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CRISPR/Cas(the clustered regularly interspaced short palindromic repeats-CRISPR associated)system exists in most bacteria and all archaea. It is an important strategy for bacteria and archaea to resist foreign nucleic acid invasion and use for self-defense. The CRISPR/Cas system is a simple, fast, and specific diagnostic tool, which is widely used in agriculture, industry, animal husbandry, and medicine. This article mainly introduces and discusses recently advantages and limitations of biosensors combining CRISPR/Cas system with fluorescence, visualization and surface enhanced raman related technologies, as well as future research directions.
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Animals , CRISPR-Cas Systems , Bacteria/genetics , ArchaeaABSTRACT
To understand the clinical characteristics of Staphylococcus aureus bloodstream infection and the main risk factors affecting clinical prognosis, providing a reference for clinical prevention and control of Staphylococcus aureus bloodstream infection. In this study, the clinical data of 152 patients with Staphylococcus aureus bloodstream infection admitted to Guangdong Provincial People's Hospital from January 2019 to December 2021 were retrospectively analyzed by reviewing the electronic medical record system, including underlying diseases, clinical characteristics, risk factors, and bacterial resistance. Statistical methods such as Chi-Squared Test and t Test were used to analyze the related risk factors that may affect the clinical characteristics and prognosis of patients with Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection, then the variables with P<0.05 in univariate analysis were included in the multivariate logistic regression model to analyze the independent risk factors of poor prognosis. The results showed among 152 patients with Staphylococcus aureus bloodstream infection, 50 patients (32.89%) were infected with MRSA. In comparison, 102 patients (67.11%) were infected with methicillin-sensitive Staphylococcus aureus (MSSA). Except for rifampicin, the resistance rate of MRSA to commonly used antibiotics was all higher than that of MSSA, and the difference was statistically significant (Chi-square values were 8.272, 11.972, 4.998, 4.776, respectively;all P-values are less than 0.05). Strains resistant to vancomycin, linezolid, and quinupristin/dalfopristin were not found. In the MRSA group, indwelling catheter and drainage tube, carbapenems, and β-lactamase inhibitor treatment were significantly higher than the MSSA group. The difference was statistically significant (P<0.05). The incidence of poor prognosis of bloodstream infection in the MRSA group was higher than that in the MSSA group (34.00% vs 13.73%), and the difference was statistically significant (χ2=8.495, P<0.05). No independent risk factors associated with poor prognosis were found in the included patients with MRSA bloodstream infection.Multivariate Logistic regression model analysis showed that solid malignant tumors (OR=13.576, 95%CI: 3.352-54.977, P<0.05), mechanical ventilation (OR=7.468, 95%CI: 1.398-39.884, P<0.05) were the most important independent risk factors for poor prognosis in patients with Staphylococcus aureus bloodstream infection. In summary, the poor prognosis rate of MRSA bloodstream infection is higher than that of MSSA. The clinical evaluation of related risk factors should be strengthened, targeted prevention and control interventions should be taken to improve the prognosis of patients with Staphylococcus aureus bloodstream infection, and the use of antibiotics should be rational and standardized, to control bacterial infection and drug resistance effectively .
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Humans , Methicillin-Resistant Staphylococcus aureus , Staphylococcus aureus , Retrospective Studies , Prognosis , Staphylococcal Infections/microbiology , Anti-Bacterial Agents/pharmacology , Methicillin/therapeutic use , SepsisABSTRACT
This study aims to develop a rapid and convenient test card for simultaneous detection of influenza A and influenza B viruses using quantum dot-based immunochromatographic assay. The test card consists of a test strip and a plastic casing. The test strip is composed of absorbent paper, a buffer pad, nitrocellulose membrane (NC membrane), sample pad, quantum dot-labeled antibody pad, and polyvinyl chloride (PVC) board. The NC membrane is coated with mouse monoclonal antibodies against influenza A and influenza B viruses for the T lines (test lines), and reference proteins A and B for the C line (control line). The quantum dot-labeled antibody pad contains mouse monoclonal antibody-quantum dot conjugates against influenza A and influenza B viruses. The results showed that the detection limit of the test card for both viruses ranged from 1.51 ×102 to 2.71×103 TCID50/ml, indicating its sensitivity for accurate detection of influenza A and influenza B viruses without being affected by various variants. The test card exhibited specific reactions with different subtypes of influenza A and influenza B virus culture fluids and showed no cross-reactivity with adenovirus, novel coronavirus, Mycoplasma pneumoniae, respiratory syncytial virus, Staphylococcus aureus, and other pathogens. Overall, the sensitivity and specificity of the test card for simultaneous detection of influenza A and influenza B viruses meet the requirements for clinical use. It offers the advantages of simplicity, rapidity, and no requirement for special equipment, enabling quick auxiliary diagnosis to prevent disease transmission.
