Objective To analyze the registered clinical trials of headache treated by TCM;To discuss the current research status;To provide reference for the optimization of subsequent clinical trial research plans.Methods All clinical trials of headache treated by TCM were retrieved from the ChiCTR and the ClinicalTrials.The retrieval time was from the database establishment to May 22,2023.The general characteristics,study types,intervention measures and outcome indicators of the trials were analyzed respectively.Results A total of 104 registered studies were included,with the number of registered studies increasing since 2004 and reaching a peak in 2020,involving 25 provincial administrative regions or countries and 69 clinical trial institutions;the funding sources were mainly scientific research funds of universities,national finance and local finance.The research type was mainly intervention research;the designing scheme was mainly randomized parallel control study;the high frequency random method was simple random method;45 registered studies used blind methods.Exploratory studies/pre-trials were the most commonly used in the phases of clinical researches.Most of the registered studies were single-center clinical trials with a total sample size of 9 648 patients.The main interventions were acupuncture and oral Chinese medicines.The high frequency outcome indicators included life quality of score,headache attack frequency,headache attack days and headache severity,etc.There were some problems in outcome indicators,such as non-standard,lack of TCM characteristic advantages,and insufficient patient participation.Conclusion The number of registered studies of headache treated by TCM has increased by year,but there are some problems in design elements,such as random method,blind method,number of research centers,sample size and the setting of outcome indicator.

Objective Quality control procedure based on the patient data in clinical chemistry was set up in laboratory information system (LIS). Methods Clinical chemistry tests results of outpatients and inpatients were collected from January 2016 to March 2017 in Zhejiang Provincial People's Hospital. Statistical results of daily patient data, including Xˉ, P2.5, P5, P10, P25, P50, P75, P90, P95 and P97.5 were calculated. Secondly, cumulative coefficients of variation (CV) of these statistical datawere calculated and compared to different criterions. Optimal analytes and related control concentrations were chosen. The minimum number of patient sample which use Xˉ as control point was calculated by PASS 11.0 software. Finally, the quality control procedure was set up base on the LIS and was verified by patient data. Results In outpatients, Xˉwas chosen as control point in AFU, APOA, APOB, CA, CL, HDL, K, MG, NA, NEFA, TP and URIC and the minimum number of sample needed were 23, 23, 30, 8, 10, 24, 34, 8, 8, 20, 13 and 22. P25 was chosen in ALP and TBIL. P50 was chosen in AST, GLU, GPDA and PHOS.P75 was chosen in ALB, CHE, CREA and DBIL. In inpatients, Xˉ was chosen as control point in AFU, ALB, APOA, APOB, CA, CL, HDL, K, Lpa, MG, NA, NEFA, TP and URIC and the minimum number of sample needed were 73, 19, 34, 18, 10, 30, 36, 21, 87, 12, 17, 51, 26 and 52;P25 was chosen in ALP, ALT, AST, CREA, DBIL, LDH, TBIL and TG. P50 in PHOS, P75 in GPDA, and P90 in CHE. 200 samples were needed in the tests which used percentiles as control points. Most CVs of these control points were higher than the commercial quality control used every day. Finally, a quality control procedure based on patient data were set up in LIS. L-J and Z score charts were used to find out systematic bias. Conclusion Patient data used in internal quality control was an economical and practical way, which can make up for the deficiency of traditional method.

OBJECTIVETo investigate the drug resistance, β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding genes of, and to explore its structure and pathogenic function.

METHODSThe strain was isolated by plate streaking method and identified by automatic bacteria detection system and 16S RNA gene PCR. Microdilution method was applied for drug susceptibility test. β-lactamases, extended spectrum β-lactamases (ESBL) and carbapenemases were detected using nitrocefin-disk, Kirby-Bauer disk, and Hodge test, respectively. Five β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene of the isolate were amplified by PCR for sequencing. Bioinformatic softwares were used to analyze the structure and function of the product of protoporphyrin ferrochelatase-encoding gene.

