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BACKGROUND: Pancreatic cancer is a common malignancy with poor prognosis and limited treatment. Here we aimed to investigate the role of host chromosomal instability (CIN) and tumor microbiome in the prognosis of pancreatic cancer patients. METHODS: One hundred formalin-fixed paraffin-embedded (FFPE) pancreatic cancer samples were collected. DNA extracted from FFPE samples were analyzed by low-coverage whole-genome sequencing (WGS) via a customized bioinformatics workflow named ultrasensitive chromosomal aneuploidy detector. RESULTS: Samples are tested according to the procedure of ultrasensitive chromosomal aneuploidy detector (UCAD). We excluded 2 samples with failed quality control, 1 patient lost to follow-up and 6 dead in the perioperative period. The final 91 patients were admitted for the following analyses. Thirteen (14.3%) patients with higher CIN score had worse overall survival (OS) than those with lower CIN score. The top 20 microbes in pancreatic cancer samples included 15 species of bacteria and 5 species of viruses. Patients with high human herpesvirus (HHV)-7 and HHV-5 DNA reads exhibited worse OS. Furthermore, we classified 91 patients into 3 subtypes. Patients with higher CIN score (n =13) had the worst prognosis (median OS 6.9 mon); patients with lower CIN score but with HHV-7/5 DNA load (n = 24) had worse prognosis (median OS 10.6 mon); while patients with lower CIN score and HHV-7/5 DNA negative (n = 54) had the best prognosis (median OS 21.1 mon). CONCLUSIONS: High CIN and HHV-7/5 DNA load were associated with worse survival of pancreatic cancer. The novel molecular subtypes of pancreatic cancer based on CIN and microbiome had prognostic value.
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Proteolysis targeting chimeras (PROTACs) is an innovative technique in targeted protein degradation. PROTACs is a heterobifunctional molecule which can bind to the E3 ligase and target protein to form a ubiquitination complex, resulting in the ubiquitin-proteasome system dependent degradation of target protein. PROTACs has been regarded as the promising method in drug discovery campaign, for its high commonality, potent degradation activity and unique selectivity profile. However, the catalytic mechanism also induces the uncontrollable protein degradation risk. Controllable PROTACs contain the responsive element in the molecular entity. In certain conditions, the element can be triggered to activate or terminate the degradation event. In this review, we will briefly summarize the strategies in controllable PROTACs and describe the representative examples according to the responsive mechanism. We hope this review could provide some insight into the further development of controllable PROTACs.
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Nrf2 is a multi-effect transcription factor, which plays a crucial role in cytoprotective system. With the deepening of research on new regulatory modes and biologic functions of Nrf2, the oncogenic role of Nrf2 in malignant transformed tumors is increasingly obvious. More and more evidences show that Nrf2 is involved in the whole process of tumor occurrence, development, metastasis and prognosis, and inhibiting Nrf2 may be a promising strategy in tumor therapy. However, the development of Nrf2 inhibitors is still in early stage. In this paper, the biological function of Nrf2 and its dual role in tumor are briefly introduced, and representative Nrf2 inhibitors are reviewed according to their structure types, so as to provide reference and ideas for the development of anti-tumor drugs centering on the regulation of Nrf2.
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For local treatment of ulcerative colitis, a new azoreductase driven prodrug CDDO-AZO from bardoxolone methyl (CDDO-Me) and 5-aminosalicylate (5-ASA) was designed, synthesized and biologically evaluated. It is proposed that orally administrated CDDO-AZO is stable before reaching the colon, while it can also be triggered by the presence of azoreductase in the colon to fragment into CDDO-Me and 5-ASA, generating potent anti-colitis effects. Superior to olsalazine (OLS, a clinically used drug for ulcerative colitis) and CDDO-Me plus 5-ASA, CDDO-AZO significantly attenuated inflammatory colitis symptoms in DSS-induced chronic colitis mice, which suggested that CDDO-AZO may be a promising anti-ulcerative colitis agent.
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Animais , Camundongos , Colite/tratamento farmacológico , Mesalamina/farmacologia , Nitrorredutases , Ácido Oleanólico/farmacologia , Pró-FármacosRESUMO
Reactive oxygen species (ROS) which were partial metabolites of oxygen are highly reactive. Different concentrations of ROS have different effects on tumor development. Tumor cells have a high level of reactive oxygen species. The antioxidant system of tumor is in highly activated state, and thus modulation of reactive oxygen species levels could be an effective strategy to target cancer cells. Treatment with small molecules that disrupt the redox balance can kill tumor cells first. This paper outlines the main ideas of developing anti-tumor drugs based on reactive oxygen species regulation, and summarizes the representative drugs and research progress according to the mechanism of action, in an effort to suggest potential reference and ideas for developing anti-tumor drugs based on reactive oxygen species regulation.
