ESCs). Phylogenomic analysis classified ST198 isolates into two clades (198.1 and 198.2), and recent occurrences of inter-/intraregion and interhost transmission were identified. Phylogenetic reconstruction with a global collection showed that one subclade of clade 198.2 was clustered with historical strains from Egypt, and the other one was clustered with strains from Southeast Asia. Isolates of clade 198.1 were clustered with strains isolated from North America. The various patterns of mutations detected in quinolone resistance-determining regions of GyrA and ParC are accordant with the phylogenetic structure. These findings indicate that our isolates may have various origins. SGI1 was exclusively detected in isolates of clade 198.2 with a highly mosaic structure, which were mainly identified as SGI1-K derivatives. Plasmid-mediated quinolone resistance genes qnrS1 and aac(6')-Ib-cr were identified in three isolates, and bla CTX-M-9 and bla CTX-M-27 were detected in 20 of 21 ESC-resistant isolates. This is the first report of the genetic and epidemiological characterization for the S Kentucky epidemic clone ST198 in China, warranting the necessity of surveillance for the high-risk clone.IMPORTANCE Ciprofloxacin and extended-spectrum cephalosporins are the choice for treatment of severe nontyphoidal S. enterica infections in adults. S. enterica serotype Kentucky ST198 has gained epidemiological importance globally, because the clone is frequently resistant to both of these high-level-resistance drug groups. The genetic and epidemiological characterization of S. Kentucky has been well studied in Western countries; however, the information is unclear for China. To fill in the gap, we here did a retrospective screening on a large collection in China, and ST198 isolates were systematically analyzed by whole-genome sequencing. Our study revealed that multidrug-resistant ST198 has spread in five provinces, and the occurrences of interregion and cross-host clonal disseminations were detected. Of note, phylogenomic analysis suggests that the Chinese isolates may have emerged with diverse origins, including Egypt, Southeast Asia, and North America. This study warrants the necessity of surveillance for the high-risk clone to prevent its further dissemination in China.

8.
Journal of Preventive Medicine ; (12): 656-660, 2021.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-881447

ABSTRACT

Objective@#To investigate the contamination status and assess the potential consumption risk of Listeria monocytogenes ( L. monocytogenes ) in cooked meat products in bulk in Zhejiang Province, so as to provide strategy for food safety supervision and management.@*Methods@#A total of 2 320 cooked meat products were sampled from eleven cities in Zhejiang Province during 2018-2020. The detection of L. monocytogenes was carried out in accordance with the national standard GB/T 4789.30-2016. Risk Ranger software was used for the semi-quantitative risk assessment on the whole population and pregnant women.@*Results@#The total detection rate of L. monocytogenes in cooked meat products in bulk in Zhejiang Province was 2.97% ( 69/2 320 ). The detection rates in stewed, smoked/roasted, fried, dried products and others were 3.85%, 1.81%, 0.59%, 0% and 0.94%, which were significantly different ( P<0.05 ). There were 28 positive samples in 1 069 samples collected in 2020, with the concentration ranging from 5 to 590 CFU/g and averaging 6.8 CFU/g. The estimated number of listeriosis cases each year caused by consumption of cooked meat products in bulk was 131 in the whole population with a risk score of 42, and 1.44 in pregnant women with a risk score of 54. The risk coefficient could reduce to approximate zero after sufficient heating before intake.@*Conclusion@#The prevalence of L. monocytogenes in cooked meat products in bulk in Zhejiang Province during 2018-2020 poses a potential risk in food safety. Pregnant women should avoid eating.

