In order to assess the cytotoxin effect of Campylobacter jejuni (C.jejuni), the whole cell lysates from 9 strains of Guillan-Barre Syndrome (GBS)- associated C.jejuni and 4 strains of diarrhea-associated C. jejuni were co-cultivated with HeLa cells at the concentration of 0.001-5.00 μg/mL in vitro. The morphologic change of HeLa cells was observed under Olympus BX51 microscope after treatment with different concentration of bacteria lysate in the following 4 days. The morphological changes including swelling, irregularity and lysis of the affected cells over 50% was selected as the cut off for positive change and C.jejuni 81-176 and Helicobacter J99 strains were chosen as the positive and negative control. It was found that the minimum concentration to induce the positive changes in 10 strains(8 GBS associated and 2 strains from diarrhea patients)was 0.1μg/mL and 1 strain with the positive change at the minimum concentration of 1 μg/mL. There was only one GBS-associated strain causing no morphologic change on HeLa cells at the concentration of 5 μg/mL. It was evident that the cytotoxic effect of C.jejuni strains on HeLa cell was strain-specific, and there was no significant difference in the cytotoxic effect on HeLa cell between GBS-associated and diarrhea associated C.jejuni strains.

Background:Helicobacter pylori(Hp)is an important pathogen for peptic ulcer and gastric cancer,and is reportedly associated with a variety of extragastrointestinal diseases. However,there is no body fluid detection technique for Hp infection in clinical practice. Aims:To identify Hp infection-associated differentially expressed proteins in urine with relative molecular mass more than 10 kDa and provide potential biomarkers for diagnosis of Hp infection through body fluid detection. Methods:Midstream urine was collected from volunteers in the morning,and 13 C-urea breath test was performed to determine Hp infection. Each of 15 Hp-negative and 15 Hp-positive urine samples were mixed respectively for protein extraction. Spectra data were acquired by high performance liquid chromatogram-mass spectrometry,and label-free technology was used for relative quantitative analysis. The other 26 urine samples(15 Hp-negative and 11 Hp-positive) were used for validation by full scan. IPA software was employed for bioinformatics analysis. Results:A total of 475 urinary proteins were detected by label-free quantitative analysis and 42 differentially expressed proteins were identified. Finally,11 significantly up-regulated differentially expressed proteins were confirmed by external scanning validation. Bioinformatics analysis revealed the molecular functions,biological pathways,and related diseases of these differentially expressed proteins. Conclusions:These 11 differentially expressed proteins more than 10 kDa identified in urine might be potential biomarkers for diagnosis of Hp infection and provide molecular evidence for the correlation of Hp infection with extragastrointestinal diseases.

Objective To research the differential expression of trace phosphorylated proteins in human gastric adenocarcinoma epithelial (AGS) cells infected by Helicobacter pylori. Methods H. pylori 26695 strain infected AGS cells 4 h and AGS cells was cultivated for 4 h as a comparison. The proteins of AGS and comparison AGS cells were extracted. Their phosphorylated proteins were enriched by metal ion af-finity adsorption enrichment techniques. After desalinated and purified the phosphorylated proteins samples were separated by 2-dimensional polyacrylamide gel electrophoresis (2-DE) technique. Computer assisted image analysis was used to analyze the differential proteomic expression. The significantly differentially ex-pressed proteins were unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF). Results Fifteen kinds of proteins were down-regulated, 4 kinds of new proteins were observed, 1 kind of proteins were up-regulated, 1 kind of proteins unexpression. The 21 proteins that were significantly differentially expressed , including cellular calcium ion homeostasis, transcription, interpretation, protein folding and transport, ribosomal assembly, centrosome replication, chromosome stability, cellular structure, cellular proliferation and apoptosis. Conclusion H. priori can cause a wide range change to human gastric adenocarcinoma epithelial cell protein pheshorylation. This change character has great significance to further comprehensive understanding of the pathogenesis of H. pylori.