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Objective To explore the effectiveness and safety of the individual-specific rapid potassium supplementation strategy, and to provide experimental basis for treating fatal severe hypokalemia. Methods An acute fatal severe hypokalemia model was reproduced in 20 healthy adult Japanese big ear white rabbits with half lethal dose (LD50) of barium chloride (BaCl2) solution 168 mg·5 mL-1·kg-1. The rabbits were divided into conventional potassium supplementation group and individual-specific rapid potassium supplementation group according to random number table method with 10 rabbits in each group. All the animals were injected with 3% KCl through the auricular marginal veins by a micro-injection pump, and the target plasma potassium concentration was 4 mmol/L. The rabbits in conventional potassium supplementation group were administered continuously potassium infusion at the standard infusion rate of 0.4 mmol·kg-1·h-1. And those in the individual-specific rapid potassium supplementation group were treated in two steps: first, a loading dose of potassium was rapidly injected within 5 minutes, and this step was repeated until the plasma potassium concentration increased to 3.5 mmol/L; second, a sustaining dose of potassium infusion was continued at the rate of 0.4 mmol·kg-1·h-1 after the increase in plasma potassium concentration. The changes in electrocardiogram, blood pressure, respiratory rate (RR), plasma potassium concentration, urine potassium concentration, urine volume, potassium content in extracellular fluid (ECF) and other parameters were monitored. The potassium supplementation, potassium excretion and potassium cross cell status were recorded. Adverse reactions and 7-day death were observed. Results Since the BaCl2 administration, the plasma potassium concentration of all experimental rabbits were significantly lower than baseline at 0.5 hour, which was decreased below 2.5 mmol/L at 2.0 hours when the ventricular arrhythmias appeared, indicating the reproduction of fatal severe hypokalemia model was successful. There was no significant difference in gender, weight, baseline heart rate (HR), RR, mean arterial pressure (MAP), blood gas analysis or K+, Na+, Cl- levels between the two groups. Compared with baseline levels, MAP was significantly decreased and RR was significantly increased before potassium supplementation in both groups, but the parameters were improved significantly and restored to the baseline after potassium supplementation. There was no significant difference in MAP or RR during potassium supplementation between the two groups. The amount of potassium supplementation in two groups showed no significant differences. However, compared with the conventional potassium supplementation group, in the individual-specific rapid potassium supplementation group, the increase in plasma potassium concentration, urine potassium concentration, and the increase in potassium content in ECF were significantly increased [the increase in plasma potassium concentration (mmol/L): 2.40±0.33 vs. 1.51±0.75, urine potassium concentration (mmol/L):164.94±18.07 vs. 108.35±19.67, the increase in potassium content in ECF (mmol): 1.17±0.16 vs. 0.73±0.35], the duration of potassium infusion was shortened (hours: 2.1±0.7 vs. 4.7±1.4), the total urine volume, renal excretion of potassium, and the amount of transcellular potassium shift were significantly decreased [total urine volume (mL):6.40±1.78 vs. 13.60±4.69, renal excretion of potassium (mmol): 1.04±0.26 vs. 1.46±0.51, amount of transcellular potassium shift (mmol): 1.39±0.21 vs. 1.84±0.62], the duration of arrhythmia was shortened (minutes: 19.60±8.92 vs. 71.80±9.84), with statistically significant differences (all P < 0.05). Hyperkalemia did not occur in both groups. The rabbits of the individual-specific rapid potassium supplementation group were all alive, while 4 died in the conventional potassium supplementation group, and statistically significant difference was found between the two groups (P < 0.01). Conclusions These data demonstrate that the individual-specific rapid potassium supplementation strategy can shorten the time for correcting hypokalemia, which is a better option to reverse life-threatening arrhythmia caused by severe hypokalemia, with a high rescue success rate. The process of potassium supplement is safe and effective.