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Animals , Mice , Humans , Influenza, Human/diagnosis , Herpesvirus 1, Cercopithecine , COVID-19 , Sensitivity and Specificity , Influenza B virusABSTRACT
This study aims to develop a rapid and convenient test card for simultaneous detection of influenza A and influenza B viruses using quantum dot-based immunochromatographic assay. The test card consists of a test strip and a plastic casing. The test strip is composed of absorbent paper, a buffer pad, nitrocellulose membrane (NC membrane), sample pad, quantum dot-labeled antibody pad, and polyvinyl chloride (PVC) board. The NC membrane is coated with mouse monoclonal antibodies against influenza A and influenza B viruses for the T lines (test lines), and reference proteins A and B for the C line (control line). The quantum dot-labeled antibody pad contains mouse monoclonal antibody-quantum dot conjugates against influenza A and influenza B viruses. The results showed that the detection limit of the test card for both viruses ranged from 1.51 ×102 to 2.71×103 TCID50/ml, indicating its sensitivity for accurate detection of influenza A and influenza B viruses without being affected by various variants. The test card exhibited specific reactions with different subtypes of influenza A and influenza B virus culture fluids and showed no cross-reactivity with adenovirus, novel coronavirus, Mycoplasma pneumoniae, respiratory syncytial virus, Staphylococcus aureus, and other pathogens. Overall, the sensitivity and specificity of the test card for simultaneous detection of influenza A and influenza B viruses meet the requirements for clinical use. It offers the advantages of simplicity, rapidity, and no requirement for special equipment, enabling quick auxiliary diagnosis to prevent disease transmission.
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Animals , Mice , Humans , Influenza, Human/diagnosis , Herpesvirus 1, Cercopithecine , COVID-19 , Sensitivity and Specificity , Influenza B virusABSTRACT
Gout is a persistent increase in blood uric acid caused by purine and uric acid metabolism disorders, resulting in monosodium urate crystal deposition induced by recurrent inflammatory diseases, mainly manifested as elevated uric acid and inflammatory attacks. Studies have shown that gout and changes in the gut miocrobiota are mutually causal, and the gut miocrobiota can not only affect purine and uric acid metabolism but also regulate inflammatory response. Therefore, regulating the gut miocrobiota is expected to become a new strategy for the treatment of gout. This article starts from the regulation of purine and uric acid metabolism and inflammatory response by gut miocrobiota, so as to clarify the specific application of gut miocrobiota regulation as a new strategy for gout treatment.