Objective:To explore the application value of evaluation and management of patients with acute chest pain in China (EMPACT) score in risk stratification for patients with acute chest pain.Methods:According to the methods of prospective cohort study, 548 patients with chest pain in the Affiliated Hospital of Jining Medical University from February to April 2021 were selected. The risk stratification was performed according to EMPACT score. The primary endpoint was the major adverse events (MAE) within 30 d, including death from all causes, acute myocardial infarction (AMI), emergency revascularization, cardiac arrest, cardiogenic shock and other life-threatening situations that need urgent attention. The receiver operating characteristic (ROC) curve was used to evaluate the predictive value of EMPACT score for MAE.Results:Among the 548 patients, 75 cases had MAE within 30 d (MAE group), and the incidence of MAE was 13.7%; 473 cases did not occur MAE (non-MAE group). The EMPACT score in MAE group was significantly higher than that in non-MAE group: 8 (12, 18) scores vs. 5 (2, 8) scores, and there was statistical difference ( Z = 8.94, P<0.01). Spearman correlation analysis result showed that EMPACT score was positively correlated with MAE ( r = 0.38, P<0.01). ROC curve analysis result showed that the area under the curve of EMPACT score in prediction within 30 d MAE was 0.820 (95% CI 0.770 to 0.871), the cut-off value was 9.5 scores (since all the scoring systems were integers, the cut-off value was 10 scores), the sensitivity was 88.6%, and the specificity was 60.0%. Conclusions:The EMPACT score has a good risk stratification capability to achieve safe and effective triage of acute chest pain.

Objective To evaluate the clinical safety of the collagen nerve scaffold with longitudinally oriented microchannels in bridging peripheral nerve defect.Methods Five patients with 8 peripheral nerve defects of 18 to 30 (mean,23.8) mm in length were involved in the pilot study and treated from July,2017 to March,2018,including 6 digital nerves and 2 medial antebrachial cutaneous nerves.The defects were repaired with the collagen nerve scaffold with longitudinally oriented microchannels independently developed.Routine therapy of prophylactic systemic antibiotics but no immunosuppressive drugs was given to all patients post-operatively.All patients were followed-up by regular review in the outpatient department combined with WeChat and telephone.The clinical safety of the nerve scaffold was preliminarily evaluated through observing the condition of the healing of the local wound and the whole body.The blood routine examineation and biochemical test were detected.The statistical analysis of the measurement data was performed by the analysis of variance of repeated measurement data,and the difference was statistically significant when P＜0.05.Results All patients were followed-up for 7 to 15 months (average,10 months).No adverse events such as infection,allergy,damage of liver and kidney function occurred.The operative incisions healed primarily,with no redness,exudation and rupture in the local area.There was no systemic symptoms such as fever,nausea,vomiting,skin itching,etc.The results of blood routine tests and biochemical tests were normal.The data of tests was compared,and the difference was not statistically significant (P＞0.05).Conclusion The preliminary study shows that it is clinically safe to bridge peripheral nerve defects with the collagen nerve scaffold with longitudinally oriented microchannels.

Objective:To compair the outcomes of repairing soft tissue defects of hand between a biodegrad- able repair patch—porcine small intestinal submucosa (SIS) and skin grafting.Methods:From December, 2017 to December, 2018, 36 cases of hand soft tissue defect were treated and analyzed retrospectively. According to the defect area and treatment methods, 36 cases were divided into 2 groups: SIS group (21 cases) and grafting group(15 cases). In SIS group, the area of soft tissue defects was 2.0 cm×1.5 cm-9.0 cm×3.5 cm with an average of 5.3 cm×2.1 cm, treat- ed with SIS; In grafting group, the area of soft tissue defects was 9.0 cm×4.0 cm-16.0 cm×9.0 cm with an average of 12.0 cm×8.5 cm, treated with autologous skin grafting after wet dressing. Wound healing was evaluated at 14, 21 and 28 days, and 3 months after the surgery according to the appearance of colour, elasticity, sensory recovery and prog- noses of partial tendon exposure.Results:All patients were followed-up for 3 to 10 months, with an average of 5 months. All wounds in both groups were completely healed; the appearance was normal, and the skin elasticity and sensation had recovered. Sensation recovery in SIS group: 14 cases were good (66.6%), 5 cases were fine (23.8%), and 2 cases were bad (9.6%); in grafting group: 9 cases were good (60.0%), 4 cases were fine (26.0%), and 2 cases were bad (14.0% ). Wound healing effect in SIS group: 14 cases were good, 5 cases were fine, and 2 cases were bad; in grafting group: 9 cases were good, 4 cases were fine, and 2 cases were bad.Conclusion:The SIS patch can be used in the reconstruction of soft tissue defects in hand. There was no significant difference in colour compared to the sur- rounding skin and left no scar. The patch is an ideal repair material for superficial skin defects.

