Objective To investigate the alterations in metabolic molecules and pathways during epithelial-mesenchymal transition (EMT) in the lungs of mice induced by silica exposure. Methods A total of 30 specific pathogen-free male C57BL/6 mice were randomly divided into a control group and a silicosis model group. Mice were intratracheally instilled with 40 µL of either 0.9% sodium chloride solution or silica suspension at a mass concentration of 125 g/L. At 7, 14, and 28 days post-exposure, five mice from each group were sacrificed to evaluate lung histopathological changes. The mRNA expression of EMT-related genes was measured by real-time quantitative polymerase chain reaction. Untargeted metabolomics was used to identify metabolites in mouse lung tissues, and differential metabolites (DMs) and metabolic pathways were analyzed. Results Following silica exposure, lung tissues of mice in the silicosis model group showed progressively aggravated pathological changes, including increased inflammatory cell infiltration, thickening of alveolar walls, collagen deposition, and progressive pulmonary fibrosis, with typical silicotic nodules forming by day 28. The Ashcroft scores and the mRNA expression of cadherin (Cdh)2 and vimentin of mice in the silicosis model group increased (all P<0.05), along with decreased Cdh1 mRNA expression compared with the control group at all the three time points (all P<0.05). The Ashcroft scores and the mRNA expression of Cdh2 and vimentin of mice in the silicosis model group increased with the increase of observation time after silica exposure (all P<0.05), while mRNA expression of Cdh1 decreased (all P<0.05). Metabolomics analysis indicated that silica exposure specifically altered the expression of lipids and disrupted multiple metabolic pathways during EMT in lung tissues of silicosis model group mice,with glycerophospholipid metabolism being most obviously affected (P<0.05). Conclusion Silica exposure may induce EMT in mouse lung tissue, resulting in pulmonary fibrosis in a time-dependent manner. The underlying mechanism may be associated with silica-induced disturbances in glycerophospholipid metabolism in mouse lung tissue.

Objective To investigate the effect of blood glucose control parameters on the prognosis of severe pneumonia patients based on continuous glucose monitoring(CGM)system.Methods A retrospective analysis was conducted on 51 severe pneumonia patients monitored by CGM at the Fourth Affiliated Hospital of Soochow University from November 2021 to August 2023.Assessed parameters included baseline clinical characteristics,glycosylated hemoglobin(HbA1c),mean glucose,standard deviation(SD),coefficient of variability(CV),mean amplitude of glycemic excursions(MAGE),maximum glucose level,minimum glucose level,and time in range(TIR)of glucose within the target range(3.9-10.0 mmol/L)as a proportion and levels of inflammatory indicators before and after treatment.Based on the 28-day follow-up results,the patients were divided into the survival group(39 cases)and the death group(12 cases).The blood glucose parameters of the two groups were compared.Multivariate Logistic regression model was used to analyze the influence of blood glucose parameters and infection indexes on the prognosis of patients with severe pneumonia.The efficacy of blood glucose parameters in the diagnosis of 28-day mortality was further evaluated by receiver operator characteristic curve(ROC curve).Results The acute physiology and chronic health evaluationⅡ(APACHEⅡ),SD and CV of blood glucose in death group were higher than those in survival group[APACHEⅡscore:20.5(14.0)vs.15.0(7.0),SD(mmol/L):2.6±0.7 vs.2.1±0.5,CV:(27.7±5.8)%vs.(23.7±4.3)%].However,the TIR(3.9-10.0 mmol/L)proportion was statistically lower than that of survival group[59.0%(17.0%)vs.68.0%(35.0%)],the differences were all statistically significant(all P<0.05).After using CGM,the white blood cell count(WBC)and hypersensitive C-reactive protein(hs-CRP)were significantly decreased[WBC(×109/L):9.2(6.5)vs.11.1(9.2),hs-CRP(mg/L):39.4(59.0)vs.56.2(133.8),both P<0.05].Multivariate Logistic regression showed that TIR(3.9-10.0 mmol/L)proportion was an independent risk factor for 28-day mortality in patients with severe pneumonia[odds ratio(OR)=0.923,95%confidence interval(95%CI)was 0.852-0.999,P=0.046].The ROC curve showed that TIR proportion was valuable in predicting the clinical outcomes of patients with severe pneumonia.Area under the curve(AUC)=0.720,95%CI was 0.563-0.878,P=0.022;when the cut-off value was 63.5%,the sensitivity and the specificity were 59.0%and 83.3%respectively.Conclusion Increase of TIR(3.9-10.0 mmol/L)proportion in patients with severe pneumonia could improve clinical outcomes,especially when TIR proportion≥63.5%.