9.
Foodborne Pathog Dis ; 17(12): 775-781, 2020 12.
Article in English | MEDLINE | ID: mdl-32639172

ABSTRACT

Campylobacter is well recognized as the leading cause of bacterial foodborne diarrheal disease worldwide with a very low outbreak reported in China. In May 2019, we investigated an outbreak of Campylobacter jejuni infections among students in a junior high school in Eastern China. Cases were interviewed to identify a common source of contamination. As cases were identified in the same school during a period of time, menus were reviewed and food items included in the questionnaire. Rectal swabs from school kitchen staff and suspected food items (raw chicken) from a local market from where the school food came were examined for C. jejuni. Pulsed-field gel electrophoresis and whole genome sequencing were performed to determine the relatedness of the isolates. To identify the source of the contamination, a case-control study was conducted. Forty-five cases were reported with diarrhea among 1696 students and staff. Stool samples for 10 of the 45 and 5 tested positive for C. jejuni. WGS analysis revealed a 0-4 single nucleotide variation in case-patient isolates. Although we were unable to identify the specific food item, a specific menu was identified as the potential source of the contamination (odds ratios = 20.82; 95% confidence interval = 6.472-66.957). In this menu, chicken was served. A food isolate collected from chicken in Zhejiang province in 2018 was positive for the same identical strain (5-7 single nucleotide polymorphisms). This is one of the few reports in China about outbreak caused by C. jejuni. This investigation illustrates the potential risk of outbreaks caused by Chinese cold dishes of chicken.


Subject(s)
Campylobacter Infections/epidemiology , Campylobacter jejuni/genetics , Meat/microbiology , Animals , Bacterial Typing Techniques , Campylobacter jejuni/isolation & purification , Case-Control Studies , Chickens/microbiology , China , Diarrhea/microbiology , Disease Outbreaks , Female , Food Contamination , Food Microbiology , Humans , Male , Students , Whole Genome Sequencing
10.
Front Microbiol ; 10: 1710, 2019.
Article in English | MEDLINE | ID: mdl-31402906

ABSTRACT

Listeria monocytogenes is an important foodborne pathogen causing public concern. A total of 3354 retail foods in bulk were sampled and screened for L. monocytogenes. Seventy-three (2.2%) samples including 21 ready-to-eat (RTE) foods and 52 raw foods were confirmed positive for L. monocytogenes. Sushi and salmon sashimi occupied the top two slots in RTE foods with relatively high presence rate of 12.9 and 6.9%, respectively. Meanwhile, L. monocytogenes was found to be distributed unequally in raw foods; the presence rates in raw meat (3.5%) and poultry (3.8%) were significantly higher than that in raw seafood (1.3%). Notably, L. monocytogenes was not detected in raw freshwater food. The L. monocytogenes isolates belonged to four serotypes, 1/2a, 1/2b, 1/2c, and 4b, with the most prevalent serotype being 1/2a (47.9%). Eighteen sequence types (STs) and eighteen virulence types (VTs) containing four newly assigned VTs (VT180, VT181, VT182, and VT183) were determined via multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST). Among the 73 L. monocytogenes isolates, 23 (31.5%) belonged to epidemic clones (ECs) including ECI, ECIV, ECV, ECVI, ECVIII and ECXI among which ECV was predominant. Antibiotic susceptibility tests revealed a high resistance rate (11.0%) to tetracycline. Moreover, we identified the distribution patterns of virulence genes of four Listeria pathogenicity islands (LIPI) in L. monocytogenes isolates. prfA, hly, plcA, plcB, mpl, actA genes in LIPI-1 and inlA, inlB, inlC, inlJ genes in LIPI-2 were detected in approximately all L. monocytogenes isolates. The distribution of both LIPI-3 genes and LIPI-4 genes exhibited association with lineage and ST. LIPI-4 genes were present exclusively in ST87 isolates. Relatedness analysis revealed the absence of distinct association between STs, ECs, LIPI-3 and LIPI-4 distribution and specific food groups. This study provided fundamental data for Chinese food safety authorities to grasp the contamination status of L. monocytogenes in foods, assess the potential risk of this pathogen and further address the safety issue of retail foods in bulk in China.

