This study was aimed at creating an engineered strain of Bacillus subtilis for efficient expression of biologically active type 2 toxin B(TcdB2)derived from a highly virulent strain of Clostridium difficile.The TcdB2 gene was cloned from ST1/RT027 strain genome DNA,incorporated into the PHT01 vector,and then transformed into B.subtilis strain WB800N for prokaryotic expression.Cell toxicity assays revealed that the recombinant TcdB2 exhibited cytotoxic effects in various cells.The engineered B.subtilis strain effectively expressed biologically active TcdB2,thus providing a basis for further exploration of the pathogenic mechanisms of highly virulent strains of C.difficile and establishing a foundation for potential vaccine can-didate targets.

Objective:To detect the pharmacokinetic(PK)parameters of coagulation factor Ⅷ(FⅧ)in adult patients with severe hemophilia A,identify the potential factors influencing FⅧ PK,and optimize the use of FⅧ in individual prophylaxis regimens.Methods:PK characteristics of FⅧ were studied in a total of 23 severe hemophilia A adults.The correlation of patients'characteristics including age,von Willebrand factor antigen(vWF:Ag),blood group,weight,body mass index(BMI)and FⅧ genotype,with FⅧ PK were evaluated.Individual prophylaxis regimens were given based on FⅧ PK parameters.Results:The mean terminal half-life(t1/2)of FⅧ was 20.6±9.3 h,ranged from 11.47 h to 30.12 h.The age(r=0.580)and vWF:Ag(r=0.814)were significantly positively correlated with t1/2 of FⅧ.The mean area under the plasma concentration curve(AUC)of FⅧ was 913±399(328-1 878)IU h/dl,and the AUC of FⅧ was positively correlated with age(r=0.557)and vWF:Ag(r=0.784).The mean residence time(MRT)of FⅧ was 24.7±12.4(13.2-62.2)h,and the MRT of FⅧ was positively correlated with age(r=0.664)and vWF:Ag(r=0.868).The mean in vivo recovery(IVR)of FⅧ was 2.59±0.888(1.5-4.29)IU/dl per IU/kg,the mean clearance(CL)of FⅧ was 3±1.58(0.97-7.18)ml/(kg·h),and there was no significant correlation of IVR and CL with age and vWF:Ag.According to the individual PK parameters,ultra low-dose,low-dose and moderate-dose FⅧ were applied to 15,6,2 adults patients with severe hemophilia A for prophylaxis,respectively.Conclusion:There are significant individual differences in the FⅧ half-life of adult patients with severe hemophilia A.The older the patient,the higher the vWF:Ag level,and the longer the FⅧ half-life.Individual administration is required based on the FⅧ PK parameters to optimize prophylaxis treatment.

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannan-binding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP. Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays. Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05). Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

The blood-brain barrier (BBB) plays a crucial role in maintaining the homeostasis of the brain's internal environment, which poses challenges to the treatment of central nervous system diseases. Drug carriers can aid in the delivery of therapeutic agents across the BBB to exert their pharmacological effects. The article reviewed the pathways for drug delivery across the BBB, the intracerebral fate and the classification of drug carriers and focuses on the functions and characteristics of liposomes, exosomes, apoptotic bodies, cell-penetrating peptides, and cell-targeting peptides. The review will provide an outlook on the future and challenge of research in the field of drug delivery across the BBB.