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The present study was designed to synthesize 2-Cyano-3, 12-dioxooleana-1, 9(11)-en-28-oate-13β, 28-olide (1), a lactone derivative of oleanolic acid (OA) and evaluate its anti-inflammatory activity. Compound 1 significantly diminished nitric oxide (NO) production and down-regulated the mRNA expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Further in vivo studies in murine model of LPS-induced acute lung injury (ALI) showed that 1 possessed more potent protective effects than the well-known anti-inflammatory drug dexamethasone by inhibiting myeloperoxidase (MPO) activity, reducing total cells and neutrophils, and suppressing inflammatory cytokines expression, and thus ameliorating the histopathological conditions of the injured lung tissue. In conclusion, compound 1 could be developed as a promising anti-inflammatory agent for intervention of LPS-induced ALI.
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Animais , Feminino , Humanos , Masculino , Camundongos , Lesão Pulmonar Aguda , Tratamento Farmacológico , Genética , Alergia e Imunologia , Anti-Inflamatórios , Líquido da Lavagem Broncoalveolar , Alergia e Imunologia , Ciclo-Oxigenase 2 , Genética , Alergia e Imunologia , Interleucina-1beta , Genética , Alergia e Imunologia , Interleucina-6 , Genética , Alergia e Imunologia , Lipopolissacarídeos , Pulmão , Alergia e Imunologia , Macrófagos , Alergia e Imunologia , Camundongos Endogâmicos BALB C , Neutrófilos , Alergia e Imunologia , Ácido Oleanólico , Peroxidase , Genética , Alergia e Imunologia , Fator de Necrose Tumoral alfa , Genética , Alergia e ImunologiaRESUMO
@#In order to search for new anti-inflammatory agents with strong activity and less toxicity relative to CDDO-Me, the ester prodrugs 2-8 of CDDO-Me were synthesized by treatment of oleanolic acid(OA)with DMF/K2CO3 to generate 1, followed by esterification of 1 with various aliphatic and aromatic carboxylic acids, respectively. All the target compounds showed strong inhibitory effects on LPS-induced NO production in RAW 264. 7 cells. Among them, compounds 2 and 7 possessed the most potent inhibitory effects with IC50=(2. 34±0. 67)and(3. 83±0. 97)nmol/L, respectively. Moreover, MTT assay indicated that all the target compounds(2-8)displayed much weaker anti-proliferative activity against RAW 264. 7 cell lines than CDDO-Me, suggesting that they may be less toxic than CDDO-Me.
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<p><b>OBJECTIVE</b>A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis).</p><p><b>METHODS</b>12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay.</p><p><b>RESULTS</b>The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively.</p><p><b>CONCLUSION</b>Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.</p>
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Antituberculosos , Farmacologia , Farmacorresistência Bacteriana , Genótipo , Immunoblotting , Métodos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Genética , Reação em Cadeia da Polimerase , Métodos , Rifampina , Farmacologia , Sensibilidade e Especificidade , Fatores de TempoRESUMO
@#The novel oleanolic acid derivatives 2a-2e were synthesized by introducing an α, β-unsaturated ketone moiety to C-ring of oleanolic acid(OA)via a nine-step reaction sequence, yielding an active CDDO-Me analogue(1), followed by coupling of C3-OH of 1 with various aliphatic and aromatic carboxylic acids, respectively. Derivatives 3a-3e were synthesized by substituting C-1 of compounds 2a-2e with bromine. The target compounds were characterized by IR, MS and 1H NMR spectra. All the target compounds showed strong inhibitory effects against two tumor cell lines(HepG2 and A549)to a varying extent. The anti-proliferative activities of active compounds 3b and 3c(IC50=6. 13±1. 16 μmol/L and IC50=5. 49±1. 03 μmol/L, respectively)against HepG2 and A549 were more potent than compound 1 and comparable to the positive control CDDO-Me. In addition, all the target compounds displayed much weaker anti-proliferative activity against the two tumor cell lines than that against normal BEAS-2B cells. Compound 3c showed ten-fold selective inhibition against HepG2 relative to BEAS-2B cells, and is thus worthy of further study.
