Objective:To explore the relationship between blood urea nitrogen to creatinine ratio and frailty in the elderly aged ≥65 years in 8 longevity areas in China.Methods:Participants were recruited from the Healthy Aging and Biomarkers Cohort Study. Based on baseline information about blood urea nitrogen and risk for frailty obtained at follow-up of the participants, blood urea nitrogen to creatinine ratio was classified according to quintiles, Cox proportional hazard regression models were used to analyze the association between blood urea nitrogen to creatinine ratio and frailty.Results:A total of 1 562 participants aged (81.0±17.0) years were included, in whom 814 (52.1%) were men, and 258 frailty events occurred during a mean follow-up of (3.73±1.43) years. Cox proportional hazards model showed that after adjusting for relevant confounders, compared with the participants in the lowest quintile group ( Q1), the risk for frailty decreased by 36%, 44%, and 40% in the participants in the third quintile group ( Q3), the fourth quintile group ( Q4) and the highest quintile group ( Q5) respectively [hazard ratio ( HR)=0.64, 95% CI: 0.43-0.94; HR=0.56, 95% CI: 0.38-0.84; HR=0.60, 95% CI: 0.41-0.88]. The risk for frailty decreased by 20% for every unit standard deviation increase in blood urea nitrogen to creatinine ratio ( HR=0.80, 95% CI: 0.70-0.91). Moreover, blood urea nitrogen to creatinine ratio and the risk for frailty showed a nearly linear dose-response relationship. Conclusions:The increase in blood urea nitrogen to creatinine ratio was associated with higher risk for frailty. Maintaining high blood urea nitrogen to creatinine ratio is important for the prevention of frailty in the elderly.

A prokaryotic expression vector for the mutant diphtheria toxin CRM197 was constructed and expressed in Esch-erichia coli cells.Anti-CRM197 antibody concentrations were detected in serum samples of healthy volunteers.The crm 197 gene was codon-optimized in E.coli and cloned into the plasmid pET28a(+)under optimized expression conditions.CRM197 was purified using Ni-NTA spin columns and ion exchange chromatography,and confirmed by western blot analysis.The puri-fied CRM197 was used to detect specific anti-CRM197 antibody levels in serum samples of different age groups.The results showed that soluble codon-optimized CRM197 was successfully expressed under optimized expression conditions.The purity of CRM197 was more than 95％,as determined with Ni-NTA spin columns and ion exchange chromatography,consistent with the single specific bands obtained by western blot analysis and detection of serum levels of the anti-CRM197 antibody.Collec-tively,these results confirmed that the proposed expression strategy achieved high-yield production of soluble CRM197,al-though high levels in human serum may affect evaluation of immune interactions with glycan-CRM197 conjugates for applica-tion as a diagnostic antigen.The diphtheria mutant toxin CRM197 is used in many conjugate vaccines.The synthetic crm 197 gene with codon optimization in pET28a was transformed into E.coli Origami B(DE3)cells.CRM197 was induced by isopro-pyl β-d-1-thiogalactopyranoside and high level accumulation of soluble CRM197 was purified using Ni-NTA spin columns and ion exchange chromatography.The purity of the final prepara-tion reached 95％.CRM197 was used to detect the concentra-tions of the anti-CRM197 antibody in serum samples of healthy volunteers of different ages.The proposed expression strategy yielded high production of CRM197,which could interfere with evaluations of induced immune interactions by glycan-CRM197 conjugates and prohibit application as a diagnostic antigen.

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.
Humans ; Enterotoxins/genetics* ; Superantigens/genetics* ; Catalytic Domain ; Selenoprotein W/metabolism* ; Receptors, Antigen, T-Cell

Humans ; Diabetes Mellitus, Type 2/epidemiology* ; Air Pollution/adverse effects* ; Air Pollutants/toxicity* ; Risk Assessment ; Particulate Matter ; Environmental Exposure

OBJECTIVE: Campylobacter jejuni NCTC11168 is commonly used as a standard strain for flagellar biosynthesis research. In this report, two distinguished phenotypic isolates (CJ1Z, flhA mutant strain, lawn; CJ2S, flhA complemented strain, normal colony) appeared during laboratory passages for NCTC11168. METHODS: Phenotypic assessments, including motility plates, transmission electron microscopy, biofilm formation assay, autoagglutination assay, and genome re-sequencing for these two isolates (CJ1Z, flhA mutant strain; CJ2S, flhA complemented strain) were carried out in this study. RESULTS: Transmission electron microscopy revealed that the flagellum was lost in CJ1Z. Phenotypic assessments and genome sequencing of the two isolates were performed in this study. The capacity for biofilm formation, colony auto-agglutination, and isolate motility was reduced in the mutant CJ1Z. Comparative genomic analysis indicated a unique native nucleotide insertion in flhA (nt, 2154) that caused the I719Y and I720Y mutations and early truncation in flhA. CONCLUSION FlhA has been found to influence the expression of flagella in C. jejuni. To the best of our knowledge, this is the first study to describe the function of the C-terminal of this protein.
Campylobacter jejuni/genetics* ; Bacterial Proteins/metabolism* ; Mutation ; Biological Variation, Population

