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: 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

Objective: To analyze the clinical epidemiological characteristics including composition of pathogens , clinical characteristics, and disease prognosis acute bacterial meningitis (ABM) in Chinese children. Methods: A retrospective analysis was performed on the clinical and laboratory data of 1 610 children <15 years of age with ABM in 33 tertiary hospitals in China from January 2019 to December 2020. Patients were divided into different groups according to age,<28 days group, 28 days to <3 months group, 3 months to <1 year group, 1-<5 years of age group, 5-<15 years of age group; etiology confirmed group and clinically diagnosed group according to etiology diagnosis. Non-numeric variables were analyzed with the Chi-square test or Fisher's exact test, while non-normal distrituction numeric variables were compared with nonparametric test. Results: Among 1 610 children with ABM, 955 were male and 650 were female (5 cases were not provided with gender information), and the age of onset was 1.5 (0.5, 5.5) months. There were 588 cases age from <28 days, 462 cases age from 28 days to <3 months, 302 cases age from 3 months to <1 year of age group, 156 cases in the 1-<5 years of age and 101 cases in the 5-<15 years of age. The detection rates were 38.8% (95/245) and 31.5% (70/222) of Escherichia coli and 27.8% (68/245) and 35.1% (78/222) of Streptococcus agalactiae in infants younger than 28 days of age and 28 days to 3 months of age; the detection rates of Streptococcus pneumonia, Escherichia coli, and Streptococcus agalactiae were 34.3% (61/178), 14.0% (25/178) and 13.5% (24/178) in the 3 months of age to <1 year of age group; the dominant pathogens were Streptococcus pneumoniae and the detection rate were 67.9% (74/109) and 44.4% (16/36) in the 1-<5 years of age and 5-<15 years of age . There were 9.7% (19/195) strains of Escherichia coli producing ultra-broad-spectrum β-lactamases. The positive rates of cerebrospinal fluid (CSF) culture and blood culture were 32.2% (515/1 598) and 25.0% (400/1 598), while 38.2% (126/330)and 25.3% (21/83) in CSF metagenomics next generation sequencing and Streptococcus pneumoniae antigen detection. There were 4.3% (32/790) cases of which CSF white blood cell counts were normal in etiology confirmed group. Among 1 610 children with ABM, main intracranial imaging complications were subdural effusion and (or) empyema in 349 cases (21.7%), hydrocephalus in 233 cases (14.5%), brain abscess in 178 cases (11.1%), and other cerebrovascular diseases, including encephalomalacia, cerebral infarction, and encephalatrophy, in 174 cases (10.8%). Among the 166 cases (10.3%) with unfavorable outcome, 32 cases (2.0%) died among whom 24 cases died before 1 year of age, and 37 cases (2.3%) had recurrence among whom 25 cases had recurrence within 3 weeks. The incidences of subdural effusion and (or) empyema, brain abscess and ependymitis in the etiology confirmed group were significantly higher than those in the clinically diagnosed group (26.2% (207/790) vs. 17.3% (142/820), 13.0% (103/790) vs. 9.1% (75/820), 4.6% (36/790) vs. 2.7% (22/820), χ²=18.71, 6.20, 4.07, all P<0.05), but there was no significant difference in the unfavorable outcomes, mortility, and recurrence between these 2 groups (all P>0.05). Conclusions: The onset age of ABM in children is usually within 1 year of age, especially <3 months. The common pathogens in infants <3 months of age are Escherichia coli and Streptococcus agalactiae, and the dominant pathogen in infant ≥3 months is Streptococcus pneumoniae. Subdural effusion and (or) empyema and hydrocephalus are common complications. ABM should not be excluded even if CSF white blood cell counts is within normal range. Standardized bacteriological examination should be paid more attention to increase the pathogenic detection rate. Non-culture CSF detection methods may facilitate the pathogenic diagnosis.
