Molecular characteristics of Acinetobacter baumannii and the changes of efflux pump expression under tigecycline pressure / 医学研究生学报

Objective　Acinetobacter baumannii (A. baumannii) is a commonly infective bacterium in the hospital. This study aims to analyze its molecular epidemiological characteristics, detect the carrying rate of efflux pump and regulatory protein genes, and investigate the effects of tigecycline on the efflux pump and expression of regulatory protein genes.　Methods　A total of 183 A. baumannii strains were collected from inpatients of the affiliated hospital of Jiangsu University from May 2017 to March 2019. They were divided into an antimicrobial-resistant group (one or more antimicrobial-resistant strains, 139 strains) and a sensitive group (the drugs in the drug sensitivity test were all non-resistant strains, 44 strains). Repeated sequence PCR was used for homology analysis of the strains, and pulse-field gel electrophoresis (PFGE) was used as the gold standard for homology analysis to verify and compare some strains. PCR was used to detect the occurrence of drug resistance-related genes. Based on homology analysis, efflux pump carrying rate detection and antibiotics sensitivity test results, 6 clinical strains carrying all efflux pump genes but different resistance phenotypes were selected as experimental strains, including sensitive strains (SAB), the multidrug resistance strain (MDRAB) and the extensively drug-resistant strain (XDRAB). All strains were induced in vitro with the minimum inhibitory concentration (MIC) of tigecycline. The induced strains were categorized as induction group, and the same strains cultured in LB agar without tigecycline was used as a control group. MIC was used to analyze the tigecycline susceptibility, and RT-qPCR was used to detect the gene expression of efflux pumps, such as TetB, AbaQ and regulatory proteins (AdeS and BaeS), in drug-resistant strains.　Results　Homology analysis showed that there were 45 clonal groups in the detected clinical isolates, with no obvious outbreak of epidemic clonal groups. Efflux pumps and regulatory proteins were widely distributed in the clinical isolates, and the expression of AdeB, TetB, AbeS, AdeS in MDRAB and XDRAB is significantly higher than that insensitive group SAB. Continuous in vitro induction with tigecycline could increase the antimicrobial resistance of some clinical strains and even significantly increase the expression levels of efflux pumps and regulatory proteins.　Conclusion　A. baumannii is widely distributed in the clinic, and efflux pumps and regulatory proteins might play an important role in drug resistance process. The unreasonable use of tigecycline could enhance the tolerance of A. baumannii by up-regulating the expression of some bacterial efflux pumps.

Tau-mediated Neurodegeneration and Potential Implications in Diagnosis and Treatment of Alzheimer's Disease / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To review recent research advances on tau, a major player in Alzheimer's disease (AD) pathogenesis, a biomarker for AD onset, and potential target for AD therapy.</p><p><b>DATA SOURCES</b>This review was based on a comprehensive search using online literature databases, including PubMed, Web of Science, and Google Scholar.</p><p><b>STUDY SELECTION</b>Literature search was based on the following keywords: Alzheimer's disease, tau protein, biomarker, cerebrospinal fluid (CSF), therapeutics, plasma, imaging, propagation, spreading, seeding, prion, conformational templating, and posttranslational modification. Relevant articles were carefully reviewed, with no exclusions applied to study design and publication type.</p><p><b>RESULTS</b>Amyloid plaques enriched with extracellular amyloid beta (Aβ) and intracellular neurofibrillary tangles comprised of hyperphosphorylated tau proteins are the two main pathological hallmarks of AD. Although the Aβ hypothesis has dominated AD research for many years, clinical Aβ-targeting strategies have consistently failed to effectively treat AD or prevent AD onset. The research focus in AD has recently shifted to the role of tau in AD. In addition to phosphorylation, tau is acetylated and proteolytically cleaved, which also contribute to its physiological and pathological functions. Emerging evidence characterizing pathological tau propagation and spreading provides new avenues for research into the molecular and cellular mechanisms underlying AD pathogenesis. Techniques to detect tau at minute levels in CSF and blood have been developed, and improved tracers have facilitated tau imaging in the brain. These advances have potential to accurately determine tau levels at early diagnostic stages in AD. Given that tau is a potential therapeutic target, anti-tau immunotherapy may potentially be a viable treatment strategy in AD intervention.</p><p><b>CONCLUSION</b>Detecting changes in tau and targeting tau pathology represent a promising lead in the diagnosis and treatment of AD.</p>

