Asenapine maleate inhibits angiotensin II-induced proliferation and activation of cardiac fibroblasts via the ROS/TGFß1/MAPK signaling pathway.

BACKGROUND: Cardiac fibrosis will increase wall stiffness and diastolic dysfunction, which will eventually lead to heart failure. Asenapine maleate (AM) is widely used in the treatment of schizophrenia. In the current study, we explored the potential mechanism underlying the role of AM in angiotensin II (Ang II)-induced cardiac fibrosis. METHODS: Cardiac fibroblasts (CFs) were stimulated using Ang II with or without AM. Cell proliferation was measured using the cell counting kit-8 assay and the Cell-Light EdU Apollo567 In Vitro Kit. The expression levels of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were detected using immunofluorescence or western blotting. At the protein level, the expression levels of the components of the transforming growth factor beta 1 (TGFß1)/mitogen-activated protein kinase (MAPK) signaling pathway were also detected. RESULTS: After Ang II stimulation, TGFß1, TGFß1 receptor, α-SMA, fibronectin (Fn), collagen type I (Col1), and collagen type III (Col3) mRNA levels increased; the TGFß1/MAPK signaling pathway was activated in CFs. After AM pretreatment, cell proliferation was inhibited, the numbers of PCNA -positive cells and the levels of cardiac fibrosis markers decreased. The activity of the TGFß1/MAPK signaling pathway was also inhibited. Therefore, AM can inhibit cardiac fibrosis by blocking the Ang II-induced activation through TGFß1/MAPK signaling pathway. CONCLUSIONS: This is the first report to demonstrate that AM can inhibit Ang II-induced cardiac fibrosis by down-regulating the TGFß1/MAPK signaling pathway. In this process, AM inhibited the proliferation and activation of CFs and reduced the levels of cardiac fibrosis markers. Thus, AM represents a potential treatment strategy for cardiac fibrosis.

Antimicrobial susceptibility of Streptococcus sp. to quinupristin-dalfopristin in China.

This study aimed to determine the in vitro activity of quinupristin-alfopristin against Streptococcus sp. isolated in China. This agent is not yet available for clinical use, but it has been tested against a high proportion of resistant Staphylococcus aureus strains. A total of 156 streptococcal isolates, which were recovered from various geographic areas and diseases, were tested using the Etest (AB Biodisk, Solna, Sweden). Quinupristin-alfopristin showed excellent activity against all of the tested streptococci isolates. These results provide useful data for the clinical use of quinupristin-alfopristin in China.

LDHA contributes to nicotine induced cardiac fibrosis through autophagy flux impairment.

Cardiac fibrosis is a typical feature of cardiac pathological remodeling, which is associated with adverse clinical outcomes and has no effective therapy. Nicotine is an important risk factor for cardiac fibrosis, yet its underlying molecular mechanism remains poorly understood. This study aimed to identify its potential molecular mechanism in nicotine-induced cardiac fibrosis. Our results showed nicotine exposure led to the proliferation and transformation of cardiac fibroblasts (CFs) into myofibroblasts (MFs) by impairing autophagy flux. Through the use of drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) technology, it was discovered that nicotine directly increased the stability and protein levels of lactate dehydrogenase A (LDHA) by binding to it. Nicotine treatment impaired autophagy flux by regulating the AMPK/mTOR signaling pathway, impeding the nuclear translocation of transcription factor EB (TFEB), and reducing the activity of cathepsin B (CTSB). In vivo, nicotine treatment exacerbated cardiac fibrosis induced in spontaneously hypertensive rats (SHR) and worsened cardiac function. Interestingly, the absence of LDHA reversed these effects both in vitro and in vivo. Our study identified LDHA as a novel nicotine-binding protein that plays a crucial role in mediating cardiac fibrosis by blocking autophagy flux. The findings suggest that LDHA could potentially serve as a promising target for the treatment of cardiac fibrosis.

In vitro protein expression profile of Campylobacter jejuni strain NCTC11168 by two-dimensional gel electrophoresis and mass spectrometry.