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<p><b>OBJECTIVE</b>To evaluate the safety and efficacy of endoscopic retrograde cholangiopancreatography (ERCP) in children with pancreaticobiliary diseases and the characteristics of pancreaticobiliary disorders in children.</p><p><b>METHOD</b>Retrospective review was conducted on the data of patients younger than 18 years who underwent ERCP between 2005 and 2012 at West China Hospital. The indications,ERCP findings, ERCP procedures, complications, and clinical outcomes were evaluated.ERCP procedures were performed using standard duodenoscopes under general anaesthesia or sedation, which included all endoscopic treatments, such as endoscopic sphincteropapillotomy, stone extraction, stent treatment and so on.</p><p><b>RESULT</b>One hundred and two ERCPs were performed on 68 patients, and all the procedures were successfully completed in 100% cases. There were 39 girls (57%), and median age at time of procedure was 14.6 years (range, 5-17 years).General anesthesia and sedation were performed in 81% and 19% of procedures, respectively. The ERCP findings were classified as follows:bile duct stone(s) (n = 37, 54%), pancreatic duct stone(s) (n = 8, 12%), bile duct benign stricture (n = 7, 10%) and other nonmalignant pancreaticobiliary diseases (n = 16, 24%).Four cases (4/102, prevalence 4%) were complicated with post-ERCP pancreatitis.Symptoms such as abdominal pain and jaundice were cured obviously after the procedures of ERCP were performed.</p><p><b>CONCLUSION</b>The main characteristics of pancreaticobiliary disorders in children were nonmalignant pancreaticobiliary diseases, such as bile duct stone, pancreatic duct stone, and bile/pancreatic duct benign stricture.When performed by well-trained endoscopists, ERCP is safe and effective in children.</p>
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Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Doenças Biliares , Diagnóstico por Imagem , Cirurgia Geral , Cálculos , Diagnóstico , Patologia , Cirurgia Geral , Colangiopancreatografia Retrógrada Endoscópica , Coledocolitíase , Diagnóstico , Patologia , Cirurgia Geral , Pancreatopatias , Diagnóstico , Patologia , Cirurgia Geral , Ductos Pancreáticos , Diagnóstico por Imagem , Cirurgia Geral , Estudos Retrospectivos , Resultado do TratamentoRESUMO
<p><b>OBJECTIVE</b>To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure.</p><p><b>METHODS</b>FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index.</p><p><b>RESULTS</b>Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.</p><p><b>CONCLUSION</b>Establishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China.</p>
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Humanos , Western Blotting , Métodos , Grupo Borrelia Burgdorferi , Virulência , China , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Doença de Lyme , Diagnóstico , MicrobiologiaRESUMO
<p><b>OBJECTIVE</b>To prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis and develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for the detection of genital C. trachomatis infections.</p><p><b>METHODS</b>The pORF5 protein was expressed in Escherichia coli and used to immunize BALB/c mice and New Zealand rabbits to produce monoclonal antibodies (mAbs) and polyclonal antibody (pAb) for DAS-ELISAs. Clinical samples from 186 urogenital infection patients (groups I) and 62 healthy donors (groups II) were detected in parallel by the DAS-ELISAs developed in this study and by IDEIA PCE commercial ELISA.</p><p><b>RESULTS</b>Two hybridoma cell lines, named 2H4 and 4E6, stably secreting specific mAbs against pORF5 were obtained. The mAb 2H4 was recognized by 32 (17.20%, positive recognition rate) and 25 (13.44%), mAb 2H4 by 0 (0%) and 2 (3.22%) samples from groups I and II, respectively. The sensitivities of mAbs 2H4 and 4E6 were 92.11% and 77.78% and the specificities were 100% and 96.88%, respectively in relation to the IDEIA PCE commercial ELISA. The sensitivities of detection for the DAS-ELISAs were 10 ng/mL (based on 2H4) and 18 ng/mL (based on 4E6).</p><p><b>CONCLUSION</b>Two DAS-ELISAs were developed in this study that provided a feasible and effective assay that could be considered alternative tools for the serodiagnosis of C. trachomatis infection.</p>
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Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Infecções por Chlamydia , Diagnóstico , Chlamydia trachomatis , Virulência , Ensaio de Imunoadsorção Enzimática , Métodos , Sistema Urogenital , MicrobiologiaRESUMO