Adolescent ; Brain Abscess ; Child ; Child, Preschool ; Escherichia coli ; Female ; Humans ; Hydrocephalus ; Infant ; Infant, Newborn ; Male ; Meningitis, Bacterial/epidemiology* ; Retrospective Studies ; Streptococcus agalactiae ; Streptococcus pneumoniae ; Subdural Effusion ; beta-Lactamases

Objective: To analyze the clinical features of patients with acute pulmonary embolism (APE) living in high altitude area of Yunnan province. Methods: This was a cross-sectional retrospective study. APE patients, hospitalized in our hospital between January 2017 and December 2019, were included. The selected patients were divided into low-risk group, medium-risk group and high-risk group according to risk stratification. The clinical data of patients, including demographic data, the main symptoms, risk factors of APE, heart rate and systolic blood pressure and laboratory testing results (D-dimer, cardiac troponin I (cTNI), N terminal B-type natriuretic peptide (NT-proBNP)) and echocardiography and electrocardiogram examination results, were obtained through the electronic medical record system. The clinical characteristics of selected patients were analyzed. Results: A total of 392 patients, aged (63.5±15.7) years, 224 males (57.14%), were included in this study and there were 59 low-risk, 304 medium-risk and 29 high-risk patients in this cohort. The main clinical manifestations were chest pain (157(40.05%)), dyspnea (107(27.30%)), hemoptysis (55(14.03%)), syncope as the first symptom (20(5.10%)), and only 6 cases (1.53%) presented with the typical "Virchow's triad". Most of the patients were accompanied by atypical chest tightness (223(56.89%)) and cough (208(53.06%)). The main risk factors were venous thrombosis of lower limbs (179(45.66%)), hypertension (138(35.20%)), surgery (63(16.07%)), and chronic obstructive pulmonary disease (COPD) (62(15.82%)). There were 57 cases (14.54%) of coronary heart disease, 57 cases (14.54%) of diabetes, 51 cases (13.01%) of cerebral infarction, 47 cases (12.00%) of advanced age, 15 cases (3.83%) of tumor, 7 cases (1.79%) of activity restriction, 6 cases (1.53%) of pregnancy and 4 cases (1.02%) of hormone use in this cohort. The proportion of lower extremity venous thrombosis was significantly higher in low-risk group than in medium-risk group (P<0.01), COPD was more common in high-risk and medium-risk groups than in low-risk group (P<0.01), hypertension was more common in high-risk group than in medium-and low-risk groups (P<0.01). The proportion of advanced age was significantly higher in medium-risk group than in low-risk group (P<0.01). There were no significant differences in RBC and hemoglobin level between low-, medium-and high-risk groups (P>0.05). The level of D-dimer was significantly higher in high-risk group than in medium-and low-risk groups (P<0.05). Levels of NT-proBNP and cTNI were significantly higher in high-risk group than in medium- and low-risk groups (P<0.05). Increased proportion of cTNI and NT-proBNP was significantly higher in high-risk group than in medium- and low-risk groups (P<0.05). There were 105 (26.79%) patients with pulmonary hypertension (PAH). The incidence of PAH was significantly higher in high-risk group than in low-risk group (P<0.01). There were 104 patients (26.53%) with right ventricular enlargement, and the incidence of right ventricular enlargement was significantly higher in high-risk group than in medium-and low-risk groups (P<0.01). Characteristic changes of electrocardiogram in patient with APE were T-wave inversion of limb leads (98(25.00%)), followed by S_ⅠQ_ⅢT_Ⅲ (83(21.17%)). Conclusions: The main clinical manifestations of APE in Yunnan high altitude area are chest pain and dyspnea, and syncope is the first symptom in some patients, but the typical "Virchow's triad" is rare. The most common risk factors are lower extremity venous thrombosis, hypertension, and COPD. Clinical symptoms, risk factors and laboratory examination results differ among patients with different risk stratification.