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Objective:To explore the protective effects and related mechanism of tea saponin (TS) on intestinal inflammation due to Shigella infection. Methods:In vitro, the antibacterial activity of TS was detected by standard broth microdilution method. The absorbance at 600 nm of the bacterial liquid was detected by Microplate Reader under different concentrations of TS, and the growth curve was drawn. Bacterial count was obtained by plate colony counting. In vivo, 15 mice were divided into 3 groups ( n=5 each): control group, Sf301 group and TS group. Sterile water or TS was applied to mice in the Sf301 group and the TS group per gavage once a day for 8 days, and the mouse model of Shigella infection was established on day 3. The disease activity index (DAI) was used to evaluate the general condition of mice. The mice were sacrificed on day 8. Colon length was measured and colon tissues were stained with HE to analyze the pathological changes. The cecal contents and feces of mice were taken for plate counting and Shigella load was obtained. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the mRNA expression levels of inflammatory factors. Results:(1) In vitro, the MIC of TS was 1 024 μg/ml. The plate counting and A600 of TS decreased in proportion to increasing concentrations (256, 512, 1 024 μg/ml) in comparison with the control group (all P<0.05). (2) In vivo, colon length was (7.70±0.24) cm in the control group and (7.35±0.41) cm in the TS group, which was significantly longer than that of the Sf301 group ([6.13±0.05] cm, P<0.05). Histopathological examination evidenced colonic epithelial cells shedding, decreased goblet cells, and inflammatory cell infiltration in the colon tissue of the Sf301 group. In the TS group, the colonic mucosa was intact without significant inflammatory cell infiltration. Bacterial load in cecal contents was significantly lower in the TS group than in the Sf301 group ( P<0.05). The level of tumor necrosis factor-α was significantly lower, and the level of interleukin-10 was significantly higher in the TS group than in the Sf301 group (all P<0.05). Conclusion:TS can effectively inhibit Shigella and alleviate Shigella infective enteritis by reducing Shigella load and inhibiting inflammation.
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Neutrophil extracellular traps (NETs) are an important part of the innate immunity. They are mainly involved in the occurrence of gout disease through NADPH oxidase 2-dependent pathways and it is related to the spontaneous remission of gouty arthritis and the formation of tophi. These pathological features can be analyzed by methods such as electron microscopy and immunohistochemical staining. In order to further explore the molecular mechanism of NETs and discover potential drug targets for gouty arthritis, this article summarizes its role in gouty diseases and related detection techniques.
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BackgroundThe recent outbreak of infections by the 2019 novel coronavirus (2019-nCoV), the third zoonotic CoV has raised great public health concern. The demand for rapid and accurate diagnosis of this novel pathogen brought significant clinical and technological challenges. Currently, metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) are the most widely used molecular diagnostics for 2019-nCoV. Methods2019-nCoV infections were confirmed in 52 specimens by mNGS. Genomic information was analyzed and used for the design and development of an isothermal, CRISPR-based diagnostic for the novel virus. The diagnostic performance of CRISPR-nCoV was assessed and also compared across three technology platforms (mNGS, RT-PCR and CRISPR) Results2019-nCoVs sequenced in our study were conserved with the Wuhan strain, and shared certain genetic similarity with SARS-CoV. A high degree of variation in the level of viral RNA was observed in clinical specimens. CRISPR-nCoV demonstrated a near single-copy sensitivity and great clinical sensitivity with a shorter turn-around time than RT-PCR. ConclusionCRISPR-nCoV presents as a promising diagnostic option for the emerging pathogen.
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OBJECTIVE@#To study the efficacy and safety of caffeine used in the early (≤72 hours after birth) and late (>72 hours after birth) stage in preterm infants with a gestational age of ≤31 weeks.@*METHODS@#A retrospective analysis was performed for 640 preterm infants (with a gestational age of ≤31 weeks) who were admitted to the neonatal intensive care unit of eight hospitals in Jiangsu Province, China. Of the 640 preterm infants, 510 were given caffeine in the early stage (≤72 hours after birth; early use group) and 130 were given caffeine in the late stage (>72 hours after birth; late use group). The clinical data were compared between the two groups.@*RESULTS@#There were no significant differences in birth weight, Apgar score, sex, gestational age, and age on admission between the two groups (P>0.05). Compared with the late use group, the early use group had a significantly younger age at the beginning and withdrawal of caffeine treatment (P0.05). Compared with the late use group, the early use group had significantly lower incidence rate of apnea (P0.05). However, significant differences were found in the incidence of bronchopulmonary dysplasia and the rate of home oxygen therapy, but there was no significant difference in the mortality rate between the two groups (P>0.05).@*CONCLUSIONS@#Early use of caffeine can shorten the duration of caffeine treatment, oxygen supply time, and length of hospital stay, with little adverse effect, in preterm infants with a gestational age of ≤31 weeks.