OBJECTIVE: To investigate the protein expression profiles of the major food-borne pathogen Campylobacter jejuni NCTC11168. METHODS: Membrane and soluble cellular proteins were extracted from the genome-sequenced C. jejuni strain NCTC11168. Protein expression profiles were determined using two-dimensional gel electrophoresis (2-DE). All the detected spots on the 2-DE map were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis. RESULTS: A total of 537 and 333 spots were detected from the whole cell and membrane-associated proteins of C. jejuni NCTC11168 cultured on Columbia agar medium at 42 °C by 2-DE and Coomassie Brilliant Blue staining, respectively. Analyses of whole cell and membrane-associated proteins included 399 and 133 spots, respectively, which included 182 and 53 functional proteins identified by MALDI-TOF/TOF analysis. CONCLUSION: The comprehensive expression protein profiles of C. jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation.

Secreted protein HP1286 of Helicobacter pylori strain 26695 induces apoptosis of AGS cells.

OBJECTIVE: Secreted proteins of Helicobacter pylori (H. pylori) interact with gastric epithelium cells and may contribute to cell damage. Considering the fact that HP0175 and hypothetical conserved protein HP1286 are included in the group and that HP0175 is a well-known apoptosis-induced factor, the present study aims to clarify whether HP1286 plays a role in bacterial pathogenicity or even functions as an apoptosis-induced factor in human stomach. METHODS: Two genes encoding HP1286 and HP0175 were cloned into pET32a vector and expressed as recombinant proteins in Escherichia coli (E. coli) BL21. Signal peptide sequences were not included. The recombinant proteins were purified with His SpinTrap and desalted by using HiTrap Desalting. Immunoreactivity of the proteins was determined by Western blot. Human gastric epithelial cell AGS was challenged with these endotoxin-free proteins; and apoptosis of cells was assayed with the Cell Death ELISA kit. RESULTS: Recombinant proteins and their respective products whose N-terminal his-tag were removed with thrombin were recognized by serum from the patient infected with H. pylori. Apoptotic AGS cells challenged by HP1286 of H. pylori 26695 were four times more than untreated cells. In addition, apoptosis-induced ability of HP1286 of SS1 was not as strong as that of H. pylori 26695 strain. CONCLUSION: HP1286 of H. pylori 26695 induces apoptosis of AGS cells in a time-dependent manner, however the apoptosis-induced ability of HP1286 may differ due to variations between different strains.

Evaluation of a new real-time PCR assay for detection of Mycoplasma pneumoniae in clinical specimens.

OBJECTIVE: To establish and evaluate a real-time PCR assay to detect Mycoplasma pneumoniae (M.pneumoniae) in clinical specimens. METHODS: By analysing the whole p1 gene sequence of 60 M.pneumoniae clinical isolates in Beijing of China, an optimized real-time PCR assay (MpP1) using p1 gene conserved region was designed. The specificity and sensitivity of this assay were evaluated and compared with other two reported assays (RepMp1 and Mp181) using 40 positive and 100 negative clinical specimens. RESULTS: The detection limit of the new assay was 8.1 fg (about 1∼3CFU) M.pneumoniae DNA. The sensitivity of MpP1, RepMp1, and Mp181 assays appeared to be 100%, 100%, and 85%, respectively. CONCLUSION: MpP1 assay is suitable for the detection of M.pneumoniae in Chinese clinical specimens.

[Acceptability of pre-exposure prophylaxis among female sex workers in Xinjiang].