Adolescent ; Altitude ; Biomarkers ; China/epidemiology* ; Cross-Sectional Studies ; Humans ; Male ; Natriuretic Peptide, Brain ; Peptide Fragments ; Prognosis ; Pulmonary Embolism/epidemiology* ; Retrospective Studies

Objective: To construct a prediction model of pathologic complete response (pCR) in locally advanced rectal cancer patients who received programmed cell death protein-1 (PD-1) antibody and total neoadjuvant chemoradiotherapy by using radiomics based on MR imaging data and to investigate its predictive value. Methods: A clinical diagnostic test study was carried out. Clinicopathalogical and radiological data of 38 patients with middle-low rectal cancer who received PD-1 antibody combined with total neoadjuvant chemoradiotherapy and underwent TME surgery from January 2019 to September 2021 in our hospital were retrospectively collected. Among 38 patients, 23 were males and 15 were females with a median age of 68 (47-79) years and 13 (34.2%) a chieved pCR. These 38 patients were stratified and randomly divided into the training group (n=26) and test group (n=12) for modeling. All the patients underwent rectal MRI before treatment. The clinical, imaging and radiomics features of all the patients were collected, and the clinical feature model and radiomics model were constructed. The receiver operating characteristic (ROC) curves of each model were drawn, and the constructed model was evaluated through the area under the curve (AUC), accuracy, sensitivity, specificity, positive predictive value and negative predictive value. Results: There were no significant differences in age, gender, primary location of tumor and postoperative pathology between the two groups (all P>0.05). Forty-one features were extracted from region of interest in each modality, including 9 first-order features, 24 gray level co-occurrence matrix features and 8 shape features. From 38 patients, 41 features were extracted from each imaging modality of baseline and preoperative DWI and T2WI images, totally 164 features. Only 4 features were preserved after correlation analysis between each pair of features and t-test between pCR and non-pCR subjects. After LASSO cross validation, only the first-order skewness of the baseline DWI image before treatment and the volume in the baseline T2WI image before treatment were retained. The area under the curve, sensitivity, specificity, positive and negative predictive values of the prediction model established by applying these two features in the training group and the test group were 0.856 and 0.844, 77.8% and 100.0%, 88.2% and 75.0%, 77.8% and 66.7%, 88.2% and 100.0%, respectively. The decision curve analysis of the radiomics model showed that the strategy of this model in predicting pCR was better than that in treating all the patients as pCR and that in treating all the patients as non-pCR. Conclusion: The pCR prediction model for rectal cancer patients receiving PD-1 antibody combined with total neoadjuvant radiochemotherapy based on MRI radiomics has the potential to be used in clinical screening or rectal cancer patients who can be spared from radical surgery.
Aged ; Antibodies/therapeutic use* ; Female ; Humans ; Magnetic Resonance Imaging/methods* ; Male ; Middle Aged ; Neoadjuvant Therapy/methods* ; Programmed Cell Death 1 Receptor ; Rectal Neoplasms/therapy* ; Retrospective Studies

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.

Objective To investigate the current distribution of ticks and predict the suitable habitats of ticks in the Yangtze River Delta urban agglomeration in 2017, so as to provide insights into tick control and management of tick-borne diseases in these areas. Methods All publications pertaining to tick and pathogen distribution in the Yangtze River Delta urban agglomeration were retrieved, and the geographical location of tick distribution was extracted. The effects of 19 climatic factors on the distribution of ticks were examined using the jackknife method, including the mean temperature of the wettest quarter, precipitation of the coldest quarter, mean temperature of the driest quarter, maximum temperature of the warmest month, precipitation of the driest month, minimal temperature of the coldest month, annual precipitation, mean daily temperature range, precipitation seasonality, annual temperature range, temperature seasonality, annual mean temperature, mean temperature of the warmest quarter, precipitation of the wettest quarter, isothermality, mean