RESULTSA strain belonging towas isolated. This isolate was sensitive to cefepime, ciprofloxacin, ofloxacin and tigecycline, but resistant to five penicillins, four cephalosporins and two carbapenems antibiotics. The isolate produced β-lactamases but did not produce ESBL and carbapenemases. The isolate had five distinct β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene. The product of protoporphyrin ferrochelatase-encoding gene contained two functional domains of protoporphyrin ferrochelatase belonging to type Ⅱ chelatase superfamily that presented the most closely genetic relationship with the protoporphyrin ferrochelatase of.

CONCLUSIONSThe isolate ofhas a higher resistance to β-lactam antibiotics and its β-lactamase-encoding genes are different with the common bacterial β-lactamase-encoding genes. Protoporphyrin ferrochelatase may act as an important virulence factor of.

Objective To develop a simple high-resolution melting ( HRM) analysis method for differentiation of Pc and P2 variants in class 1 integron.Methods DNA fragments containing Pc and P2 variants were amplified from plasmids pACW ( PcW ) and pACWP2 ( PcW-P2 ) respectively , then these purified PCR products and P 2 promoters were analyed full-length amplicon by HRM .Eight DNA fragments containing different Pc promoters were amplified and site-specific mutated from plasmids pACS ( PcS ) , pACH2 ( PcH2 ) , pACH1 ( PcH1 ) , pACW ( PcW ) , genomic DNA of Klebsiellar pneumonia HS07-68 (PcWTGN-10)and HS05-1792(PcH2TGN-10)respectively.The purified PCR products and eight Pc variants were characterized by HRM analyses of an unlabeled probe and full-length amplicon.This assay was applied to the differentiate Pc and P2 variants in 109 class 1 integrons from 95 urine clinical Escherichia coli isolates in Huashan Hospital during 2004 -2007.The differentiation results were compared with that determined by direct sequencing .Results P2 promoter with a significant higher melting temperature ( Tm ) can be identified by HRM analysis clearly .P2 promoters were identified in 2 class 1 integrons and consistent with direct sequencing results .Eight Pc variants were classified into three groups: PcS, PcSTGN-10 , PcW, PcWTGN-10, PcH1, PcH1TGN-10.Using direct HRM analysis.PcH2, PcH2TGN-10 were classified into four groups:PcS, PcH1, PcH2, PcW, PcSTGN-10 , PcH1TGN-10 , PcH2TGN-10 , PcWTGN-10 according to the melting curves of the unlabeled probe .Combined the HRM analyses of the whole amplicon and unlabeled probe , the eight Pc variants can be differentiated from each other .Five different Pc variants, PcS, PcW, PcH1, PcH2TGN-10 and PcWTGN-10 , were identified and consistent with direct sequencing results .Conclusions This developed a simple Pc and P 2 variants differentiation method via simultaneous HRM analyses of an unlabeled probe and full-length amplicon .This method is cost-effective and accurate , could be used in differentiation of Pc and P2 variants of class 1 integrons in clinical isolates .

We isolated and identified the bacterial pathogen in a pyogenic balanoposthitis patient and investigated the drug resistance and its mechanism of the isolate.Urethral secretions and balanus pustule liquids were collected for microscopic examination after Gram-staining and detection of mycoplasma using Mycoplasma IST 2 kit.The two samples were inoculated on Columbia blood plate,N.gonorrhoeae selective plate and chromID Candida plate for isolation.The obtained colonies were identified by VITEK 2-compact automatic bacterial detection and analysis system.Moreover,PCR was performed to detect 16S rRNA gene of N.gonorrhoeae in the samples and colonies.KB method was applied for detecting susceptibility of five common antibiotics against the isolate.The β-lactamase and extended spectrum β-lactamase confirmatory tests were used to investigate the enzyme production of the isolate as well as drug resistance-associated tetM,TEM,mefA and ermF genes in the isolate were detected by PCR.Results showed that all the clinic samples showed negative for mycoplasma.All the isolating cultivation results of urethral secretions were negative while the balanus pustule liquids provided positive isolating cultivation in the blood and selective plates.The VITEK 2-compact system and 16S rRNA-PCR revealed that the isolated strain belongs to N.gonorrhoeae.The isolate can produce β-lactamases and resist to penicillin G,ciprofloxacin and tetracycline.The tetM,TEM,mefA and ermF genes could be found in the isolate's genome.The patient's balanoposthitis is caused by infection of N.gonorrhoeae.The multidrug resistance of Neisseria gonorrhoeae isolate is closely associated with its carried resistant genes.