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The emergence of drug resistance and limitation of antifungal agents complicate the management of fungal infection. Candida albicans, as the most common fungal infection pathogen, causes candidiasis via developing its virulence factors. In this study, we found trans-cinnamaldehyde (TC), known as a "Generally Regarded As Safe" (GRAS) molecule, had moderate antifungal activities against various Candida species and could retard the virulence of C. albicans in a dose-dependent manner by inhibiting the adhesion, morphological transition and biofilms formation. The mechanisms investigation revealed that the inhibition of hyphae and biofilms development was caused by the increasing farnesol secretion induced by Dpp3 expression. Since drug resistance restricted the treatment of clinical fungal infection, we explored the capacity of TC to develop drug-resistance under a long time TC treatment. Results showed that TC had little chance to form resistance by a serial passage experiment. Our work illustrates the underlying mechanism of TC inhibition of morphological transition and provides a optional application in treating the relevant fungal infections by targeting fungal virulence factors.
Subject(s)
Acrolein/analogs & derivatives , Antifungal Agents/pharmacology , Candida albicans/drug effects , Drug Resistance, Fungal , Farnesol/metabolism , Acrolein/pharmacology , Biofilms , Candida albicans/metabolism , Candida albicans/pathogenicity , Cell Adhesion , Cell Proliferation , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Inhibitory Concentration 50 , Kinetics , Virulence/drug effects , Virulence Factors/metabolismABSTRACT
Objective@#To investigate the clinical and microbiological characteristics of Myroides odoratimimus producing MUS-1 carbapenemase. @*Methods@#A strain of gram-negative bacterium isolated from the urine sample of one patient hospitalized in the oncology department of Affiliated Hospital of Xuzhou Medical University was identified by the Vitek 2 automatic microbial analyzer and 16S RNA sequencing, and its bla MUS-1 gene was detected with PCR. The minimum inhibitory concentrations (MICs) of antimicrobial drugs were determined by the broth dilution method. @*Results@#One strain of MUS-1 carbapenemase producing Myroides odoratimimus was found. The drug susceptibility test showed that it was resistant to most of antibiotics conventionally used, but sensitive to minocycline and meropenem, the MIC of imipenem was 8 μg/mL, which was judged as intermediate. @*Conclusion@#The bla MUS-1 gene may be the cause leading Myroides odoratimimus to resistant carbapenems drugs.
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Objective@#To find the differential expression profiles of circRNA in whole blood and predict its target genes in blood of patients with tuberculosis in Xinjiang, and explore the relationship between circRNA and the development of tuberculosis. @*Methods@#The circRNAs expression in peripheral blood from 3 pulmonary tuberculosis patients and 3 healthy individuals were tested by using circRNA microarray assay. The whole blood from 43 patients with tuberculosis, 40 healthy individuals and 43 patients with pneumonia were collected to verify the results by real-time quantitative PCR system. The possibility of differentially expressed circRNA target genes were predicted by circRNA target gene prediction database. @*Results@#In the results of microarray assay 835 circRNAs were found to be expressed differentially in whole blood between the tuberculosis patients and healthy controls of Xinjiang area, of which 249 circRNAs were up-regulated and 586 circRNAs were down-regulated in the patients. The expressions of four significantly different circRNA were verified by real time quantitative PCR and the results showed that hsa_circ_0008276, hsa_circ_0003452, hsa_circ_0001846 and hsa_circ_0090508 were down-regulated (P<0.05), and hsa_circ_0090508 was the more specific than the other three circRNAs. The results of circRNA target genes prediction suggested that has-miR-1294, has-miR-604, has-miR-616, has-miR-663b and has-miR-486-3p may be the potential target genes of hsa_circ_0090508. @*Conclusion@#The differentially expressed circRNA hsa_circ_0090508 was significantly downregulated in the patients with tuberculosis and may affect the regulation mechanism of tuberculosis through target genes.