11.
Foodborne Pathog Dis ; 14(10): 564-572, 2017 10.
Article in English | MEDLINE | ID: mdl-28753035

ABSTRACT

Bacillus cereus is an important foodborne pathogen, which can cause severe food poisoning. The aim of this study was (i) to evaluate the quantitative prevalence of B. cereus in retail prepackaged infant formula and ready-to-eat rice flour in China and (ii) to gain the basic information on pheno- and genotypic characteristics of B. cereus isolates. We found that 40 out of the 587 samples were positive for B. cereus. B. cereus in 3.5% of infant formula samples and 1.0% of rice flour samples outnumbered 100 Colony-Forming Units (CFU)/g. B. cereus level even attained 103-104 CFU/g in four infant formula samples and one rice flour sample. Furthermore, we identified the distribution patterns of toxin genes in B. cereus isolates. The results showed that 97.5% of B. cereus isolates harbored at least one enterotoxin gene. Antibiotic susceptibility tests revealed that all isolated B. cereus strains were resistant to penicillin and 50% of them were multidrug resistant. Thirteen new sequence types (STs) and four new alleles were identified via multilocus sequence typing. Clonal Complex (CC) ST-205 and CC ST-142 were predominant clonal complexes. Interestingly, we revealed the special relationship between STs of B. cereus isolates and the geographical distributions of infant food manufacturers for the first time. The data implied that B. cereus of different STs might have a distinct ecological niche in China. In view of relatively high contamination level of enterotoxin- producing B. cereus in a proportion of infant foods, especially in those suitable for the ≤6-month-old infant group, appropriate safety criteria and hygienic control measures for infant foods should be drafted in China to prevent B. cereus infection.


Subject(s)
Bacillus cereus/isolation & purification , Enterotoxins/genetics , Food Contamination/analysis , Food Microbiology , Foodborne Diseases/microbiology , Infant Formula/microbiology , Bacillus cereus/genetics , Bacterial Typing Techniques , China/epidemiology , Colony Count, Microbial , Flour/microbiology , Foodborne Diseases/epidemiology , Genotype , Humans , Infant , Multilocus Sequence Typing , Oryza/microbiology , Phenotype , Prevalence
12.
Food Microbiol ; 62: 46-50, 2017 Apr.
Article in English | MEDLINE | ID: mdl-27889164

ABSTRACT

Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles were newly found in PubMLST database. All isolates except for one singleton (ST-1049), were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and 13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands were compared. It was found the brands might be potentially tracked by the variety of STs, such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they could not be easily tracked just by clonal complex types of the isolates.


Subject(s)
Bacillus cereus/classification , Bacillus cereus/genetics , Infant Formula/microbiology , Milk/microbiology , Alleles , Animals , Bacillus cereus/isolation & purification , China , DNA, Bacterial , Genotype , Humans , Infant , Multilocus Sequence Typing/methods , Product Surveillance, Postmarketing
13.
PLoS One ; 10(11): e0142881, 2015.
Article in English | MEDLINE | ID: mdl-26587591

ABSTRACT

Treg cells play a crucial role in immune tolerance, but mechanisms that induce Treg cells are poorly understood. We here have described eosinophils in lamina propria (LP) that displayed high aldehyde dehydrogenase (ALDH) activity, a rate-limiting step during all-trans retinoic acid (ATRA) synthesis, and expressed TGF-ß1 mRNA and high levels of ATRA. Co-incubation assay confirmed that LP eosinophils induced the differentiation of naïve T cells into Treg cells. Differentiation promoted by LP eosinophils were inhibited by blocked either TGF-ß1 or ATRA. Peripheral blood (PB) eosinophils did not produce ATRA and could not induce Treg differentiation. These data identifies LP eosinophils as effective inducers of Treg cell differentiation through a mechanism dependent on TGF-ß1 and ATRA.