temperature of the coldest quarter, precipitation of the wettest month, precipitation of the driest quarter and precipitation of the warmest quarter. The distribution of ticks was analyzed in 2020 using the maximum entropy (MaxEnt) model, and the potential suitable habitats of ticks were predicted in 2070 using the MaxEnt model based on climatic data. Results A total of 380 Chinese and English literatures were retrieved, and 148 tick distribution sites were extracted, with 135 sites included in the subsequent analysis. There were 7 genera (Haemaphysalis, Rhipicephalus, Ixodes, Dermacentor, Boophilus, Hyalomma and Amblyomma) and 27 species of ticks detected in the Yangtze River Delta urban agglomeration. The climatic factors affecting the distribution of ticks in the Yangtze River Delta urban agglomeration mainly included the mean temperature of the wettest quarter and the precipitation of the coldest quarter, with 26.1% and 23.6% contributions to tick distributions. The high-, medium- and low-suitable habitats of ticks were 20 337.08, 40 017.38 km² and 74 931.43 km² in the Yangtze River Delta urban agglomeration in 2020, respectively. The climate changes led to south expansion of the suitable habitats of ticks in the Yangtze River Delta urban agglomeration in 2070, and the total areas of suitable habitats of ticks was predicted to increase by 18 100 km². In addition, the high-, medium- and low-suitable habitats of ticks were predicted to increase to 24 317.84, 45 283.02 km² and 83 766.38 km² in the Yangtze River Delta urban agglomeration in 2070, respectively. Conclusions Multiple tick species are widespread in the Yangtze River Delta urban agglomeration, and the future climate changes may lead to expansion of tick distribution in these areas.

OBJECTIVE: To investigate the mechanism of Tojapride, a Chinese herbal formula extract, on strengthening the barrier function of esophageal epithelium in rats with reflux esophagitis (RE). METHODS: Ten out of 85 SD rats were randomly selected as the sham group (n10), and 75 rats were developed a reflux esophagitis model (RE) by the esophageal and duodenal side-to-side anastomosis. Fifty successful modeling rats were divided into different medicated groups through a random number table including the model, low-, medium-, and high-dose of Tojapride as well as omeprazole groups (n10). Three doses of Tojapride [5.73, 11.46, 22.92 g/(kg•d)] and omeprazole [4.17 mg/(kg•d)] were administrated intragastrically twice daily for 3 weeks. And the rats in the sham and model groups were administered 10 mL/kg distilled water. Gastric fluid was collected and the supernatant was kept to measure for volume, pH value and acidity. Esophageal tissues were isolated to monitor the morphological changes through hematoxylin-eosin (HE) staining, and esophageal epithelial ultrastructure was observed by transmission electron microscopy. The expressions of nuclear factor kappa-light-chain-enhancer of activated B cells p65 (NF-KBp65), κB kinase beta (IKKß), occludin, and zonula occludens-1 (ZO-1) in the esophageal tissues were measured by immunohistochemistry and Western blot, respectively. RESULTS: The gastric pH value in the model group was significantly lower than the sham group (P<0.05). Compared with the model group, gastric pH value in the omeprazole and medium-dose of Tojapride groups were significantly higher (P<0.05). A large area of ulceration was found on the esophageal mucosa from the model rats, while varying degrees of congestion and partially visible erosion was observed in the remaining groups. Remarkable increase in cell gap width and decrease in desmosome count was seen in RE rats and the effect was reversed by Tojapride treatment. Compared with the sham group, the IKKß levels were significantly higher in the model group (P<0.05). However, the IKKß levels were down-regulated after treatment by all doses of Tojapride (P<0.01 or P<0.05). The occluding and ZO-1 levels decreased in the model group compared with the sham group (Ps0.01 or Ps0.05), while both indices were significantly up-regulated in the Tojapride-treated groups (P<0.01 or P<0.05). CONCLUSIONS Tojapride could improve the pathological conditions of esophageal epithelium in RE rats. The underlying mechanisms may involve in down-regulating the IKKß expression and elevating ZO-1 and occludin expression, thereby alleviating the inflammation of the esophagus and strengthening the barrier function of the esophageal epithelium.