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Objective:To construct oxaliplatin (L-OHP) drug-resistant cell line HCT-116/L-OHP in human colon cancer, in order to observe the reversal effect of curcumin (cur) on its drug resistance, and preliminarily explore the possible drug resistance mechanism. Method:The concentration gradient increasing method was used to gradually increase the L-OHP concentration of HCT-116 in parental colon cancer cells, and the cell line HCT-116/L-OHP resistant to L-OHP was established. The cytotoxicity of L-OHP and curcumin to HCT-116 and HCT-116/L-OHP cells was detected by cell counting kit-8(CCK-8) method to observe whether curative resistance could be reversed. Western blot was used to detect the expressions of drug-resistance-related proteins. Real-time PCR was used to detect changes in related genes. Result:Human colon cancer cell line resistant to L-OHP were successfully established and named as HCT-116/L-OHP, with a drug resistance index of 12.6.Compared with HCT-116 cell lines, the expression levels of resected and repaired cross complementation gene 1 (ERCC1) protein and gene in HCT-116/L-OHP cell lines were significantly increased (PP-1), the expression of ERCC1 decreased (PPConclusion:HCT-116/L-OHP cell lines have a stable drug resistance, and its drug resistance mechanism may be up-regulated with the expression of ERCC1, which leads to the up-regulation of Bcl-2,GST-π,MRP,P-gp,Survivin and other related proteins, and enables tumor cells to acquire drug resistance. Curcumin can reverse the drug resistance of HCT-116/L-OHP, and its mechanism may be to reduce the expression of ERCC1, thereby down-regulating the expressions of Bcl-2,GST-π,MRP,P-gp,Survivin and other drug-resistant related genes and proteins, and increase the sensitivity of tumor to L-OHP, so as to reverse the drug resistance of tumor cells.
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Objective To study the effect of [Gly14]-Humanin on oxidative stress and neuron apoptosis after focal cerebral ischemia reperfusion injury in rats. Methods A rat model of acute middle cerebral artery occlusion(MCAO)and reperfusion was established by suture embolism. Ninety-six healthy male Sprague-Dawley rats were randomly divided into sham-operation group, model group, normal saline group and HNG group. And 5 μL HNG(100 nmol/L)in the rats of the HNG group, normal saline in the rats of the sham-operation group and normal saline group were given to rats 3 days before operation respectively(iv, three times, qd). After cerebral ischemia 3 h and 24 h of reperfusion, neurological deficit score(NDS) were performed for each group, and the activity of sod, levels of GSH and MDA in cerebral tissue were detected. TUNEL staining were used to detect neuronal apoptosis. Results Median(Interquartile range)of NDS was 0(0)in sham-operation group, 1.5(1)in model group, 3(1)in normal saline group and 3(1)in HNG group, respectively, and the NDS of sham-operation group was significantly lower than the other three groups. The NDS of HNG group was significantly lower than the model group and normal saline group(all P<0.05). Compared with sham-operation group, the activity of SOD of model group, normal saline group and HNG group rats[(11.65±1.66),(12.15±1.56)and (19.43±1.47)U/mL]and levels of GSH[(8.84±1.23),(7.51±1.16)and (11.17±1.67)mg/L]were decreased, and levels of MDA[(42.61±1.79), (40.33±1.24)and (35.39±1.29)nmol/mL]were increased(P<0.05). The activity of SOD and levels of GSH of HNG group were significantly higher than model group and normal saline group, but the levels of MDA was significantly lower than model group and normal saline group(all P<0.05). Compared with sham-operation group[(0.13±0.01)%], percentage of neuronal apoptosis of model group, normal saline group and HNG group rats[(0.59 ±0.07)%,(0.51 ±0.08)% and (0.22 ±0.03)%, respectively]were increased(P<0.05), and percentage of neuronal apoptosis of HNG group was significantly lower than model group and normal saline group(all P<0.05). Conclusion By increasing the activity of SOD and levels of GSH,[Gly14]-Humanin can resist focal cerebral ischemia reperfusion injury, and can decrease the neuro apoptosis of ischemic area, thus mitigate the neurologic deficit.