Humans ; Asthma/drug therapy* ; Biological Therapy

ObjectiveTo investigate the therapeutic effect of rhubarb decoction （RD） retention enema on a rat model of minimal hepatic encephalopathy （MHE） and its mechanism of action based on bile acid （BA） metabolomics. MethodsA total of 55 male Sprague－Dawley rats were randomly divided into blank group （NC group with 10 rats）， hepatic encephalopathy group （HE group with 15 rats）， MHE group with 15 rats， and MHE+rhubarb decoction treatment group （MHEY group with 15 rats）. Intraperitoneal injection of carbon tetrachloride （CCl₄） and thioacetamide （TAA） was performed to establish a rat model of MHE or HE， and the rats were sacrificed after 2 weeks of administration. The serum levels of aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， alkaline phosphatase （ALP）， total bilirubin （TBil）， and total bile acid （TBA） and the concentration of blood ammonia were measured； the colonic contents were collected to measure pH value； liver and brain tissue samples were collected， and HE staining was used to observe the histopathological changes of the liver； the bile was collected， and liquid chromatography－mass spectrometry was used to perform BA－targeted metabolomics analysis. Continuous data were expressed as mean±standard deviation； a one－way analysis of variance was used for comparison between multiple groups， and the least significant difference t－test was used for further comparison between two groups. ResultsCompared with the NC group， the HE group and the MHE group had a significant increase in searching platform latency （after modelling and after administration） and a significant reduction in the number of platform crossings （all P<0.05）； compared with the MHE group， the MHEY group had a significant reduction in searching platform latency （after administration） and a significant increase in the number of platform crossings， and the HE group had a significant increase in searching platform latency and a significant reduction in the number of platform crossings （all P<0.05）. Compared with the NC group， the HE group and the MHE group had significant increases in AST， ALT， ALP， TBil， TBA， blood ammonia， and colon pH value （all P<0.05）； compared with the MHE group， the MHEY group had significant reductions in AST， ALT， ALP， TBil， TBA， blood ammonia， and colon pH value （all P<0.05）， and the HE group had significant increases in AST， ALT， ALP， TBil， TBA， blood ammonia， and colon pH value （all P<0.05）. The MHE group had significantly lower TBA， primary BA， and secondary BA than the NC group （all P<0.05）； compared with the MHE group， the HE group had significantly lower TBA and primary BA （all P<0.05）， and the MHEY group had significantly higher TBA and primary BA （all P<0.05）. Compared with the NC group， the MHE group had significant reductions in GCDCA， GUDCA， GHDCA， TCDCA， TUDCA， GLCA， and TLCA （all P<0.05） and significant increases in γ－MCA， THCA， 7－KDCA， AlloLCA， and α－MCA （all P<0.05）， and compared with the MHE group， the MHEY group had significant increases in THDCA， TMCA， TCDCA， TUDCA， and TLCA （all P<0.05）. ConclusionRD retention enema can improve liver injury and cognitive function in a rat model of MHE induced by CCl₄ and TAA by regulating the enterohepatic circulation of BA， possibly by increasing the synthesis of taurine－binding BA.

