Objective To investigate the mechanism of carbapenems resistance in Serratia marces-cens strains isolated from Wenzhou and their epidemiological characteristics.Methods 147 non-duplicated Serratia marcescens isolates were collected from the First Affiliated Hospital of Wenzhou Medical University during 2006 to 2012.The antimicrobial susceptibility test for all isolates was performed by using Vitek2 Compact to screen carbapenems-resistant Serratia marcescens strains.The minimum inhibitory concentrations ( MICs) of 10 commonly used antibiotics against carbapenems-resistant Serratia marcescens strains were de-termined by agar dilution method.The phenotypes of carbapenemase were analyzed by using the modified Hodge test.PCR analysis was used to detect the genes encoding carbapenemase, AmpC enzyme, efflux pump and outer membrane proteins.The changes of MICs before and after using CCCP efflux pump inhibitor were measured by agar dilution method.Outer membrane proteins were detected by SDS-PAGE.Carbapene-ms resistance genes were transferred from carbapenems-resistant Serratia marcescens strains to recipient strains by conjugation.The transconjugants were amplified by PCR and measured for MICs.Pulsed-field gel electrophoresis ( PFGE) was used to analyze homology among strains.Results 11 isolates resistant to car-bapenems were screened out from 147 Serratia marcescens isolates and all of them were resistant to penicil-lins, cephalosporins and ertapenem.10 out of the 11 isolates were both resistant to imipenem and meropen-em, but remained susceptible to fluoroquinolones and aminoglycoside.Among the 11 isolates, 10 carried blaKPC-2 gene, 1 carried blaIMP-1 gene, 8 harbored both blaEBC and blaMOX genes, 1 harbored both blaEBC and blaDHA genes, and 1 carried blaEBC , blaMOX and blaDHA genes.No additional genes were identified by PCR.The MICs of imipenem to 7 isolates and the MICs of ertapenem to 3 isolates were respectively decreased by 4-64 folds and 8-256 folds after using CCCP.CCCP had no effects on the MICs of meropenem.Loss of outer membrane protein was not detected among the 11 isolates.The blaKPC-2 genes were successfully transferred from 7 isolates into recipient strains.The MICs against the transconjugants were higher than those against the recipient strains in varying degrees.PFGE analysis demonstrated that 8 out of 11 Serratia marcescens strains belonged to one clonotype.Conclusion KPC-2 carbapenemase played an essential role in carbapenems re-sistance in Serratia marcescens strains isolated from Wenzhou.Attention should be paid to the clonal spread of KPC-2 and its horizontal transmission in Wenzhou.

Objective To investigate the effect of thyroid hormone(T3)on the expressions of cytochrome c (CytC) and apoptosis?inducing factor(AIF) after cerebral ischemia reperfusion injury in rats and its mechanism. Methods SD male rats were randomly divided into four groups: sham operation group(sham1),sham operation group + T3(sham2) ,ischemia?reperfusion group (IR) ,and thyroid hormone treatment group(T3). A rat model of cerebral ischemia?reperfusion injury was established by right middle cerebral artery occlusion for 2 h,followed by reperfusion for 24 h. Thyroid hormones (10μg/100 g) or normal saline were given at 1 h after onset of ischemia and 6 h after reperfusionby intraperitoneal injection. Neurological deficit scores were evaluated 24 h after reperfusion. Cerebral infarction volume was evaluated by TTC staining. Histological changes was observed by HE staining. Expressions and mRNA levels of CytC and AIF in ischemic brain tissue were evaluated by immunohistochemistry and real?time fluorescent quantitative RT?PCR. Results Ascompared with those in sham operation groups ,the expressions and mRNA levels of CytC and AIFincreased significantlyin IR groups. As compared with those in IR groups ,the indexes were remarkably decreasedin T3 groups (P < 0.01). Nerve function was markedly improved andinfarction area narrowed.Conclusions Thyroid hormone plays a certain role in protection of cerebral ischemia?reperfusion injury in rats,whose mechanism may be associated with inhibition of the expression of apoptosis factors?CytC and AIF.

Objective To investigate the effect of thyroid hormone on the expression of NF-κB and TNF-oα in the ischemic cortex of rats after focal cerebral ischemia-reperfusion.Methods 96 male SD rats were randomly divided into sham-operation group,sham-operation+T3 group,IR group and IR+T3 group.Using suture legal method to establish a rat model of middle cerebral artery occlusion for 2 h followed by reperfusion.In sham-operation+T3 group and IR+T3 group,T3 was given 3 days before ischemia and 1 hour after ischemia,respectively,intraperitoneal injection T3 10 μg/100 g for rats.Rats in other groups were given the same volume normal saline at the same time.The infarct size was determined by TTC staining at 24 h after reperfusion.HE staining was used to observe the morphological and structural changes of brain tissue.Using Real-time PCR method and immunohistochemical staining method to detect the expression of NF-κB mRNA,TNF-α mRNA and protein in ischemic cortex of rats.Results Compared with sham-operation group and sham-operation+T3 group,the pathological damage of brain tissue in IR group was obvious,while the pathologic damage of IR +T3 group was less than that in IR group.Immunohistochemistry assay showed that the expression of NF-κB was(49.19±5.55)in sham-operation group,(45.75±2.12) in sham-operation+T3 group,(56.88±2.23)in IR group and(50.25±1.67)in IR +T3 group,the expression of TNF-α was (22.50±3.07) in sham-operation group,(24.13±2.03) in sham-operation+T3 group,(37.25±2.82) in IR group and (30.25±1.67) in IR +T3 group,and the NF-κB,TNF-α in IR group were obviously higher than that in sham-operation group and sham-operation+T3 group(P＜0.05),while IR+T3 group were lower than that in IR group(P＜0.05).Real-time PCR showed that NF-κB mRNA,TNF-α mRNA level in IR group was the highest,which was higher than that of sham-operation group and sham-operation+T3 group(P＜0.05),and the NF-κB mRNA,TNF-oα mRNA expression in IR+T3 group were significantly decreased compared with that in IR group(P＜0.05).Conclusion Thyroid hormone has a protective effect on cerebral ischenia reperfusion injury,which may be achieved by reducing the expression of inflammatory factor NF-κB and TNF-oα.