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Objective To carry out innovative practice as well as questionnaire survey and conduct promotion analysis of clinical molecular biology testing based on problem-based learning (PBL). Methods 137 Students of medical laboratory technology discipline were chosen as subjects to conduct the innovative practice of PBL method. The teaching results were shared and promoted by video online through WeChat. Micro-questionnaire was conducted in 335 students, and the results of the survey were statistically analyzed. Result The improved PBL teaching based on speech contest can boost students to integrate into classroom teaching actively in which they had a better understanding of the knowledge so that the expected teaching quality of clinical molecular biology testing was achieved effectively. Questionnaire showed that the impact of PBL teaching method on students was not extensive currently, indicating that there was still more space to promoting such teaching methods. Although traditional teaching methods still held a place in students' minds (43.9%, 147 cases), the innovative teaching of PBL, which is novel, interesting and colorful, is more in line with the expectations of medical laboratory students in the new era and has the highest acceptance (49.6%, 166 cases). Conclusion The innovative practice of PBL teaching based on speech contest can achieve the purpose of strengthening students' knowledge acquisition and flexibility in the practical use of knowledge, which is worthy of promoting. Teachers could gradually integrate the new PBL teaching model into the tradi-tional teaching methods in the future, making students study efficiently in a relaxed and active atmosphere
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Objective To investigate the distribution of hypervirulent capsular serotypes and virulence genes of Klebsiella pneumoniae associated with pyogenic liver abscess(PLA),and analyze the homology of strains.Methods Twelve strains of liver abscess-related Klebsiella pneumoniae were isolated in Wuxi Second People's Hospital during January 2016 and August 2017.Among them,five were also detected in blood samples.All the strains were performed the viscous thread test,and the hypervirulent capsular serotypes and main virulence genes were detected by PCR.The homology of the strains was analyzed by the multilocus sequence types(MLST) and pulsed-field gel electrophoresis(PFGE).Results The positive rate of the viscous thread test for 12 strains of liver abscess-related Klebsiella pneumoniae was 75%.Three kinds of hypervirulent capsular serotypes,including K1 (6/12,50%),K54(1/12,8.3%) and K57(5/12,41.7%),were detected.The positive rates of virulence genes,such as wcaG,rmpA,ureA,fimH,mrkD,uge,Aer and iroNB,were all 100%,while that of iucB was 83.3%.The cf29a gene was not found,and the magA,allS and kfuBC genes were only detected in K1 serotype strains.MLST found that ST23(4/12,33.3%) and ST25(3/12,25%) were main sequence types,and then were ST412(2/12,16.7%),ST1660(1/12,8.3%),ST1049(1/t2,8.3%) and ST11 (1/12,8.3%).PFGE showed that 12 strains of Klebsiella pneumoniae were divided into 8 types,and that only 3 strains of K1 serotype belong to the same clonotype.Conclusion All the isolated liver abscess-related Klebsiella pneumoniae strains are highly virulent.ST23 and ST25 are main sequence types,and ST1049 is first reported.PFGE results show genetic diversity,and Kl-type Klebsiella pneumoniae has a certain epidemic tendency.
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Objective To understand the prevalence of resistance gene and homology of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from ICU of emergency.Methods A total of 19 CRKP isolates were obtained from emergency ICU of the Affiliated Hospital of Xuzhou Medical University from July 2015 to August 2016.PCR was performed to screen the genes encoding carbapenemase,extended spectrum beta-lactamase (ESBL) and AmpC.Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used for molecular typing of these bacterial strains.Results Among the 19 CRKP,carbapenemase-resistant genes were detectable in 18 CRKP isolates,including 17 isolates of harboring blaKPC gene and 1 strain of harboring blaNDM gene.All the 18 strains carried ESBLs genes which were identified as 8 blaSHV-12,3 blaSHV-11,5 blaSHV-2a,15 blaTEM-1,10 blaCTX-M-65,3 blaCTX-M-15,1 blaCTX-M-14 and 1 blaCTX-M-27.The 13 strains harboring cephalosporin-resistant genes were all identified as blaDHA-1.PFGE results revealed that the 19 CRKP strains were grouped into 4 types (A,B,C and D) and 4 subtypes(A1,2,3 and 4):A1 (n =12),A2(n =1),A3 (n=1),A4(n=1),B(n=2),C(n=1) and D(n=1).MLST showed that ST11 was the predominant sequence type (n=15) among the 19 CRKP strains,and ST48 (n =2),ST37 (n =1) and untyped (n =1) were also identified.The 15 blaKPC-2-producing CRKP ST11 clone shared the A type of PFGE pattern.Conclusion The report on CRKP suggested the dissemination of blaKPC-2-producing ST11 clone was existed in the ICU of emergency department in this hospital.The surveillance for drug-resistance and effective disinfectant quarantine measures should be strengthened.