Objective: To explore the characteristics and correlations of vaginal flora in women with cervical lesions. Methods: A total of 132 women, including 41 women diagnosed with normal cervical (NC), 39 patients with low-grade cervical intraepithelial neoplasia (CIN 1), 37 patients with high-grade cervical intraepithelial neoplasia (CIN 2/3) and 15 patients with cervical squamous cell carcinoma (SCC), who came from the gynecological clinic of Second Hospital of Shanxi Medical University during January 2018 to June 2018, were enrolled in this study according to the inclusive and exclusive criteria strictly. The vaginal flora was detected by 16S rDNA sequencing technology. Co-occurrence network analysis was used to investigate the Spearman correlations between different genera of bacteria. Results: The dominant bacteria in NC, CIN 1 and CIN 2/3 groups were Lactobacillus [constituent ratios 79.4% (1 869 598/2 354 098), 63.6% (1 536 466/2 415 100) and 58.3% (1 342 896/2 301 536), respectively], while Peptophilus [20.4% (246 072/1 205 154) ] was the dominant bacteria in SCC group. With the aggravation of cervical lesions, the diversity of vaginal flora gradually increased (Shannon index: F=6.39, P=0.001; Simpson index: F=3.95, P=0.012). During the cervical lesion progress, the ratio of Lactobacillus gradually decreased, the ratio of other anaerobes such as Peptophilus, Sneathia, Prevotella and etc. gradually increased, and the differential bacteria (LDA score >3.5) gradually evolved from Lactobacillus to other anaerobes. The top 10 relative abundance bacteria, spearman correlation coefficient>0.4 and P<0.05 were selected. Co-occurrence network analysis showed that Prevotella, Peptophilus, Porphyrinomonas, Anaerococcus, Sneathia, Atopobium, Gardnerella and Streptococcus were positively correlated in different stages of cervical lesions, while Lactobacillus was negatively correlated with the above anaerobes. It was found that the relationship between vaginal floras in CIN 1 group was the most complex and only Peptophilus was significantly negatively correlated with Lactobacillus in SCC group. Conclusions: The increased diversity and changed correlations between vaginal floras are closely related to cervical lesions. Peptophilus is of great significance in the diagnosis, prediction and early warning of cervical carcinogenesis.
Female ; Humans ; Vagina/microbiology* ; Uterine Cervical Neoplasms/genetics* ; Uterine Cervical Dysplasia ; Cervix Uteri ; Lactobacillus/genetics* ; Papillomavirus Infections

OBJECTIVE: This study was aimed at investigating the carrier rate of, and molecular variation in, α- and β-globin gene mutations in Hunan Province. METHODS: We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province. Hematological screening was performed, and molecular parameters were assessed. RESULTS: The overall carrier rate of thalassemia was 7.1%, including 4.83% for α-thalassemia, 2.15% for β-thalassemia, and 0.12% for both α- and β-thalassemia. The highest carrier rate of thalassemia was in Yongzhou (14.57%). The most abundant genotype of α-thalassemia and β-thalassemia was -α ^3.7/αα (50.23%) and β ^IVS-II-654/β ^N (28.23%), respectively. Four α-globin mutations [CD108 (ACC>AAC), CAP +29 (G>C), Hb Agrinio and Hb Cervantes] and six β-globin mutations [CAP +8 (C>T), IVS-II-848 (C>T), -56 (G>C), beta nt-77 (G>C), codon 20/21 (-TGGA) and Hb Knossos] had not previously been identified in China. Furthermore, this study provides the first report of the carrier rates of abnormal hemoglobin variants and α-globin triplication in Hunan Province, which were 0.49% and 1.99%, respectively. CONCLUSION Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population. The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.
Humans ; beta-Thalassemia/genetics* ; alpha-Thalassemia/genetics* ; Hemoglobinopathies/genetics* ; China/epidemiology* ; High-Throughput Nucleotide Sequencing

In this study, the Web of Science and China National Knowledge Infrastructure(CNKI) were searched comprehensively for the literature about the research on Polygalae Radix. After manual screening, 1 207 Chinese articles and 263 English articles were included in this study. Excel was used to draw the line chart of the annual number of relevant publications. CiteSpace 6.1.R3 was used for the visual analysis of author cooperation, publishing institutions, keyword co-occurrence, keyword clustering, and bursts in the research on Polygalae Radix. The results showed that the number of articles published in Chinese and English increased linearly, which indicated the rising research popularity of Polygalae Radix. WANG J and LIU X were the authors publishing the most articles in Chinese and English, respectively. Shanxi University of Chinese Medicine and Chinese Academy of Medical Sciences were the research institutions with the largest number of Chinese and English publications in this field, respectively. The institutions publishing the relevant articles in English formed a system with the Chinese Academy of Medical Sciences as the core. According to the keywords, the research hotspots of Polygalae Radix included variety selection and breeding, quality standard, extraction and identification of active chemical components, prescription compatibility, processing, clinical medication rules, and pharmacological mechanism. The research frontiers were the molecular mechanisms of Polygalae Radix and its active components in exerting the protective effect on brain nerve, regulating receptor pathways, alleviating anxiety and Alzheimer's disease, as well as data mining and clinical medication summary. This study has reference significance for the topic selection and frontier identification of the future research on Polygalae Radix.
Plant Breeding ; China ; Plant Roots/chemistry* ; Brain ; Publications