Objective To discuss the correlations between hemoperfusion(HP) times and therapeutic effects/prognosis in patients with severe acute organophosphorus poisoning(AOPP). Methods According to the frequency of HP,82 patients with severe AOPP were divided into three groups:non HP(25 cases),HP1(27 cases) and HP2(30 cases)groups. The non HP group received only routine treatment,on the basis of routine treatment,the HP1 group accepted once HP within 12 hours after poisoning and the HP2 group underwent twice or more times of HP,the interval between each time being 24 hours. The comparisions of clinical indexes,incidences of complications and rates of mortality among the three groups were performed. Results With the increase of HP times,the dosages of atropine and pralidoxime chloride were significantly reduced,the times of serum cholinesterase(ChE)activity recovery,consciousness recovery,hospitalization and mechanical ventilation were significantly shortened,the score of acute physiology and chronic health evaluation Ⅱ(APACHE Ⅱ)score in 48 hours after admission,incidence of complications and mortality were evidently decreased(all P<0.05). Compared with those in HP1 group,the dosages of atropine(mg:164.57±68.82 vs. 256.81±97.06)and pralidoxime chloride(mg:6.95±1.40 vs. 8.76±1.64) in HP2 group were significantly reduced,the times of ChE activity recovery(day:9.03±2.46 vs. 10.96±3.44), consciousness recovery(hour:23.83±6.29 vs. 39.93±8.24),hospitalization(hour:9.57±2.39 vs. 11.52±3.02) and mechanical ventilation(hour:40.50±16.55 vs. 65.74±18.88)in HP2 group were significantly shortened;APACHEⅡscore during 48 hours after admission(11.97±3.47 vs. 14.26±2.88)was obviously decreased,and the incidences of complications,such as intermediate syndrome(10.0% vs. 18.5%),rebound phenomenon(3.3% vs. 25.9%),arrhythmia(13.3%vs. 44.4%),multiple organ dysfunction syndrome(MODS,6.7%vs. 29.6%)and mortality rate(6.7% vs. 18.5%)in HP2 group were markedly decreased(all P<0.05). Conclusion It is recommendable that combined with routine treatment,early and multiple HP application would enhance the therapeutic effect and decrease the mortality in patients with severe AOPP.
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Objective:To evaluate the predictive value of 3 methods, namely plasma paraquat concentration, severity index of paraquat poisoning (SIPP), and acute physiology and chronic health evaluation (APACHE II) in severity evaluation and prognosis of acute paraquat poisoning. Methods:A total of 73 acute paraquat poisoning patients with oral administration were collected. Paraquat concentration in the plasma and other parameters on admission for SIPP and APACHE II were taken from medical records. According to the clinical outcome in the hospital or 7 days atfer the discharge, discrimination and calibration test were performed to evaluate the prognosis of the 3 methods. Results:Discrimination of the 3 methods was greater than 0.8, and the area under the receiver operator curve for SIPP (0.938) was greater than paraquat concentration in the plasma (0.857) and APACHE II (0.801) with statistical signiifcance (z=2.429, 2.021;P=0.015, 0.043). Difference in plasma paraquat concentration (0.857) and APACHE II (0.801) had no statistical signiifcance (z=0.755, P=0.450). Hosmer-Lemeshow good fit test suggested better calibration value with statistical signiifcance for the 3 methods (P>0.05). Conclusion:hTe 3 methods are valid in the severity evaluation and prognosis of acute paraquat poisoning. SIPP is the most preferred method, followed by paraquat concentration on admission. When there is no facility to measure paraqut concentration, APACHE II can be used as a reference for the death risk in acute paraquat poisoning.
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<p><b>OBJECTIVE</b>This paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumoniae) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPVD2-VD3) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae.</p><p><b>METHODS</b>MOMPVD2-VD3 were overexpressed in Escherichia coli and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors).</p><p><b>RESULTS</b>In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively.</p><p><b>CONCLUSION</b>The novel MOMPVD2-VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.</p>
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Animais , Humanos , Camundongos , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa , Alergia e Imunologia , Infecções por Chlamydophila , Diagnóstico , Microbiologia , Chlamydophila pneumoniae , Ensaio de Imunoadsorção Enzimática , Métodos , Estrutura Terciária de ProteínaRESUMO
<p><b>OBJECTIVE</b>To clone the plasmid protein pORF8 of Chlamydia trachomatis and localize its expression in Chlamydia-infected cells.</p><p><b>METHODS</b>pORF8 gene was amplified and cloned into pGEX-6p vector, and the pORF8 fusion protein was expressed in E.coli XL1 Blue. After purification with glutathione-conjugated agarose beads, the pORF8 fusion protein was used to immunize BALB/c mice to generate polyclonal antibodies against pORF8 protein. The antibodies obtained were used to localize the plasmid protein pORF8 in Chlamydia-infected cells with immunofluorescence assay (IFA).</p><p><b>RESULTS</b>The pORF8 gene 744 bp in length was successfully cloned and the GST fusion protein with a relative molecular mass of 54 000 was obtained. The cellular distribution pattern of the plasmid protein pORF8 was similar to that of the major outer membrane protein (MOMP), a known C. trachomatis inclusion body protein, but not to that of chlamydial protease-like activity factor (CPAF, a secreted protein).</p><p><b>CONCLUSION</b>The plasmid protein pORF8 is localized on the bacterial organism as an inclusion body protein in C. trachomatis-infected cells. The cellular location of pORF8 protein can potentially provide important insights into the pathogenesis of C. trachomatis.</p>