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This article introduced the main biological mechanisms of acupuncture promoting nerve function recovery after spinal cord injury, which include inhibition of inflammation and oxidative stress, alleviation of neuropathic pain, increase of neurotrophic active sub-stance, regulation of cell survival/apoptosis gene and neural regeneration pathway.
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Objective To isolate and identify exosomes from serum samples of the patients with polymyositis / dermatomyositis (PM/ DM),and analyze their protein composition preliminarily.Methods Exosomes from serum samples of the patients with PM/DM were isolated and purified by the ExoQuickTM kit.The morphological characteristics and particle size of exosomes were determined by transmission electron microscope (TEM) and NanoSight analyzer,respectively.The surface markers of exosomes such as CD9,CD81 and Flotillin-2 were identified by western blot.The concentration and composition of exosome protein were determined by the BCA method and SDS-PAGE,respectively.Results The exosomes from serum samples of PM/DM patients displayed round or oval vesicles with membrane structure under TEM,and their diameter range was about (92 ± 67) nm.western blot showed that these exosomes expressed CD9,CD81 and Flotillin-2.The total protein concentrations of exosomes in the patients with PM/DM and healthy controls were 14.68 (6.00,32.55) μg/μL and 14.09 (8.00,23.28) μg/μL,respectively.SDS-PAGE showed that high-abundance proteins enriched in 55-70 kD in both PM/DM patients and healthy controls,and that there were different bands in 40-55 kD between them.Conclusion Exosomes are isolated from serum samples of the patients with PM/DM successfully,and their protein concentration and composition are analyzed preliminarily,which provides the experimental evidences for further finding differential proteins.
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Objective To identify the drug resistance-related genes in a clinically isolated strain of Enterobacter cloacae.Methods A strain of Enterobacter cloacae was isolated from sputum of a patient with chronic obstructive pulmonary disease from the Affiliated Hospital of Xuzhou Medical University in March 2013.Modified Hodge test and metal enzyme inhibition test were performed for drug-resistant phenotype screening.Carbapenemase genes blaMUS-1, blaVIM-1, blaVIM-2, blaIMP, blaKPC-2, blaNDM-1, blaOXA-48 and blaGESwere amplified by polymerase chain reaction (PCR), and the positive products were sequenced and analyzed.Plasmid conjugation and transformation experiments were used to confirm that the resistance gene mediated by plasmids.Agar dilution method was used for antibiotic susceptibility test.Results Both modified Hodge test and metal enzyme inhibition test were positive in this strain of Enterobacter cloacae.blaNDM-1 gene and blaKPC-2 gene were detected by PCR, and further confirmed by sequencing.blaNDM-1 gene was carried by IncX plasmid with 54×103 bp, KPC-2 gene was carried by untyping plasmid with 42×103 bp.The strain was only sensitive to tetracycline (MIC=2 μg/mL) and tigecycline (MIC=1 μg/mL).The symptoms were improved after the patient was treated by tigecycline combined with Piperacillin/Tazobactam.Conclusion blaNDM-1 and blaKPC-2 genes in Enterobacter cloacae can be mediated by plasmids, and appropriate therapy for its infection should be based on the result of antibiotic susceptibility test.