A recombinant adenovirus(rAd)for expression of Mycobacterium tuberculosis(M.tb)c-di-AMP phosphodiesterase CnpB was constructed,and its induced humoral immune response was detected.The codon-optimized gene of M.tb CnpB was cloned into the adenoviral plasmid pcADV.The recombinant plasmid pcADV-CnpB was transfected into HEK293T cells,and expression was detected with Western blot.The recombinant plasmid pcADV-CnpB and the backbone plasmid were co-transfected into HEK293T cells to obtain the recombinant adenovirus rAd-CnpB.rAd-CnpB was amplified in HEK293T cells,and the target protein expression of rAd-CnpB was detected with Western blot and immunofluorescence.Mice were immunized with rAd-CnpB intranasally,and their sera and bronchoalveolar lavage fluid(BALF)were collected.ELISA was used to detect levels of antigen-specific antibodies.Restriction enzyme digestion and sequencing indicated that the recombinant plasmid pcADV-CnpB was successfully constructed and led to protein expression in eukaryotic cells.rAd-CnpB was packaged and produced in HEK293T cells.After amplification and purification,rAd-CnpB with a titer of 5.53×1010 PFU/mL was obtained.rAd-CnpB led to CnpB expression in HEK293T cells.Intranasal immunization with rAd-CnpB increased levels of IgG and secretory IgA in BALF and led to high levels of IgG in sera.rAd-CnpB,the recombinant adenovirus for expression of c-di-AMP phosphodiesterase CnpB was successfully constructed,and was found to induce antigen-specific humoral and mucosal immune responses through mucosal immunization.Thus,rAd-CnpB may be used in further research on new TB vaccine strategies.

A recombinant adenovirus(rAd)for expression of Mycobacterium tuberculosis(M.tb)c-di-AMP phosphodiesterase CnpB was constructed,and its induced humoral immune response was detected.The codon-optimized gene of M.tb CnpB was cloned into the adenoviral plasmid pcADV.The recombinant plasmid pcADV-CnpB was transfected into HEK293T cells,and expression was detected with Western blot.The recombinant plasmid pcADV-CnpB and the backbone plasmid were co-transfected into HEK293T cells to obtain the recombinant adenovirus rAd-CnpB.rAd-CnpB was amplified in HEK293T cells,and the target protein expression of rAd-CnpB was detected with Western blot and immunofluorescence.Mice were immunized with rAd-CnpB intranasally,and their sera and bronchoalveolar lavage fluid(BALF)were collected.ELISA was used to detect levels of antigen-specific antibodies.Restriction enzyme digestion and sequencing indicated that the recombinant plasmid pcADV-CnpB was successfully constructed and led to protein expression in eukaryotic cells.rAd-CnpB was packaged and produced in HEK293T cells.After amplification and purification,rAd-CnpB with a titer of 5.53×1010 PFU/mL was obtained.rAd-CnpB led to CnpB expression in HEK293T cells.Intranasal immunization with rAd-CnpB increased levels of IgG and secretory IgA in BALF and led to high levels of IgG in sera.rAd-CnpB,the recombinant adenovirus for expression of c-di-AMP phosphodiesterase CnpB was successfully constructed,and was found to induce antigen-specific humoral and mucosal immune responses through mucosal immunization.Thus,rAd-CnpB may be used in further research on new TB vaccine strategies.