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Animais , Humanos , Camundongos , Anticorpos , Alergia e Imunologia , Proteínas de Bactérias , Genética , Infecções por Chlamydia , Metabolismo , Chlamydia trachomatis , Química , Genética , Metabolismo , Clonagem Molecular , Escherichia coli , Genética , Metabolismo , Vetores Genéticos , Genética , Células HeLa , Camundongos Endogâmicos BALB C , Plasmídeos , Genética , Proteínas Recombinantes de Fusão , Genética , Alergia e ImunologiaRESUMO
Mycoplasmas, the smallest free-living, self-replicating bacteria with diameters of 200 to 800 nm, have been reported to be associated with human diseases. It is well known that the mycoplasma lipoprotein/peptide is able to modulate the host immune system, whose N-terminal structure is an important factor in inducing immunity and distinguishing Toll-like receptors (TLRs). However, there is still no clear elucidation about the pathogenic mechanism of mycoplasma lipoprotein/peptide and the signaling pathway. Some researchers have focused on understanding the structures of these proteins and the relationships between their structure and biological function. This review provides an update on the research in this field.
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Lipoproteínas , Química , Metabolismo , Modelos Biológicos , Mycoplasma , Química , Metabolismo , Receptores Toll-Like , Química , MetabolismoRESUMO
<p><b>BACKGROUND</b>Acinetobacter baumannii has emerged as an important pathogen related to serious infections and nosocomial outbreaks around the world. However, of the frequently used methods, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) in Acinetobacter baumannii genotyping lack the direct molecular proof of drug resistance. This study was conducted to establish a typing method based on drug resistant gene identification in contrast to traditional PFGE and AFLP in the period of nosocomial epidemic or outbreak.</p><p><b>METHODS</b>From January 2005 to October 2005, twenty-seven strains of Acinetobacter species from Intensive Care Units, the Second Affiliated Hospital in Ningbo were isolated, including both epidemic and sporadic events. Susceptibility test, PFGE, AFLP and drug resistance gene typing (DRGT) were carried out to confirm the drug resistance and analyze the genotyping, respectively. PFGE was used as a reference to evaluate the typeability of DRGT and AFLP.</p><p><b>RESULTS</b>Twenty-seven strains of Acinetobacter displayed multiple antibiotic resistance and drug resistant genes, and beta-lactamase genes were detected in 85.2% strains. The result of DRGT was comparable to PFGE in Acinetobacter strains with different drug resistance though a little difference existed, and even suggested a molecular evolution course of different drug-resistant strains. AFLP showed great polymorphism between strains and had weak ability in distinguishing the drug resistance.</p><p><b>CONCLUSION</b>Compared to AFLP and PFGE, DRGT is useful to analyze localized molecular epidemiology of nosocomial infections and outbreaks, which would benefit clinical diagnosis and therapy.</p>
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Infecções por Acinetobacter , Microbiologia , Acinetobacter baumannii , Classificação , Genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Antibacterianos , Farmacologia , Farmacorresistência Bacteriana Múltipla , Genética , Fisiologia , Eletroforese em Gel de Campo Pulsado , Genótipo , Testes de Sensibilidade Microbiana , Reação em Cadeia da PolimeraseRESUMO
Induced pluripotent stem cells(iPS)are derived from the differentiated somatic cells that regain the differentiation capabilities by the direct nuclear reprogramming with the exogenous factors.The methods for iPS induction evolved from transcription factors to RNA binding proteins,small molecules,and extracellular signals with the efforts in improving biosafty.The cellular and epigenetic characterization for iPS generation is a progressive and time-dependent process,which is tightly associated with the cellular differentiation status.However,epigenetics of iPS is not same as the embryonic stem cells.In combination with gene therapy and cellular transplantation therapy,the achievements of iPS had been applied in animal disease model treatment.