Objective:To illustrate the characteristics of the distribution of prophages among the Group A Streptococcus(GAS) by mining the existing whole genome sequencing of the GAS, performing bioinformatic analyses, extracting data about prophages, and analyzing the state of prophages in the genome and genetic composition of some prophages. Methods:It was a retrospective study.Genome assembly sequences of GAS reported in GenBank till May 2020 were collected, and the important background information of these strains was sorted out to create a local genomic database.A phylogenetic tree of the whole genome of GAS was conducted using the bioinformatics software.The core genome was analyzed, and potential prophages and their integrity in the genome were predicted to obtain the characteristics of the distribution of prophages.Genotype types, number of core genes, and number, length and carrying rate of prophages in the database for GAS were analyzed.Results:A database containing the genome sequence of 2 529 GAS strains was established, involving 140 emm genotypes.These strains were isolated from 19 countries from East Asia, Europe, America and Oceania.Stratified by the disease background, these strains were mainly divided into invasive infection, non-invasive infection and immune sequelae.Prophage analysis of 1 798 genomes showed that at least one complete prophage was detected in 1 366 (76.0%) genomes.The number of complete prophages of each strain ranged from 0 to 6, and the length ranged from 32.8 to 62.6 kb, which was mainly 30-40 kb in length.The phiHKUssa, phiHKUvir and phiHKU488 were the most common prophages present in dominant clones circulated in China in recent years, which mainly carried virulence genes like the speC, spd1 and ssa. Conclusions:Prophages are widely distributed in the genome of GAS, which are of great significance in the evolution and expansion of dominating clones and thus reshape the population structure within the emm genotype.The establishment of a local genome database provides important baseline data for molecular epidemiological surveillance.

Objective:To establish a nucleic acid detection and genotyping method for Mycoplasma pneumoniae ( Mp) based on nucleic acid in clinical samples. Methods:Through genomic comparison, the specific target sequences of Genotype 1 and Genotype 2 Mp strains were selected to design synthetic primers and probes, and a PCR detection and classification method for Mp dual fluorescent probe was established, and the specificity, accuracy, detection limit and repeatability of the method were evaluated. The established fluorescence PCR method was used to detect the nucleic acid of clinical specimens and compared with the reported fluorescent PCR methods. Results:The nucleic acid of 18 pathogens, including other species of Mycoplasma and common respiratory bacteria and viruses, which were closely related to the Mp species, were detected, and the results showed that there was no cross-reactivity. The accuracy of detection and typing of 90 Mp nucleic acid was 100%. The detection limits of Genotype 1 and Genotype 2 Mp samples were 1.0 copy/μl, and the experimental coefficient of variation of repeatability within groups and between groups was less than 2.5%. In the detection of 88 nucleic acid of clinical specimens, the Kappa value was 0.675 and the P value was 0.267 compared with the reported real-time PCR method, showing a high degree of agreement. Conclusions:The method for detecting and genotyping Mp in this study has high sensitivity, specificity, and accuracy, which can be applied to the monitoring and prevention and control of Mp in the disease control system of provinces and cities at all levels in China. This method promotes the improvement of the Mp prevention and control system in China, strengthens the surveillance ability, and is of great significance for the early warning and prediction of Mp.

Objective:To analyze the amino acid polymorphism of truncated Staphylococcal enterotoxin-like toxin X (tSElX), and to evaluate its related emetic activities.Methods:Sequence of tselx was compared with both the genome sequence of 145 CC398 strains completed in our research group and the NCBI database. Primers were designed to amplify the target gene of tselx, and the fragment was recombined into pMD18-T vector and sequenced. PCR product was digested with BamHⅠ and EcoRⅠ, and constructed into plasmid pGEX-6P-1 and pET-28a (+). After recombinant plasmid was identified, the protein expression was induced by IPTG. Proteins expressed in the form of inclusion bodies were denatured and renatured, then purified by affinity chromatography and ultrafiltration. Purified tSElX protein was then fed to common marmosets with the dose of 250 μg/kg to observe the vomiting reaction. Results:tselx gene was present in 145 strains of CC398 strains from the different origins (patients, healthy people and animals) in China. Homology of the amino acid sequence of the protein from the Chinese strains appeared 100.0 %, while the homology with the four American strains were 97.8 %(1) and 98.9 %(3), respectively. Through two sets of expression systems and different induction conditions, tSElX was expressed in the form of inclusion bodies. The high purity soluble recombinant tSElX was thus obtained by denaturated and renaturated processes. At the dose of 250 μg/kg, tSElX protein did not cause vomiting in common marmosets. Conclusions:Results of this study showed that the amino acid sequence of tSElX was highly conserved and was universally present in a particular clone group. We obtained soluble recombinant tSElX protein with high purity. We also noticed that tSElX did not have the animal emetic activity at a dose of 250 μg/kg.