Subject(s)
Aldehyde Dehydrogenase/biosynthesis , CD4-Positive T-Lymphocytes/metabolism , T-Lymphocytes, Regulatory/cytology , Transforming Growth Factor beta1/biosynthesis , Tretinoin/metabolism , Aldehyde Dehydrogenase/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Eosinophils/immunology , Eosinophils/metabolism , Immune Tolerance , Lymphocyte Activation/immunology , Mice , Mucous Membrane/immunology , Mucous Membrane/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/immunology , T-Lymphocytes, Regulatory/immunology , Transforming Growth Factor beta1/immunology
14.
PLoS One ; 8(10): e75652, 2013.
Article in English | MEDLINE | ID: mdl-24124502

ABSTRACT

BACKGROUND: Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca(2+) concentration ([Ca(2+)]i) elevation induced by infection can cause cell death, but [Ca(2+)]i changes and high [Ca(2+)]i-induced death of macrophages due to infection of Leptospira have not been previously reported. METHODOLOGY/PRINCIPAL FINDINGS: We first used a Ca(2+)-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca(2+)]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca(2+)]i elevation was caused by both extracellular Ca(2+) influx through the purinergic receptor, P2X7, and Ca(2+) release from the endoplasmic reticulum, as seen by suppression of [Ca(2+)]i elevation when receptor-gated calcium channels were blocked or P2X7 was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3), which in turn induces intracellular Ca(2+) release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S(-1). Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca(2+)]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca(2+)]i. Moreover, PI-PLCs (PI-PLC-ß3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca(2+)]i increases induced apoptosis and necrosis of macrophages, while mild [Ca(2+)]i elevation only caused apoptosis. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that L. interrogans infection induced [Ca(2+)]i elevation through extracellular Ca(2+) influx and intracellular Ca(2+) release cause macrophage apoptosis and necrosis, and the LB361 gene product was shown to be a novel PI-PLC of L. interrogans responsible for the [Ca(2+)]i elevation.


Subject(s)
Leptospira interrogans/enzymology , Leptospira interrogans/pathogenicity , Macrophages/metabolism , Type C Phospholipases/metabolism , Virulence Factors/metabolism , Animals , Apoptosis , Calcium/metabolism , Cell Line , Humans , Mice , Phosphorylation
15.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 39(4): 350-6, 2010 07.
Article in Chinese | MEDLINE | ID: mdl-20731032

ABSTRACT

OBJECTIVE: To construct, express and identify the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma encoding bifunctional protein sflk1-IFN-gamma (soluble fetal liver kinase 1 and interferon-gamma). METHODS: sflk1 and IFN-gamma gene fragments were cloned by RT-PCR, and then inserted into pcDNA3.1(+) plasmid between BamHI-EcoRI and XhoI-XbaI restriction sites to form the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma. The recombinant sflk1-IFN-gamma transiently expressed in COS-7 cells was detected by ELISA and Western blotting. Bioactivities of sflk1-IFN-gamma fusion protein were identified by proliferation inhibition assay with H5V cells and NK activity assay. RESULTS: pcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in COS-7 cells. Concentrations of sflk1 and IFN-gamma in culture supernatants of pcDNA3.1(+)/sflk1-IFN-gamma transfected COS-7 cells were (20.85+/-2.48) ng/ml and (1.08+/-0.09) ng/ml, respectively. Western blotting showed that the molecular weight of sflk1-IFN-gamma fusion protein was about 130 kDa, while that of sflk1 was 115 kDa. The supernatants of transfected cells significantly inhibited the proliferation of H5V cells stimulated by mouse VEGF 164 and enhanced the NK activity of splenocytes, demonstrating that sflk1-IFN-gamma fusion protein possessed the bioactivities of both sflk1 and IFN-gamma. CONCLUSION: The constructed plasmid pcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in eukaryotes. The expressed sflk1-IFN-gamma fusion protein has the biological activities of both sflk1 and IFN-gamma.


Subject(s)
Interferon-gamma/biosynthesis , Recombinant Proteins/biosynthesis , Vascular Endothelial Growth Factor Receptor-2/biosynthesis , Animals , COS Cells , Chlorocebus aethiops , Female , Interferon-gamma/genetics , Mice , Mice, Inbred C57BL , Plasmids , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Vascular Endothelial Growth Factor Receptor-2/genetics
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