Objective: To evaluate the safety and effectiveness of a vaccine based on latent membrane protein 2 (LMP2) modified dendritic cells (DCs) that boosts specific responses of cytotoxic T lymphocytes (CTLs) to LMP2 before and after intradermal injection in patients with nasopharyngeal carcinoma (NPC). Methods: DCs were derived from peripheral blood monocytes of patients with NPC. We prepared LMP2-DCs infected by recombinant adenovirus vector expressing LMP2 (rAd-LMP2). NPC patients were immunized with 2 × 10 Results: We demonstrated that DCs derived from monocytes displayed typical DC morphologies; the expression of LMP2 in the LMP2-DCs vaccine was confirmed by immunocytochemical assay. Twenty-nine patients with NPC were enrolled in this clinical trial. The LMP2-DCs vaccine was well tolerated in all of the patients. Boosted responses to LMP2 peptide sub-pools were observed in 18 of the 29 patients with NPC. The follow-up data of 29 immunized patients from April, 2010 to April 2015 indicated a five-year survival rate of 94.4% in responders and 45.5% in non-responders. Conclusion In this pilot study, we demonstrated that the LMP2-DCs vaccine is safe and effective in patients with NPC. Specific CTLs responses to LMP2 play a certain role in controlling and preventing the recurrence and metastasis of NPC, which warrants further clinical testing.
Adult ; Aged ; Cancer Vaccines/therapeutic use* ; China ; Dendritic Cells/immunology* ; Female ; Humans ; Immunotherapy/methods* ; Injections, Intradermal ; Male ; Middle Aged ; Nasopharyngeal Carcinoma/therapy* ; Nasopharyngeal Neoplasms/therapy* ; T-Lymphocytes, Cytotoxic/immunology* ; Viral Matrix Proteins/therapeutic use* ; Young Adult

OBJECTIVE: To study two novel CD36 gene mutations at the CD36 splicing sites found in Guangxi population, as well as the molecular basis and population incidence of them. METHODS: DNA sequencing and cDNA clonal sequencing were used to detect CD36 exon sequence and the protein coding region sequence of CD36 mRNA for 2 CD36 deficient individuals (HHC and WGM) found in Guangxi population. Eukaryotic expression cell lines were established for the discovery of CD36 mRNA abnormal transcripts and Western blot assay was used to verify the effect of abnormal CD36 mRNA transcripts on CD36 expression. A DNA PCR-SSP genotyping method was established for the two CD36 novel mutations, and the population distribution was investigated among 110 CD36 deficient individuals in Guangxi region and 296 random individuals in Guangxi population. RESULTS: Novel mutation of c.430 -1G>C was found at the CD36 splicing site in HHC and WGM individuals, and novel mutation of c.1006 +2T>G at the CD36 splicing site was also found in the WGM individual. CD36 cDNA clonal sequencing showed that CD36 c.430 -1G>C could lead to the production of the two CD36 mRNA transcript variants: c.429_430ins［430-17_430-2;C］(p.Ala144fsTer1) and c.430_609del(p.Ala144_Pro203del)(GenBank:HM 217023.1); and CD36 c.1006 +2T>G could lead to the production of CD36 mRNA transcript variant of c.819_1006 del (p.Ser274GlufsTer16) (GenBank: HM217025.1). It was verified that all the three transcript variants could lead to CD36 deficiency by establishment of eukaryotic expression cell lines and Western blot assay. A study of the population incidence of two novel CD36 splicing site mutations found showed that in 110 CD36 deficient individuals and in 296 random individuals in Guangxi region, the mutation rate of CD36 c.430 -1G>C was 10.91% (12/110) and 1.35% (4/296), respectively, while CD36 c.1006 +2T>G was 2.73% (3/110) and 0 (0/296), respectively. CONCLUSION This study identifies two novel CD36 mutations at CD36 splicing site, and preliminary clarified their molecular basis for the CD36 deficiency and the distribution characteristics in Guangxi population as well. It provides an experimental and theoretical basis for studying the molecular mechanism and characteristics of CD36 deficiency in Chinese population.
Blood Platelet Disorders ; China ; Genetic Diseases, Inborn ; Humans ; Mutation ; RNA Splicing