Objective:To investigate the clinical effects and related mechanism of antibiotic bone cement in treating diabetic foot ulcer (DFU).Methods:A prospective randomized controlled study was conducted. From August 2020 to August 2022, 24 patients with DFU who met the inclusion criteria were admitted to the First Affiliated Hospital of Air Force Medical University. According to the block randomization, the patients were divided into 2 groups, with 12 patients in each group. In antibiotic bone cement group, there were 7 male and 5 female patients, aged (64±8) years, with the ulcer area of (41±21) cm 2. In silver sulfadiazine group, there were 8 male and 4 female patients, aged (62±8) years, with the ulcer area of (38±19) cm 2. Under the condition of ensuring the patency of at least one main inferior genicular artery in each patient, the continuous vacuum sealing drainage was performed for 3-5 days after thorough debridement. Thereafter, the wounds in antibiotic bone cement group were treated with gentamicin-laden bone cement, and the wounds in silver sulfadiazine group were treated with silver sulfadiazine cream for dressing change. After 3 weeks of dressing change, the wound was covered with split-thickness skin graft from the lateral thigh on the affected side. Before debridement and after 3 weeks of dressing change, the blood flow intensities of wound tissue and normal skin tissue in foot were measured using laser Doppler flowmeter, and then, the percentage of relative blood flow intensity of wound and the change rate of blood flow intensity were calculated. After 3 weeks of dressing change, the wound margin tissue was taken, the number of CD31-positive neovascular and the vascular morphology were observed and detected by immunohistochemical staining, the morphology of blood vessels surrounded by CD31 and α-smooth muscle actin (α-SMA) double-positive cells was observed by immunofluorescence staining, the cell proliferation activity was evaluated by immunofluorescence staining (denoted as the ratio of Ki67 positive cells), and the protein expression of vascular endothelial growth factor receptor 2 (VEGFR2) was detected by Western blotting. The skin graft survival was observed 3-5 days after skin grafting, and the wound healing time was recorded. Data were statistically analyzed with independent sample t test and Fisher's exact probability test. Results:The percentages of relative blood flow intensity of wounds of patients before debridement were similar between the two groups ( P>0.05). After 3 weeks of dressing change, the percentage of relative blood flow intensity of wounds and the change rate of blood flow intensity of patients in antibiotic bone cement group were (44.7±2.0)% and (129±12)%, respectively, which were significantly higher than (28.3±1.2)% and (41±8)% in silver sulfadiazine group (with t values of 24.15 and 20.97, respectively, P<0.05). After 3 weeks of dressing change, compared with those in silver sulfadiazine group, the number of CD31-positive neovascular in the wound margin tissue of patients in antibiotic bone cement group was significantly increased ( t=33.81, P<0.05) with larger diameter and more regular arrangement, the vascular wall continuity surrounded by CD31 and α-SMA double-positive cells was better, and the ratio of Ki67 positive cells and protein expression of VEGFR2 were significantly increased (with t values of 40.97 and 47.38, respectively, P<0.05). On post skin grafting day 3-5, all the patients in antibiotic bone cement group and 8 patients in silver sulfadiazine group had good skin graft survival, while 4 patients in silver sulfadiazine group showed spotted/patchy skin graft necrosis, which were cured after corresponding treatment. The wound healing time of patients in antibiotic bone cement group was (47.1±2.9) d, which was significantly shorter than (58.8±2.3) d in silver sulfadiazine group ( t=10.86, P<0.05). Conclusions:Compared with silver sulfadiazine, clinical application of antibiotic bone cement for treating DFU has the characteristics of accelerating wound healing and better reconstruction of local blood flow, which may be closely related to the fact that antibiotic bone cement promoted the local angiogenesis effectively in the wound through enhancing the expression of VEGFR2.

Primary familial brain calcification (PFBC) is an inherited neurodegenerative disorder mainly characterized by progressive calcium deposition bilaterally in the brain, accompanied by various symptoms, such as dystonia, ataxia, parkinsonism, dementia, depression, headaches, and epilepsy. Currently, the etiology of PFBC is largely unknown, and no specific prevention or treatment is available. During the past 10 years, six causative genes (SLC20A2, PDGFRB, PDGFB, XPR1, MYORG, and JAM2) have been identified in PFBC. In this review, considering mechanistic studies of these genes at the cellular level and in animals, we summarize the pathogenesis and potential preventive and therapeutic strategies for PFBC patients. Our systematic analysis suggests a classification for PFBC genetic etiology based on several characteristics, provides a summary of the known composition of brain calcification, and identifies some potential therapeutic targets for PFBC.
Animals ; Brain Diseases/therapy* ; Xenotropic and Polytropic Retrovirus Receptor ; Brain/pathology*