OBJECTIVE: To assess the acceptability of pre-exposure prophylaxis (Pr-EP) among the female sex workers in Xinjiang. METHODS: A volunteer-based, anonymous and one-to-one questionnaire survey was conducted in 762 female sex workers (FSW) in Urumqi and Kelamayi of Xinjiang Uyghur Autonomous Region. RESULT: Among 762 FSW surveyed, 673 (88.32%) was not aware of pre-exposure prophylaxis with an awareness rate of 11.55%. The awareness rate of FSWs working in high-end entertainment venues was higher than that of FSWs working in medium-low end entertainment venues(P<0.001). Five hundred and twenty eight FSWs (69.29%) were willing to take Pr-EP, 145 (19.03%) were unwilling to take the medicine and 89 (11.68%) were possible to use the Pr-EP. There was no significant difference in willingness of using Pr-EP among FSWs working in high and medium-low end entertainment venuew (P=0.285). The subjects who were willing to take Pr-EP mainly concerned of the drug security, effectiveness and cost. The main reasons for not willing to take Pr-EP were: not having risk of infecting HIV, suspecting effectiveness of Pr-EP and worrying about side effects. CONCLUSION: The acceptability to use Pr-EP in female sex workers of Xinjiang is relatively high and the drug security, effectiveness and cost will influence the promotion and application of Pr-EP in the future.

A Conjugative MDR pMG1-Like Plasmid Carrying the lsa(E) Gene of Enterococcus faecium With Potential Transmission to Staphylococcus aureus.

lsa(E) is a pleuromutilin, lincosamide, and streptogramin A (PLSA phenotype) resistance gene that was first described in S. aureus and was thought to have been transferred from Enterococcus sp. This study aimed to elucidate the prevalence of the lsa(E) gene among E. faecium isolates at a tertiary teaching hospital and to evaluate the transferability of the lsa(E) gene from E. faecium to S. aureus in vitro. A total of 96 E. faecium strains isolated from one hospital in Beijing in 2013 were analysed for quinupristin-dalfopristin (QDA) resistance genes, and multilocus sequence typing (MLST) was performed. The transferability of QDA resistance between ten E. faecium strains and four S. aureus strains was determined by filter mating. Genome sequencing of the transconjugant was performed. A total of 46 E. faecium isolates (46/96, 47.92%) tested positive for lsa(E), while two isolates (2/96, 2.08%) tested positive for lsa(A). Thirty-six lsa(E)-positive strains (36/46, 78.3%) belonged to ST78. Among 40 mating tests, lsa(E) was successfully transferred through one conjugation at a frequency of 1.125 × 10-7 transconjugants per donor. The QDA resistance of the transconjugant N7435-R3645 was expressed at a higher level (MIC = 16 mg/L) than that of the parent S. aureus strain (MIC = 0.38 mg/L). Next-generation sequencing (NGS) analysis of the transconjugant N7435-R3645 showed that the complete sequence of the lsa(E)-carrying plasmid pN7435-R3645 had a size of 92,396 bp and a G + C content of 33% (accession no. MT022086). The genetic map of pN7435-R3645 had high nucleotide similarity and shared the main open reading frame (ORF) features with two plasmids: E. faecium pMG1 (AB206333.1) and E. faecium LS170308 (CP025078.1). The rep gene of pN7435-R3645 showed 100% identity with that of pMG1, although it did not belong to the rep1-19 family but instead a unique rep family. Multiple antibiotic resistance genes, including lsa(E), aadE and lnu(B), erm(B), ant6-Ia, and lnu(B), were present on the plasmid. In conclusion, an lsa(E)-carrying plasmid that can be transferred by conjugation from E. faecium to S. aureus in vitro was identified. This multidrug resistance (MDR) pMG1-like plasmid may act as a vector in the dissemination of antimicrobial resistance among species.

Nicotine Causes Mitochondrial Dynamics Imbalance and Apoptosis Through ROS Mediated Mitophagy Impairment in Cardiomyocytes.