The aim of this study is to determine the diversity of virulence genes carried by different vancomycin-resistant enterococci (VRE),which will provides a basis for studying pathogenic mechanism of VRE.Microdilution-based drug sensitivity test was applied to detect the vancomycin resistance of 490 Enterococcus faecium isolates and 862 Enterococcus faecalis isolates in Zhejiang area.The seven virulence genes (ace,asa1,cylA,efaA,esp,gelE and hyl) in the isolates of VRE were detected by PCR.According to the results of drug sensitivity test,10％ of the E.faecium isolates (49/490) and 0.8％ of the E.faecalis (7/862) were identified as VRE.In the vancomycin-resistant E.faecium isolates,five isolates were negative for any of the target genes and the other 44 isolates were positive for asa1,esp,gelE and hyl genes alone,in which the esp (73.5％,36/49) and hyl (53.1％,26/49) were the predominant genes and single or double virulence genes acted as the major carrying models.Except for the hyl gene,the vancomycin-resistant E.faecalis isolates were positive for the other six pathogenic genes,and the isolates could carry 3-6 pathogenic genes.All the data indicate that E.faeciurn is the major species of VRE in the local area,and the carrying rate,types and models of virulence genes in the vancomycin-resistant E.faecium and E.faecalis isolates are obviously different.

We investigated correlation between the level of miR-16 expression and the severity of Staphylococcus aureus sepsis,and further explored its potentially clinical significance.Blood samples were collected from 32 patients,including each 8 cases of septic shock,severe sepsis and general sepsis,as well as 8 cases of healthy volunteers.Blood samples from 24 cases of healthy subjects with different ages were measured,and additionally 8 cases of blood samples from gram negative bacteria sepsis were also determined in current study as a control.Trizol solution for the whole blood lysis was added into blood samples,and followed by the extraction of microRNA.The expression levels of miR-16 in different groups were measured by fluorescence quantitative PCR,in which 2-Delta Ct method was used.SPSS software (version 13.0) was used to analyze the statistical differences between the groups,and further analyze the correlation between miR-16 value and the corresponding CRP and PCT values.Results showed that the expression level of miR-16 was negatively correlated with the severity of Staphylococcus aureus sepsis.There were statistically significant differences in experimental groups when compared with the control (P＜0.001),and there was also a statistically significant difference between each experimental group (P＜0.01).We found that the expression level of miR-16 was negatively correlated with CRP and PCT,the correlation coefficients were-0.561 and-0.769 respectively,and trend analysis showed that there was a significantly negative correlation.A significantly negative correlation was found between the miR-16 expression level and severity of sepsis,suggesting that miR-16 may serve as a biomarker for the severity of Staphylococcus aureus sepsis.

Objective To investigate the relationship between hypoxia and insulin resistance in males with metabolic syndrome ( MS ). Methods Parameters at physical, hematological, glucose metabolism, lipid metabolism, and insulin resistance were measured in 295 middle-aged men who took health examination. The trends of laboratory indexes grouped by hemoglobin quartile and MS components were compared. The correlation between hypoxia and insulin resistance was analyzed. Results With the increase of hemoglobin, the occurrence rates of MS, TG, INS, and HOMA-IR were also significantly increased ( P < 0. 05 ), while HDL-C and HOMA-IS reduced remarkably(P<0. 05). With the increase of MS components, hematological parameters(RBC, Hb, Hct), INS, and HOMA-IR were significantly increased( P<0. 05) while HOMA-IS were significantly decreased( P<0. 05). Blood lactate and HIF-1αwere significantly increased with the increase of hemoglobin as well as MS components(P<0. 05);Hematological parameters were positively correlated with blood lactate and HIF-1α, and Hypoxia related indicators including hematological parameters, Lac, HIF-1α showed a positive correlation with HOMA-IR and a negative correlation with HOMA-IS( P < 0. 05). Conclusion Chronic hypoxia does exist in metabolic syndrome, and the degree of hypoxia is associated with insulin resistance. Hematological parameters are also significantly correlated with insulin resistance which could be as a result of chronic hypoxia.