Expression of SLC25A38 in leukemic cells from children with acute lymphoblastic leukemia / 中国实验血液学杂志

This study was aimed to investigate the SLC25A38 expression in pediatric patients with acute lymphoblastic leukemia (ALL) and its clinical significance. A total of 23 newly diagnosed ALL pedictric patients were enrolled in test group, 10 pediatric patients with non-hematologic malignancies were selected as control group. The expression in protein and mRNA levels of SLC25A38 were detected by Western blot and real-time PCR respectively. The results showed that the SLC25A38 protein was positive in 8 of 23 pediatric ALL patients (34.78%), while no positive case was found in 10 controls. The relative expression level of SLC25A38 mRNA was 0.4673 ± 0.05344 in SLC25A38-protein positive group of ALL patients, while that was 1.296 ± 0.2517 in SLC25A38-protein negative group of ALL patients. The expression level of SLC25A38 mRNA in SLC25A38-protein positive group was significantly lower than that in negative group (P = 0.001) . No statistically significant difference was found in comparison of SLC25A38-protein negative group of ALL patients with the control group (P = 0.1097). The analysis of clinical data showed that there were significantly differences in sex, immunophenotype, initial peripheral white blood cell count and LDH between the SLC25A38-protein positive and SLC25A38-protein negative groups (P < 0.05). It is concluded that as a novel protein, SLC25A38 highly expressed in pediatric ALL patients, indicating that SLC25A38 may serve as a molecular marker and potential therapeutic target for acute lymphoblastic leukemia in children.

Immunoregulation effect of transmission of T-bet gene on T cell subgroups in asthmatic Balb/c mice / 中华实用儿科临床杂志

Objective To construct the adeno-associated virus (AAV)vector of mouse carrying T-bet gene,which was applied to mouse model of asthma by nose,and to investigate the immunomodulatory effects of T-bet delivery on asthma.Methods Forty healthy Balb/c mice (aged 6-8 weeks) were randomly divided into 4 groups:control group (group A),asthma model group(group B),model/control recombinant adeno-associated virus carrying enhanced green fluorescent protein(rAAV-eGFP) intervention group (group C) and model/rAAV-T-bet virus intervention group(group D),with 10 mice in each group.In group B,group C,and group D,Balb/c mice were sensitized by intraperitoneal injection with OVA and challenged by nebulized OVA.In group C and group D,Balb/c mice were intervened with the isodose rAAV-eGFP and rAAV-T-bet,while in group B,the mice were intervened with the same amount of saline,both of them were dropped into the nasal cavity.Twenty-four hours after the last injection,the mice were sacrificed and the samples were obtained.The total cells in bronchoalveolar lavage fluid (BALF) were counted,and the types of cells were analyzed by Wright-Giemsa staining.The levels of interferon-γ (IFN-γ),IL-4 and IL-5 in BALF were determined by enzyme-linked immunosorbent assay.The expressions of T-bet,GATA-3 and Foxp3 protein in the lungs of asthmatic mice were detected with immunohistochemical technique.Results 1.The level of specific transcription factor T-bet in group B and group C were distinctly lower than that of group A (all P ＜ 0.05),while the level of GATA-3 was significantly higher than that of group A(all P ＜ 0.05).In group D,the expression of T-bet protein was stronger than that in group B and group C,however,the expression of GATA-3 decreased obviously (all P ＜ 0.05).The results of Foxp3 were similar to T-bet.2.In group D,the mice had suppressed levels of IL-4 [(158 ± 55) ng/L] and IL-5 [(68-± 22) ng/L] compared with those in group B and group C(all P ＜0.05).The level of IFN-γ[(113-±35) ng/L] increased compared with those in group B and group C (all P ＜ 0.05).At the same time,the number of total cells and eosinophil in BALF were both obviously lower than those of group B and group C (all P ＜ 0.05).Conclusions On the basis of building mouse model of asthma,intranasal administration of rAAV-T-bet can deliver T-bet gene to airway successfully and efficiently,and plays an immunomodulatory role in immune confusion of asthma.