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective To establish a brucellosis monitoring and testing technique applicable for the rapid field screening of natural epidemic diseases.Methods A rapid testing technique for Brucella was developed based on a double-antibody sandwich testing model using gradient-variant quantum dots as fluorescent tracers.The sensitivity,linearity,precision,and specificity of the technique were evaluated using suspensions of standard Brucella strains.Methodological comparisons across different sample types were conducted to assess the consistency of the test results.Results The gradient-variant quantum dots detection method was evaluated with standard Brucella strains,exhibiting a sensitivity of 1×103 CFU/mL and a linear correlation coefficient(r)of 0.994(95%CI,0.933-1.055).The maximum coefficient of variation was 12.94%in repeated tests,showing good specificity.A comparative assessment of 305 clinical samples was conducted using the Brucella gradient-variant quantum dots detection method,the Rose Bengal plate agglutination test(RBT),and the serum agglutination test(SAT),yielding a Kappa value of 0.95,indicating almost perfect agreement.Additionally,a comparative assessment of 110 environmental samples collected on-site was conducted using the Brucella gradient-variant quantum dots detection method and quantitative real-time PCR(qPCR).The Kappa values for aerosol collection fluid,surface wipes,and wool samples were all above 0.83,demonstrating near-perfect agreement.For fecal and soil samples,the Kappa values were above 0.62,indicating substantial agreement.Conclusion The Brucella detection method based on gradient-variant quantum dots technology is simple and can be conducted rapidly.The detection method demonstrates high sensitivity,linearity,precision,and specificity.It shows consistent performance in clinical sample testing.It is well-suited for field rapid screening of natural epidemic diseases in field settings and shows good application prospects in the monitoring,prevention,and rapid detection of zoonotic diseases.

BACKGROUND: Spicy food consumption has been reported to be inversely associated with mortality from multiple diseases. However, the effect of spicy food intake on the incidence of vascular diseases in the Chinese population remains unclear. This study was conducted to explore this association. METHODS: This study was performed using the large-scale China Kadoorie Biobank (CKB) prospective cohort of 486,335 participants. The primary outcomes were vascular disease, ischemic heart disease (IHD), major coronary events (MCEs), cerebrovascular disease, stroke, and non-stroke cerebrovascular disease. A Cox proportional hazards regression model was used to assess the association between spicy food consumption and incident vascular diseases. Subgroup analysis was also performed to evaluate the heterogeneity of the association between spicy food consumption and the risk of vascular disease stratified by several basic characteristics. In addition, the joint effects of spicy food consumption and the healthy lifestyle score on the risk of vascular disease were also evaluated, and sensitivity analyses were performed to assess the reliability of the association results. RESULTS: During a median follow-up time of 12.1 years, a total of 136,125 patients with vascular disease, 46,689 patients with IHD, 10,097 patients with MCEs, 80,114 patients with cerebrovascular disease, 56,726 patients with stroke, and 40,098 patients with non-stroke cerebrovascular disease were identified. Participants who consumed spicy food 1-2 days/week (hazard ratio [HR] = 0.95, 95% confidence interval [95% CI] = [0.93, 0.97], P <0.001), 3-5 days/week (HR = 0.96, 95% CI = [0.94, 0.99], P = 0.003), and 6-7 days/week (HR = 0.97, 95% CI = [0.95, 0.99], P = 0.002) had a significantly lower risk of vascular disease than those who consumed spicy food less than once a week ( Ptrend <0.001), especially in those who were younger and living in rural areas. Notably, the disease-based subgroup analysis indicated that the inverse associations remained in IHD ( Ptrend = 0.011) and MCEs ( Ptrend = 0.002) risk. Intriguingly, there was an interaction effect between spicy food consumption and the healthy lifestyle score on the risk of IHD ( Pinteraction = 0.037). CONCLUSIONS Our findings support an inverse association between spicy food consumption and vascular disease in the Chinese population, which may provide additional dietary guidance for the prevention of vascular diseases.