Objective:To establish and validate an LC-MS/MS method for simultaneous determination of Aβ 1-42, Aβ 1-40, and Aβ 1-38 in cerebrospinal fluid. Additionally, the consistency between this method and three mainstream detection methods was evaluated.Methods:This study involved method establishment, validation, and consistency evaluation. The N15 labeled β-amyloid protein was used as the internal standard. Extraction was performed using Waters MCX 96-wells solid phase extraction plate, and the eluent was collected to QuanRecovery MaxPeak 700 μl plate. At the positive ion mode, the multi-reaction ion monitoring mode based on electric spray ionization is chosen for the determination of CSF Aβ 1-42, Aβ 1-40, and Aβ 1-38. Referring to the CLSI C62-A and EP-15A3 guidelines, the method is evaluated and verified, including quantitation of limit (LOQ), linearity, recovery, precision, and accuracy. In addition, a total of 57 clinical residual CSF samples were collected and the concentrations of Aβ 1-42 and Aβ 1-40 were determined based on manual INNOTEST ELISA assay and Lumipulse G and Roche Elecsys fully automated biochemical analyzers. The comparison analysis and deviation evaluation were conducted by passing-bablok and Bland Altman methods.Results:The analysis time of this method is 8 min, and the LOQ of Aβ 1-42, Aβ1-40 and Aβ1-38 is 0.1 ng/ml, 0.5 ng/ml, and 0.1 ng/ml, respectively, and the linear range can meet the needs of clinical detection. Respectively, the recovery is 86.2%-93.8%, 100.9%-103.9% and 103.3%-107.1%; the total imprecision is 4.7%-7.4%, 3.5%-4.6% and 5.2%-10.9%. The measured values of Aβ 1-42 certified reference materials are all within the allowable uncertainty requirements. Moreover, the carryover rate of three analytes was all≤0.11%. In addition, the correlations of Aβ 1-42 and Aβ1-40 in CSF between this LC-MS/MS method and the INNOTEST ELISA method, Lumipulse G and Roche Elecsys fully automated biochemical analyzers were all deemed good, with correlation coefficient (r) ranging from 0.920 to 0.970. However, the measured values between the four methods were remarkably different.Conclusion:We established and validated a robust method based on LC-MS/MS technology for simultaneous determination of Aβ 1-42, Aβ 1-40, and Aβ 1-38 in CSF. The method is accurate, simple, and suitable for clinical measurements. However, despite good correlations, there were substantial differences in the measurement results of Aβ 1-42 and Aβ 1-40 among different analytical platforms, indicating the need for further promotion of harmonization and standardization processes for AD classic biomarkers.

Objective To determine the distribution of two important virulence factors [lipooligosaccharide (LOS) and capsular polysaccharide (CPS)] in Campylobacter jejuni (C. jejuni) isolated from different sources in China and to develop a rapid screening method for Guillain–Barré syndrome (GBS)-associated strains. Methods Whole-genome sequencing was carried out for 494 C. jejuni strains. The OrthoMCL software was used to define the LOS/CPS gene clusters. CPS genotyping was performed with serotype-specific sequence alignment using the BLAST software. Real-time Polymerase chain reaction (PCR) was developed with the unique sequences of specific CPS types. Results Nine novel and 29 previously confirmed LOS classes were identified. LOS classes A, B, and C were the most common (48.2％, 238/494) among the 494 strains. Twenty-six capsular types were identified in 448 strains. HS2, HS4c, HS5/31, HS19, and HS8/17 were the most frequent CPS genotypes (58.7％, 263/448). Strains of 17 CPS genotypes (strain number > 5) had one or two prevalent LOS classes (P < 0.05). Multiplex real-time PCR for rapid identification of HS2, HS19, and HS41 was developed and validated with strains of known serotypes. Conclusion Our results describe the genetic characteristics of the important virulence factors in C. jejuni strains in China. The multiplex real-time PCR developed in this study will facilitate enhanced surveillance of GBS-associated strains in China.

OBJECTIVES: Safe and effective anticoagulation is essential for hemodialysis patients who are at high risk of bleeding. The purpose of this trial is to evaluate the effectiveness and safety of two-stage regional citrate anticoagulation (RCA) combined with sequential anticoagulation and standard calcium-containing dialysate in intermittent hemodialysis (IHD) treatment. METHODS: Patients at high risk of bleeding who underwent IHD from September 2019 to May 2021 were prospectively enrolled in 13 blood purification centers of nephrology departments, and were randomly divided into RCA group and saline flushing group. In the RCA group, 0.04 g/mL sodium citrate was infused from the start of the dialysis line during blood draining and at the venous expansion chamber. The sodium citrate was stopped after 3 h of dialysis, which was changed to sequential dialysis without anticoagulant. The hazard ratios for coagulation were according to baseline. RESULTS: A total of 159 patients and 208 sessions were enrolled, including RCA group (80 patients, 110 sessions) and saline flushing group (79 patients, 98 sessions). The incidence of severe coagulation events of extracorporeal circulation in the RCA group was significantly lower than that in the saline flushing group (3.64% vs. 20.41%, P<0.001). The survival time of the filter pipeline in the RCA group was significantly longer than that in the saline flushing group ((238.34±9.33) min vs. (221.73±34.10) min, P<0.001). The urea clearance index (Kt/V) in the RCA group was similar to that in the saline flushing group with no statistically significant difference (1.12±0.34 vs. 1.08±0.34, P=0.41). CONCLUSIONS Compared with saline flushing, the two-stage RCA combined with a sequential anticoagulation strategy significantly reduced extracorporeal circulation clotting events and prolonged the dialysis time without serious adverse events.
Humans ; Citric Acid/adverse effects* ; Prospective Studies ; Sodium Citrate ; Hemorrhage/chemically induced* ; Citrates/adverse effects* ; Anticoagulants/adverse effects* ; Renal Dialysis/adverse effects*