Nicotine contained in traditional cigarettes, hookahs, and e-cigarettes is an important risk factor for cardiovascular disease. Our previous study showed that macroautophagic flux impairment occurred under nicotine stimulation. However, whether nicotine influences mitochondrial dynamics in neonatal rat ventricular myocytes (NRVMs) is unclear. The purpose of this study was to explore the effects and potential mechanism of nicotine on mitophagy, mitochondrial dynamics, apoptosis, and the relationship between these processes in NRVMs. Our results showed that nicotine exposure increased mitochondria-derived superoxide production, decreased mitochondrial membrane potential, and impaired PINK1/Parkin-mediated mitophagic flux in NRVMs. Interestingly, nicotine significantly promoted dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and suppressed mitofusin (MFN)-mediated fusion, which was also observed in the bafilomycin A1-treated group. These results suggest that mitophagic flux impairment may contribute to Drp-1-mediated mitochondrial fission. Finally, nicotine caused excessive mitochondrial fission and contributed to apoptosis, which could be alleviated by mdivi-1, an inhibitor of Drp1. In addition to CTSB, as we previously reported, the enzyme activity of cathepsin L (CTSL) was also decreased in lysosomes after stimulation with nicotine, which may be the main cause of the hindered mitophagic flux induced by nicotine in NRVMs. Pretreatment with Torin 1, which is an inhibitor of mTOR, activated CTSL and ameliorated nicotine-induced mTOR activation and mitophagy impairment, decreased mitochondria-derived superoxide production, and blunted mitochondrial fission and apoptosis. Pretreatment with the ROS scavenger N-acetyl-cysteine (NAC) or inhibitors of p38 and JNK, which could also alleviate mitophagy impairment, exhibited similar effects as Torin1 on mitochondria. Taken together, our study demonstrated that nicotine treatment may lead to an increase in Drp1-mediated mitochondrial fission by blocking mitophagic flux by weakening the enzyme activity of CTSL and activating the ROS/p38/JNK signaling pathway. Excessive mitochondrial fission induced by nicotine ultimately leads to apoptosis. Torin1 restored the decreased CTSL enzyme activity by removing excessive ROS and alleviated the effects of nicotine on mitophagic flux, mitochondrial dynamics, and apoptosis. These results may provide new evidence on the relationship between mitophagic flux and mitochondrial dynamics and new perspectives on nicotine's effects on mitochondrial dynamics in cardiomyocytes.

Proteome analysis of Neisseria meningitidis serogroup strains C associated with outbreaks in China.

OBJECTIVE: During 2003-2005, an outbreak of meningitis due to Neisseria meningitidis serogroup C occurred in China. With the aim to find strain clues result in the final epidemics, the ancestral strain 053442, a clinical isolate, and a carrier strain 053426 with different gene type were analyzed. METHODS: Clinical strain 053442 and carrier strain 053426 were cultured on GC agar plates under the same condition. Two-dimensional electrophoresis was performed using the pH 3-10 nonlinear IPG strips of 24 cm length, and all the protein spots were identified by matrix-assisted laser desorption/ionization time of flight spectrometry. RESULTS: 502 and 380 protein spots were identified in 053426 and 053442 respectively, relating to 266 and 202 different genes covering a wide range of cellular functions. The express volume and number of proteins involved in energy metabolism, protein synthesis and amino acid biosynthesis in 053426 were higher than in 053442. Virulence factor Opa, Opc and a series of proteins involved in pilus assembly and retraction were identified in 053442, which appear to be of primary importance in colonization and invasion of human cells. Compared to 053442, virulence protein species were less in 053426, with lower express volumes too. No Opa and Opc were detected in 053426. CONCLUSIONS: The different protein expression profiles of the clinical strain 053442 and carrier strain 053426 in the present study provide some clues of the different pathogenicity of the two strains, which may account for result in the final epidemics.

Cilostazol alleviate nicotine induced cardiomyocytes hypertrophy through modulation of autophagy by CTSB/ROS/p38MAPK/JNK feedback loop.