Humans ; Male ; Female ; Prospective Studies ; Middle Aged ; Aged ; Vascular Diseases/etiology* ; Risk Factors ; China/epidemiology* ; Adult ; Proportional Hazards Models ; Cerebrovascular Disorders/epidemiology* ; East Asian People

Objective To investigate the effect of blood glucose control parameters on the prognosis of severe pneumonia patients based on continuous glucose monitoring(CGM)system.Methods A retrospective analysis was conducted on 51 severe pneumonia patients monitored by CGM at the Fourth Affiliated Hospital of Soochow University from November 2021 to August 2023.Assessed parameters included baseline clinical characteristics,glycosylated hemoglobin(HbA1c),mean glucose,standard deviation(SD),coefficient of variability(CV),mean amplitude of glycemic excursions(MAGE),maximum glucose level,minimum glucose level,and time in range(TIR)of glucose within the target range(3.9-10.0 mmol/L)as a proportion and levels of inflammatory indicators before and after treatment.Based on the 28-day follow-up results,the patients were divided into the survival group(39 cases)and the death group(12 cases).The blood glucose parameters of the two groups were compared.Multivariate Logistic regression model was used to analyze the influence of blood glucose parameters and infection indexes on the prognosis of patients with severe pneumonia.The efficacy of blood glucose parameters in the diagnosis of 28-day mortality was further evaluated by receiver operator characteristic curve(ROC curve).Results The acute physiology and chronic health evaluationⅡ(APACHEⅡ),SD and CV of blood glucose in death group were higher than those in survival group[APACHEⅡscore:20.5(14.0)vs.15.0(7.0),SD(mmol/L):2.6±0.7 vs.2.1±0.5,CV:(27.7±5.8)%vs.(23.7±4.3)%].However,the TIR(3.9-10.0 mmol/L)proportion was statistically lower than that of survival group[59.0%(17.0%)vs.68.0%(35.0%)],the differences were all statistically significant(all P<0.05).After using CGM,the white blood cell count(WBC)and hypersensitive C-reactive protein(hs-CRP)were significantly decreased[WBC(×109/L):9.2(6.5)vs.11.1(9.2),hs-CRP(mg/L):39.4(59.0)vs.56.2(133.8),both P<0.05].Multivariate Logistic regression showed that TIR(3.9-10.0 mmol/L)proportion was an independent risk factor for 28-day mortality in patients with severe pneumonia[odds ratio(OR)=0.923,95%confidence interval(95%CI)was 0.852-0.999,P=0.046].The ROC curve showed that TIR proportion was valuable in predicting the clinical outcomes of patients with severe pneumonia.Area under the curve(AUC)=0.720,95%CI was 0.563-0.878,P=0.022;when the cut-off value was 63.5%,the sensitivity and the specificity were 59.0%and 83.3%respectively.Conclusion Increase of TIR(3.9-10.0 mmol/L)proportion in patients with severe pneumonia could improve clinical outcomes,especially when TIR proportion≥63.5%.

Objective:To study the comparative genomic characteristics of Marmota himalayana-derived (referred to as marmota-derived) Brucella abortus (B.ab). Methods:The species and types of one strain of marmota-derived Brucella and one strain of human-derived Brucella isolated from the brucellosis epidemic area in Qinghai Province in the same year were identified. Meanwhile, DNA was extracted for whole genome sequencing and comparative genomics analysis (including phylogenetic tree construction, gene family clustering analysis, common/specific gene analysis, and genomic structural variation analysis, etc.). Results:Two Brucella strains from different hosts were identified as B.ab. By constructing a phylogenetic tree, the marmota-derived B.ab strain was grouped with strains from Heilongjiang Province and showed genetic correlation with strains from Russia. Human-derived B.ab strain was classified as a strain in Inner Mongolia Autonomous Region, Hebei Province, Beijing City, and Gansu Province. The multilocus sequence typing (MLST) of the two strains belonged to the ST2 type. Multiple locus variable-number tandem repeat analysis (MLVA) belonged to two new MLVA-8 and MLVA-11 genotypes, which were clustered in two subclusters of the same cluster and clustered with the strains from Inner Mongolia Autonomous Region, Xinjiang Uygur Autonomous Region, and Hebei Province. The pan-genome numbers of the marmota-derived B.ab and human-derived B.ab were 283 and 8, respectively; the number of core genes (common genes) was 68 and 2, respectively; and the number of unique genes was 3 and 4, respectively. The unique gene encoded proteins were inconsistent. In marmota-derived B.ab, the main ones were the ABC transporter ATP-binding protein, N-terminal acetyltransferase, and glucose/galactose transporter. The number of homologous genes of the marmota-derived B.ab and human-derived B.ab was 16 and 20, respectively; the number of translocation and inversion genes was 13 and 8, respectively; the number of deletion mutation genes was 11 and 14, respectively. Pathogenicity analysis showed that both strains had the mprF resistance gene, and the marmota-derived B.ab strain also carried bacitracin and macrolide resistance genes. Conclusions:Brucella exhibits cross-species genetic diversity. The proteins encoded by the unique genes of the marmota-derived B.ab mainly play a role in metabolic and epigenetic regulation. The strains cluster with B.ab strains from northern China, providing a reference for molecular epidemiology and pathogen tracing of B.ab infection.