The purpose of this study was to investigate the intervention effect and mechanism of Lycium barbarum leaves on letrozole-induced polycystic ovary syndrome (PCOS) mice. The PCOS model was prepared by letrozole combined with high-fat diet. After successful modeling, 40 mice were randomly divided into PCOS group, positive drug metformin group, low-dose Lycium barbarum leaves group, and high-dose Lycium barbarum leaves group. The corresponding drugs were given by gavage for 29 days. At the end of the experiment, the eyeballs were removed for blood collection and ovarian tissue was collected. The ovarian mass, fasting blood glucose (FBG), fasting insulin (FINS), testosterone (T), anti-Mullerian hormone (AMH), luteinizing hormone (LH), follicle stimulating hormone (FSH), and estradiol (E₂) levels were measured in each group. The morphology of ovarian tissue was observed by hematoxylin-eosin staining, and the oocytes, cystic follicles and corpus luteum were counted. Cecal contents of mice were collected for analysis of intestinal flora composition and differential flora. The animal experiment process was approved by the Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine. The results showed that the estrous cycle of PCOS mice was disordered. Compared with the PCOS group, the Lycium barbarum leaves group can significantly reduce the ovarian damage of mice, reduce the number of cystic dilated follicles, and normalize the estrous cycle. After the intervention of Lycium barbarum leaves, the levels of FBG, FINS, T, AMH, LH, FSH and LH/FSH were significantly decreased (P < 0.05), while the level of E₂ was significantly increased (P < 0.001). In addition, Lycium barbarum leaves can regulate the disorder of intestinal flora diversity in PCOS mice, increase the abundance of Bacteroidetes, and reduce the abundance of Firmicutes, Ileibacterium, Romboutsia and Faecalibaculum. In summary, Lycium barbarum leaves can play a therapeutic role in PCOS mice by improving insulin resistance, regulating reproductive hormone disorders and gut microbiota imbalance. It provides scientific basis and useful reference for the rational utilization and development of Lycium barbarum leaves.

OBJECTIVES: To investigate the incidence of extrauterine growth retardation (EUGR) and its risk factors in very preterm infants (VPIs) during hospitalization in China. METHODS: A prospective multicenter study was performed on the medical data of 2 514 VPIs who were hospitalized in the department of neonatology in 28 hospitals from 7 areas of China between September 2019 and December 2020. According to the presence or absence of EUGR based on the evaluation of body weight at the corrected gestational age of 36 weeks or at discharge, the VPIs were classified to two groups: EUGR group (n=1 189) and non-EUGR (n=1 325). The clinical features were compared between the two groups, and the incidence of EUGR and risk factors for EUGR were examined. RESULTS: The incidence of EUGR was 47.30% (1 189/2 514) evaluated by weight. The multivariate logistic regression analysis showed that higher weight growth velocity after regaining birth weight and higher cumulative calorie intake during the first week of hospitalization were protective factors against EUGR (P<0.05), while small-for-gestational-age birth, prolonged time to the initiation of total enteral feeding, prolonged cumulative fasting time, lower breast milk intake before starting human milk fortifiers, prolonged time to the initiation of full fortified feeding, and moderate-to-severe bronchopulmonary dysplasia were risk factors for EUGR (P<0.05). CONCLUSIONS It is crucial to reduce the incidence of EUGR by achieving total enteral feeding as early as possible, strengthening breastfeeding, increasing calorie intake in the first week after birth, improving the velocity of weight gain, and preventing moderate-severe bronchopulmonary dysplasia in VPIs.
Female ; Fetal Growth Retardation ; Gestational Age ; Hospitalization ; Humans ; Incidence ; Infant ; Infant, Newborn ; Infant, Premature ; Infant, Very Low Birth Weight ; Prospective Studies ; Risk Factors

Dozens of genes are associated with idiopathic hypogonadotropic hypogonadism (IHH) and an oligogenic etiology has been suggested. However, the associated genes may account for only approximately 50% cases. In addition, a genomic systematic pedigree analysis is still lacking. Here, we conducted whole exome sequencing (WES) on 18 unrelated men affected by IHH and their corresponding parents. Notably, one reported and 10 novel variants in eight known IHH causative genes (AXL, CCDC141, CHD7, DMXL2, FGFR1, PNPLA6, POLR3A, and PROKR2), nine variants in nine recently reported candidate genes (DCAF17, DCC, EGF, IGSF10, NOTCH1, PDE3A, RELN, SLIT2, and TRAPPC9), and four variants in four novel candidate genes for IHH (CCDC88C, CDON, GADL1, and SPRED3) were identified in 77.8% (14/18) of IHH cases. Among them, eight (8/18, 44.4%) cases carried more than one variant in IHH-related genes, supporting the oligogenic model. Interestingly, we found that those variants tended to be maternally inherited (maternal with n = 17 vs paternal with n = 7; P = 0.028). Our further retrospective investigation of published reports replicated the maternal bias (maternal with n = 46 vs paternal with n = 28; P = 0.024). Our study extended a variant spectrum for IHH and provided the first evidence that women are probably more tolerant to variants of IHH-related genes than men.