Objective To investigate the prognostic value of regulatory B cells (Bregs) in patients with acute pancreatitis .Methods Flow cytometry analysis was performed to detected the percentages of CD19+IL-10+and CD19+CD24hiCD27hi Bregs in peripheral blood samples collected from patients with acute pancreatitis (36 cases with mild acute pancreatitis and 15 cases with severe acute pancreatitis ) as well as the surface costimulatory molecules including CD80 and CD86 on CD19+CD24hiCD27hi Bregs.Their correlations with lymphocytes and C-reactive protein ( CRP) were further analyzed .Results The numbers of lympho-cytes, CD19+lymphocytes, CD19+IL-10+and CD19+CD24hi CD27hi Bregs in peripheral blood samples col-lected from patients with severe and mild acute pancreatitis as well as the mean fluorescence intensities ( MFI) of CD80 and CD86 were significantly lower than those from healthy subjects .Compared with patients with mild acute pancreatitis , the numbers of lymphocytes and CD 19+lymphocytes , the absolute numbers of CD19+IL-10+and CD19+CD24hiCD27hi Bregs as well as the mean fluorescence intensities (MFI) of CD80 and CD86 in patients with severe acute pancreatitis were significantly decreased .The percentages of CD19+IL-10+and CD19+CD24hiCD27hi Bregs in patients with mild acute pancreatitis were significantly increased af-ter an initial drop , but in patients with severe acute pancreatitis those values were continuously decreased along with the disease progression .The percentage of CD19+IL-10+Bregs was positively correlated with the percentage of CD19+CD24hiCD27hi Bregs and the absolute number of CD19+lymphocytes, but was negatively correlated with CRP .Conclusion The abnormal number and function of CD 19+IL-10+and CD19+CD24 hi CD27hi Bregs might be one of the important reasons causing immune dysfunction in patients with acute pan -creatitis.

Objective To construct a prokaryotic expression system for tlyA gene of Leptospira in-terrogans ( L.interrogans) strain and to investigate the effects of the expressed rTlyA protein on the hemolysis of sheep erythrocytes and the secretion of pro-inflammatory cytokines by human THP-1 cells and murine J774A.1 macrophages.Methods The fragment of tlyA gene of L.inetrrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR.The PCR product was sequenced after T-A cloning.A prokary-otic expression system for the tlyA gene was constructed by using pET-42a as the expression vector and E.coli BL21DE3 strain as the host strain.The expression of rTlyA protein was detected by SDS-PAGE.Ni-NTA af-finity chromatography was performed for purification.The lytic activity of the rTlyA protein on sheep erythro-cytes was determined by plate hemolytic test and spectrophotometry.Real-time fluorescence quantitative PCR and Western blot assay were performed to respectively detect the expression of tlyA gene in THP-1 and J774A.1 cells at mRNA and protein levels after infection with L.interrogans strain Lai.ELISA was per-formed to evaluate the effects of the rTlyA protein on the secretion of several pro-inflammatory cytokines ( IL-β, IL-6 and TNF-α) by THP-1 and J774A.1 cells.Results The nucleotide and amino acid sequences of the cloned tlyA gene were 99.2% and 99.8% identical to those corresponding sequences in GenBank, re-spectively.The constructed prokaryotic expression system for tlyA gene successfully expressed the rTlyA pro-tein.The rTlyA protein at the concentration of 10μg/ml showed stronger lytic activity on sheep erythrocytes. The transcription levels of tlyA gene (P<0.05) and the secretion of TlyA protein in THP-1 and J774A.1 cells were significantly increased after infecting with L.interrogans strain Lai for 1 to 8 hours.The rTlyA pro-tein at concentrations of 0.1, 1 and 10 μg/ml significantly enhanced the secretion of IL-1β, IL-6 and TNF-αby THP-1 and J774A.1 cells (P<0.05).Conclusion The TlyA protein, encoded by the tlyA gene of L.interrogans strain, was confirmed to be a hemolysin with the ability to induce macrophages to secret IL-1β, IL-6 and TNF-α.This study suggested that the TlyA protein played an important role in inflammatory responses during leptospirosis.