Nicotine is proved to be an important factor for cardiac hypertrophy. Autophagy is important cell recycling system involved in the regulation of cardiac hypertrophy. Cilostazol, which is often used in the management of peripheral vascular disease. However, the effects of cilostazol on nicotine induced autophagy and cardiac hypertrophy are unclear. Here, we aim to determine the role and molecular mechanism of cilostazol in alleviating nicotine-induced cardiomyocytes hypertrophy through modulating autophagy and the underlying mechanisms. Our results clarified that nicotine stimulation caused cardiomyocytes hypertrophy and autophagy flux impairment significantly in neonatal rat ventricular myocytes (NRVMs), which were evidenced by augments of LC3-II and p62 levels, and impaired autophagosomes clearance. Interestingly, cathepsin B (CTSB) activity decreased dramatically after stimulation with nicotine in NRVMs, which was crucial for substrate degradation in the late stage of autophagy process, and cilostazol could reverse this effect dramatically. Intracellular ROS levels were increased significantly after nicotine exposure. Meanwhile, p38MAPK and JNK were activated after nicotine treatment. By using ROS scavenger N-acetyl-cysteine (NAC) could reverse the effects of nicotine by down-regulation the phosphorylation of p38MAPK and JNK pathways, and pretreatment of specific inhibitors of p38MAPK and JNK could restore the autophagy impairment and cardiomyocytes hypertrophy induced by nicotine. Moreover, CTSB activity of lysosome regained after the treatment with cilostazol. Cilostazol also inhibited the ROS accumulation and the activation of p38MAPK and JNK, which providing novel connection between lysosome CTSB and ROS/p38MAPK/JNK related oxidative stress pathway. This is the first demonstration that cilostazol could alleviate nicotine induced cardiomyocytes hypertrophy through restoration of autophagy flux by activation of CTSB and inhibiting ROS/p38/JNK pathway, exhibiting a feedback loop on regulation of autophagy and cardiomyocytes hypertrophy.

Genetic analysis of group A streptococcus isolates recovered during acute glomerulonephritis outbreaks in Guizhou Province of China.

In this study, 68 group A streptococcus (GAS) isolates associated with two outbreaks of acute glomerulonephritis (AGN) in China were analyzed by emm typing. A total of 11 different emm types were identified. Analysis of emm type distribution suggested that AGN outbreaks in two counties were caused by emm60.1- and emm63.0-type GAS. These two types were further characterized by pulsed-field gel electrophoresis, multilocus sequence typing, sof sequence typing, and PCR-based identification of streptococcal pyrogenic exotoxin A, B, and C (speA, speB, and speC) genes. In antimicrobial susceptibility tests, all outbreak strains were resistant to erythromycin and tetracycline, and the rates of resistance of nonoutbreak strains to the two antibiotics were 63.6% and 90.9%. This study is also the first to report a nephritogenic M63 GAS strain.

Comparative proteomic analysis of passaged Helicobacter pylori.

In order to identify the proteins associated with Helicobacter pylori colonization in mice, we used 2-dimensional gel electrophoresis (2-DE) to analyze the membrane- and soluble-cellular proteins extracted from H. pylori strain 26695 and the mouse-passaged homolog 88-3887. We defined 2- and 3-fold changes in protein expression as the threshold values for differential expression in the membrane-protein and whole-cell-protein fractions, respectively. The differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF). A total of 29 proteins, including 16 membrane- or membrane-associated proteins (13 upregulated, 3 downregulated) and 13 cellular proteins (10 upregulated, 3 downregulated) were differentially expressed between the strains 26695 and 88-3887. Among the upregulated proteins, 10 proteins had been previously shown to be associated with the mouse colonization, and 13 upregulated proteins were shown to be associated with the adaptation of H. pylori in murine hosts for the first time in this study. The identified proteins were classified as proteins related to metabolism, stress response, virulence, or adhesion. The data presented in this report indicated that there were subsets of upregulated proteins in mouse-adapted H. pylori. In particular, the adhesins, virulence factors, and stress-response proteins are likely to contribute to colonization in mice.

Adherence and invasion of mouse-adapted H pylori in different epithelial cell lines.

AIM: To assess the adhesion and invasion abilities of different mouse adapted H pylori strains in different cell lines in vitro and investigate their effects on the virulence factors cagA and vacA. METHODS: The adherence and invasion abilities of different H pylori strains in different epithelial cell lines were examined by the gentamycin protection assay. The null mutants of cagA and vacA were processed by direct PCR mutation method. The morphologic changes of different cell lines after H pylori attachment were examined by microscopy. RESULTS: The densities of adherence to and invasion into cells in vitro were different from those in the mouse infection experiments. 88-3887 strain could invade and adhere to cells stronger than SS1 and X47. All tested strains had better adhering and invasive abilities in SCG-7901 cell. CagA and vacA minus mutants had the same invasion and adherent abilities as their wild types. In all strains and cell lines tested, only AGS cell had the significant hummingbird phenotype after inoculation with the 88-3887 wild-type. CONCLUSION: Both the host cells and the bacteria play important parts in the invasion and adhesion abilities of H pylori. CagA and VacA are not related to the ability of invasion and adhesion of H pylori in different cell lines in vitro.