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective:To study the comparative genomic characteristics of Marmota himalayana-derived (referred to as marmota-derived) Brucella abortus (B.ab). Methods:The species and types of one strain of marmota-derived Brucella and one strain of human-derived Brucella isolated from the brucellosis epidemic area in Qinghai Province in the same year were identified. Meanwhile, DNA was extracted for whole genome sequencing and comparative genomics analysis (including phylogenetic tree construction, gene family clustering analysis, common/specific gene analysis, and genomic structural variation analysis, etc.). Results:Two Brucella strains from different hosts were identified as B.ab. By constructing a phylogenetic tree, the marmota-derived B.ab strain was grouped with strains from Heilongjiang Province and showed genetic correlation with strains from Russia. Human-derived B.ab strain was classified as a strain in Inner Mongolia Autonomous Region, Hebei Province, Beijing City, and Gansu Province. The multilocus sequence typing (MLST) of the two strains belonged to the ST2 type. Multiple locus variable-number tandem repeat analysis (MLVA) belonged to two new MLVA-8 and MLVA-11 genotypes, which were clustered in two subclusters of the same cluster and clustered with the strains from Inner Mongolia Autonomous Region, Xinjiang Uygur Autonomous Region, and Hebei Province. The pan-genome numbers of the marmota-derived B.ab and human-derived B.ab were 283 and 8, respectively; the number of core genes (common genes) was 68 and 2, respectively; and the number of unique genes was 3 and 4, respectively. The unique gene encoded proteins were inconsistent. In marmota-derived B.ab, the main ones were the ABC transporter ATP-binding protein, N-terminal acetyltransferase, and glucose/galactose transporter. The number of homologous genes of the marmota-derived B.ab and human-derived B.ab was 16 and 20, respectively; the number of translocation and inversion genes was 13 and 8, respectively; the number of deletion mutation genes was 11 and 14, respectively. Pathogenicity analysis showed that both strains had the mprF resistance gene, and the marmota-derived B.ab strain also carried bacitracin and macrolide resistance genes. Conclusions:Brucella exhibits cross-species genetic diversity. The proteins encoded by the unique genes of the marmota-derived B.ab mainly play a role in metabolic and epigenetic regulation. The strains cluster with B.ab strains from northern China, providing a reference for molecular epidemiology and pathogen tracing of B.ab infection.

Objective:To analyze the surveillance results of human brucellosis in Qinghai Province, to study the epidemic characteristics of brucellosis, and to provide scientific basis for formulating brucellosis prevention and control strategies and measures.Methods:By using descriptive epidemiological method, surveillance data from 4 national and 26 provincial brucellosis surveillance sites in Qinghai Province in 2022 and brucellosis related information from the "Disease Surveillance Information Reporting and Management System" in 2022 were collected and summarized. The population, time, regional distribution and epidemic characteristics of brucellosis in Qinghai Province were analyzed.Results:In 2022, a total of 12 483 people were monitored at 4 national and 26 provincial brucellosis surveillance sites. In Rose Bengal plate agglutination test (RBPT), 714 patients were positive, and the positive rate was 5.72% (714/12 483). In tube agglutination test (SAT), 508 individuals tested positive, and the positive rate was 4.07% (508/12 483). There were 1 156 reported cases in 2022, including 910 males and 246 females, with a gender ratio of 3.70 ∶ 1.00. The age range was mainly between 30 and 59 years old, accounting for 77.68% (898/1 156). Most of them were farmers engaged in breeding and transportation, accounting for 81.40% (941/1 156). The onset time was concentrated from June to August, accounting for 50.26% (581/1 156). The distribution area was mainly in the northeast of Qinghai Province (Haibei Tibetan Autonomous Prefecture, Xining City, Haidong City), accounting for 86.33% (998/1 156).Conclusions:The epidemic of human brucellosis in Qinghai Province is still relatively serious, especially in the northeastern agricultural area. Comprehensive measures should be taken to further curb the outbreak of human brucellosis.