Expression of TLR8 in human cervical cancer HeLa cells and the effect of TLR8 agonist on the cell proliferation and apoptosis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the expression of Toll-like receptor 8 (TLR8) in human cervical cancer cell-line HeLa cells, and the effects of TLR8 agonist CL075 on the survival and proliferation of HeLa cells.</p><p><b>METHODS</b>PCR and RT-PCR were used to detect the expression of TLR8 in 13 cancer cell lines, and the expression of COX-2, Bcl-2, VEGF mRNA in the HeLa cells stimulated by TLR8 agonist CL075 were also measured by RT-PCR. Immunofluorescence technique was used to determine the exact location of TLR8 in the cells. The percentage of viable cells was determined by trypan blue exclusion after the HeLa cells were stimulated with TLR8 agonist CL075 (0.1 µg/ml, 0.5 µg/ml, 1.0 µg/ml, 2.5 µg/ml), and cell cycle and apoptosis were analyzed by flow cytometry, and the proliferation was measured by MTT.</p><p><b>RESULTS</b>Compared with the other cancer cell lines, the expression of TLR8 in HeLa cells was the highest (703.7 ± 20.6). After stimulation by CL075, the cells had a remarkable increase of the percentage of cells in G(2)/M + S phases. In the control group, the percentage of cells in G(2)/M +S phases was (39.02 ± 2.33)%, whereas after stimulated with 1.0 µg/ml CL075, the percentage of cells in G(2)/M + S phases reached the highest ratio (57.67 ± 1.73)%, and the percentage of cells in G(2)/M + S phases had a less decrease after 2.5 µg/ml CL075 stimulation and the percentage was (56.14 ± 3.73)%. After the CL075 treatment, there was no significant changes of apoptosis compared with that of the control cells (P > 0.05), but after DDP treatment the apoptosis had a significant change (P < 0.01). After stimulation by 1.0 µg/ml CL075 for 24 h, no significant difference (P > 0.05) was found by MTT test, but a significant difference was found at 48 h and 72 h (P < 0.01). An increased expression of COX-2, Bcl-2 and VEGF mRNA was observed in HeLa cells after stimulation by TLR8 agonist CL075 for 24 h and 48 h (P < 0.05).</p><p><b>CONCLUSIONS</b>Expression of TLR8 is significantly increased in HeLa cells. The proportion of cells at different phases has a significant change after CL075 stimulation, which may up-regulate the proliferation of HeLa cells. These data suggested that TLR8 agonist may influence the tumor development and TLR8 may become a potential target in the treatment for cervical cancer.</p>

Detection and significance of transcription factors and cytokines of Th17/Treg cells in peripheral blood in the gastric cancer patients / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect the expression levels of transcription factors and associated cytokines of Th17 and Treg cells in peripheral blood mononuclear cells (PBMC) of patients with gastric cancer, and explore the possible pathological mechanism of these cells involved in the development of gastric cancer.</p><p><b>METHODS</b>The mRNA levels of RORgammat, FoxP3 in PBMC were determined by quantitative real-time PCR (QRT-PCR) from 57 patients with gastric cancer, 31 patients with benign gastric illness and 40 healthy people. The concentration of IL-17, IL-23, TGF-beta, IL-10 in plasma were detected by enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Compared with healthy volunteers, patients with gastric cancer showed higher levels of RORgammat and FoxP3 in PBMC (P < 0.05). The ratio of FoxP3/RORgammat in gastric cancer group was higher than that in the volunteer group and benign gastric illness group (P < 0.05). The ratio of FoxP3/RORgammat was higher in advanced disease than early disease (P < 0.05). The expressions of IL-17, IL-23, TGF-beta and IL-10 were higher in patients with gastric cancer than that in healthy volunteers (P < 0.05). In addition, The expression of TGF-beta and IL-10 were significantly increased in the advanced disease group than that in the early group (P < 0.05), but IL-17 and IL-23 was not significantly changed between the two groups (P > 0.05).</p><p><b>CONCLUSION</b>There are higher levels of Th17 and Treg cells in gastric cancer patients, and it also shows a persistent predominant tendency of Treg cells and a reduced tendency of Th17 cells in advanced disease. Detecting the expression of Th17/Treg transcription factor and related cytokines would contribute to the diagnosis and prediction of the disease development and prognosis.</p>