Berberine(BBR)is an isoquinoline alkaloid extracted from Coptis chinensis that improves diabetes,hyperlipidemia and inflammation.Due to the low oral bioavailability of BBR,its mechanism of action is closely related to the gut microbiota.This study focused on the CYP51 enzyme of intestinal bacteria to elucidate a new mechanism of BBR transformation by demethylation in the gut microbiota through multiple analytical techniques.First,the docking of BBR and CYP51 was performed;then,the pharma-cokinetics of BBR was determined in ICR mice in vivo,and the metabolism of BBR in the liver,kidney,gut microbiota and single bacterial strains was examined in vitro.Moreover,16S rRNA analysis of ICR mouse feces indicated the relationship between BBR and the gut microbiota.Finally,recombinant E.coli con-taining cyp51 gene was constructed and the CYP51 enzyme lysate was induced to express.The metabolic characteristics of BBR were analyzed in the CYP51 enzyme lysate system.The results showed that CYP51 in the gut microbiota could bind stably with BBR,and the addition of voriconazole(a specific inhibitor of CYP51)slowed down the metabolism of BBR,which prevented the production of the demethylated metabolites thalifendine and berberrubine.This study demonstrated that CYP51 promoted the deme-thylation of BBR and enhanced its intestinal absorption,providing a new method for studying the metabolic transformation mechanism of isoquinoline alkaloids in vivo.

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipe-line and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to gen-erate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.

OBJECTIVE: To retrospectively analyze the results of treatment outcome by surgery combined with ₁₂₅I brachytherapy and correlative factors of adenoid cystic carcinoma (ACC). METHODS: In the study, 75 patients with primary ACC of oral and maxillofacial region were treated by surgery combined with ₁₂₅I seeds brachytherapy. Radical resection or subtotal resection was applied for the tumor. The brachytherapy treatment planning system was used to create implant plans with the prescribed dose of 60 Gy to 120 Gy. The ₁₂₅I seeds were implanted intraoperatively or postoperatively. The regular follow-up was required. The Kaplan-Meier method was used to assess the tumor control rate and the patients' survival rates. Meanwhile, the Cox regression analysis was used to find out the prognostic factors. RESULTS: Local control rates at the end of 3 and 5 years were as follows: T1-T2, 92.2% and 82.0%; T3-T4, 82.6% and 82.6%; and overall, 90.0% and 78.8%. The disease-free survival rates were 74.9% and 54.3%, respectively. The overall survival rates for all the patients were 86.0% and 79.6%, respectively at the end of 3 and 5 years and were 91.3% and 91.3% for T1-T2 patients vs. 73.9% and 59.7% for T3-T4 patients. Distant metastasis-free survival rates at the end of 3 and 5 years were 84.4% and 76.7%, respectively. The distant metastasis-free survival rates at the end of 3 and 5 years were 83.4% and 79.6% with T1-T2 lesion compared with 86.0% and 67.8% with T3-T4 lesion. According to the COX univariate analysis and multivariate analysis, the risk of local recurrence would be raised by the age. Tumor stage and tumor site were the prognostic factors of the overall survival rates. CONCLUSION ₁₂₅I brachytherapy conducted as an adjuvant therapy postoperatively of ACC of oral and maxillofacial region can acquire satisfactory localregional control, distant metastasis-free survival, disease-free survival and overall survival. Tumors are prone to recur on the older patients. Patients having advanced tumor stage or tumor located in the nasal cavity or sinuses will suffer lower survival rates.
Brachytherapy ; Carcinoma, Adenoid Cystic ; Combined Modality Therapy ; Humans ; Iodine Radioisotopes ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Retrospective Studies ; Survival Rate