[Effects of docosahexaenoic acid on cell apoptosis, invasion and migration of cervical cancer cells in vitro].

OBJECTIVE: To investigate the effect of docosahexaenoic acid (DHA) on apoptosis, migration and invasion of cervical cancer cell lines. METHODS: cervical cancer cell lines Hela and Siha in logarithmic phase were treated different concentrations of DHA. The morphological changes of the cells were observed microscopically and cell apoptosis was observed using Hoechst 33258 fluorescent staining. MTT assay was used to evaluate the effect of DHA in suppressing cell growth, and flow cytometry was employed to analyze the changes of cell apoptotic rate following DHA stimulations. Wound healing assay and Transwell migration assay were used to evaluate the migration of the cell lines. The expression levels of Bax, Bcl-2 cleaved caspase3, MMP-9 and VEGF proteins were detected by Western blotting. RESULTS: DHA exposure of the cells caused obvious morphological changes and dose-dependently increased the number of apoptotic bodies in the cells. MTT assay showed that DHA inhibited the growth of the cancer cells in a time- and concentration-dependent manner. DHA also effectively suppressed migration and invasion of the cancer cells. The cells exposed to DHA showed significantly down-regulation of Bcl-2, MMP-9 and VEGF proteins and up-regulation of cleaved-caspase 3 and Bax. CONCLUSION: DHA can promote cervical carcinoma cell apoptosis by down-regulating the anti-apoptotic proteins Bax, Bcl-2 and cleaved-caspase3 and suppress cell invasion by decreasing MMP-9 and VEGF expressions.

Analysis of the urinary peptidome associated with Helicobacter pylori infection.

AIM: To investigate the relationship between urinary peptide changes and Helicobacter pylori (H. pylori) infection using urinary peptidome profiling. METHODS: The study was performed in volunteers (n=137) who gave informed consent. Urinary peptides were enriched by magnetic beads based weak cation exchange chromatography and spectrums acquired by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). ClinProTools bioinformatics software was used for statistical analysis and the recognition of peptide patterns. The marker peptides were identified by LTQ Obitrap XL tandem MS. RESULTS: Approximately 50 proteins or peptides which loaded onto the magnetic beads were detected by MALDI-TOF MS. By optimizing the parameters of the model, the Genetic Algorithm model had good recognition capability (97%) and positive predictive value (94%). Based on the model, 2 markers with molecular masses of 6788 and 1912 Da were found that differentiated between H. pylori positive and negative volunteers. The m/z 1912 sequence was parsed as SKQFTSSTSYNRGDSTF. The peptide was identified as isoform 1 of the fibrinogen α chain precursor, whose concentration in urine was markedly higher in H. pylori infected volunteers than in H. pylori non-infected ones. CONCLUSION: The appearance of urinary fibrinogen degradation products is caused by an active H. pylori-induced process.

Time-series gene expression profiles in AGS cells stimulated with Helicobacter pylori.

AIM: To extend the knowledge of the dynamic interaction between Helicobacter pylori (H. pylori) and host mucosa. METHODS: A time-series cDNA microarray was performed in order to detect the temporal gene expression profiles of human gastric epithelial adenocarcinoma cells infected with H. pylori. Six time points were selected to observe the changes in the model. A differential expression profile at each time point was obtained by comparing the microarray signal value with that of 0 h. Real-time polymerase chain reaction was subsequently performed to evaluate the data quality. RESULTS: We found a diversity of gene expression patterns at different time points and identified a group of genes whose expression levels were significantly correlated with several important immune response and tumor related pathways. CONCLUSION: Early infection may trigger some important pathways and may impact the outcome of the infection.