OBJECTIVE: To evaluate the effectiveness of integrative medicine (IM) on patients with coronary artery disease (CAD) and investigate the prognostic factors of CAD in a real-world setting. METHODS: A total of 1,087 hospitalized patients with CAD from four hospitals in Beijing, China were consecutively selected between August 2011 and February 2012. The patients were assigned to two groups based on the treatment: Chinese medicine (CM) plus conventional treatment, i.e., IM therapy (IM group); or conventional treatment alone (CT group). The endpoint was major adverse cardiac events [MACE; including cardiac death, myocardial infarction (MI), and revascularization]. RESULTS: A total of 1,040 patients finished the 2-year follow-up. Of them, 49.4% (514/1,040) received IM therapy. During the 2-year follow-up, the total incidence of MACE was 11.3%. Most of the events involved revascularization (9.3%). Cardiac death/MI occurred in 3.0% of cases. For revascularization, logistic stepwise regression analysis revealed that age ⩾ 65 years [odds ratio (OR), 2.224], MI (OR, 2.561), diabetes mellitus (OR, 1.650), multi-vessel lesions (OR, 2.554), baseline high sensitivity C-reactive protein level ⩾ 3 mg/L (OR, 1.678), and moderate or severe anxiety/depression (OR, 1.849) were negative predictors (P<0.05); while anti-platelet agents (OR, 0.422), β-blockers (OR, 0.626), statins (OR, 0.318), and IM therapy (OR, 0.583) were protective predictors (P<0.05). For cardiac death/MI, age ⩾ 65 years (OR, 6.389) and heart failure (OR, 7.969) were negative predictors (P<0.05), while statin use (OR, 0.323) was a protective predictor (P<0.05) and IM therapy showed a beneficial tendency (OR, 0.587), although the difference was not statistically significant (P=0.218). CONCLUSION In a real-world setting, for patients with CAD, IM therapy was associated with a decreased incidence of revascularization and showed a potential benefit in reducing the incidence of cardiac death or MI.
Aged ; Coronary Artery Disease ; drug therapy ; Female ; Humans ; Integrative Medicine ; Logistic Models ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Prognosis

BACKGROUND: The recombinant adenovirus has certain toxic reactions to cell growth and survival, but the determination of the optimal multiplicity of infection (MOI) is not well documented. OBJECTIVE: To study the effectiveness and toxicity of transfection of bone marrow mesenchymal stem cells (BMSCs) by adenovirus vectors with different MOIs, and to explore the effect on osteogenic capability of the cells. METHODS: BMSCs at passage 5 were infected with enhanced green fluorescent protein (EGFP) via adenovirus vectors with different MOI (0, 50, 100, 150, 200 and 250). Cell morphology and mortality rate were observed and calculated using trypan blue staining method at 24 hours after cell transfection. The transfection efficiency and the relative mRNA expression of EGFP and Runx2 were analyzed respectively by inverted florescent microscopy and real-time quantitative PCR at 48 hours after cell transfection. The activity of BMSCs transfected with different MOIs was evaluated by MTT, and the optimal MOI was determined thereafter. RESULTS AND CONCLUSION: With the increase of MOI, BMSCs showed decreased adherent ability, and even some cells aged and died. The mortality rate of BMSCs transfected for 24 hours at a MOI of 0 to 250 was 5.80%, 6.67%, 7.95%, 7.76%, 10.35% and 11.18%, respectively, indicating that the mortality rate of BMSCs is positively correlated with the MOI. The transfection efficiency changed insignificantly when the MOI was greater than 100. On the contrary, the cellular viability and osteogenic differentiation capability of rabbit BMSCs were receded when the MOI level was up to 200-250. The study discovered that the suitable scope of MOI to transfect BMSCs is 50-150, and the optimal MOI is 100.