Objective To investigate the prevalence and plasmid size of qnrD determinant in Morganella morganii (M.morganii) isolates.Methods A total of 100 non-duplicated M.morganii clinical isolates were collected from inpatients.Standard ager dilution method was used to determine the minimum inhibitory concentrations (MICs) of fluoroquinolones against M.morganii isolates.PCR were performed to detect plasmid-mediated quinolone resistance determinants (PMQRs) in M.morganii isolates and the prevalence of extended-spectrum β-lactamase (ESBL) genes and AmpC β-lactamase genes in PMQRs-positive M.morganii strains.The homology analysis among qnrD-positive M.morganii strains were conducted by using pulsed-field gel electrophoresis (PFGE).The location of qnrD gene and the size of plasmid carrying it were determined by southern hybridization.The transferability of qnrD gene was determined by conjugation experiment.Results Thirty out of 100 M.morganii isolates (30％) were found carrying PMQRs including 17 qnrD-positive strains,14 aac (6')-Ib-cr-positive strains and 5 qepA-positive strains.PCR and sequencing confirmed that thirty PMQRs-positive isolates carried blaDHA-1.Among them,six isolates were positive for ESBLs genes (four for blaCTX-M-14,one for blaCTX-M-3 and one for blaCTX-M-24) and four isolates were positive for blaTEM-1.Almost all PMQRs-positive M.morganii isolates showed reduced susceptibility to fluoroquinolones.Moreover,seventeen qnrD-positive M.morganii isolates harbored blaDHA-1 including five (29.4％) harboring aac(6')-Ib-cr gene,four (23.5％) harboring blaCTX-M-14,two (11.8％) harboring blaTEM-1 and one harboring aac(6')-Ib-cr gene,blaCTX-M-14 and blaDHA-1.PFGE analysis showed that the 17 qnrD-positive M.morganii isolates were divergent from each other and not clone-related.Southern hybridization analysis showed that qnrD genes of all M.morganiiis isolates were mainly located in a 2.7 kb plasmid,but only a few of them were located in a size of 5.1 kb plasmid.M.morganiiis isolates failed to transfer qnrD gene to E.coli EC600 through conjugation.Conclusion PMQRs were widely distributed in M.morganiiis isolates.qnrD gene was the predominant determinants with a high prevalence rate of 17.0％,followed by aac(6')-Ib-cr gene.qnrD gene was located on a non-conjugative plasmid of approximately 2.7 kb or 5.1 kb.One qnrD-positive M.morganii isolate carrying aac(6')-Ib-cr gene,blaCTX-M-14 and blaDHA-1 was detected.

Objective To investigate the drug resistant genes against carbapenems,aminoglycosides and quinolones and the molecular epidemiology of clinical isolates of Acinetobacter baumannii.Methods Forty non-duplicate strains of Acinetobacter baumannii were collected from clinical specimens in First Affiliated Hospital of Wenzhou Medical University.The identification of strains was conducted by Vitek 2 Compact system.The susceptibilities to antimicrobials commonly used were determined by agar plate dilution method and broth microdilution method.The presence of class B metalloenzyme-encoding genes (blaIMP,blaVIM,blaNDM,blaSIM,blaGIM),class D cabapenemase-encoding genes (blaOXA-23,blaOXA-48,blaOXA-58),16S rRNA methylase genes (armA,rmtB) and quinolone resistance-determining regions (QRDR) in gyrA and parC were detected by polymerase chain reaction (PCR) and sequenced.Chromosomal or plasmid location of blaOXA-23 and armA genes were assessed by Southern blot.Multiple loci sequence classification (MLST) was performed to analyze the molecular epidemiology of these strains.Results All of the 40 isolates were multi-drug resistant Acinetobacterbaumannii (MDR-AB) and showed high level resistance to all of the tested antimicrobial agents excluding colistin and tigecycline.The positive rates of blaOXA-23 and armA were 90％ and 95％,respectively.All of the 40 isolates carried QRDR mutations in gyrA and parC genes,leading to the Ser83→ Leu and the Ser80→ Leu amino-acid substitutions,respectively.Southern blot showed the chromosomal location of blaOXA-23 and armA genes.Six different ST (ST191,ST381,ST373,ST426,ST208 and ST207) were assigned for these isolates by MLST and the most dominant clones were ST191 (23/40) and ST381 (10/40).Conclusions The predominant cabapenemase-encoding gene and 16S rRNA methylase gene of Acinetobacter baumannii isolates in First Affiliated Hospital of Wenzhou Medical University are blaOXA-23 and armA,respectively,which may be located on the chromosome and vertically transmit the drug resistance.ST191 MDR-AB with blaOXa-23 and armA gene clonally spread in this hospital.

Objective To investigate the clinical,imaging,pathological and molecular biological features of mitochondrial encephalomyopathy with lactic acidosis and stroke -like episodes(MELAS)in children.Methods The clinical,imaging,pathological and molecular biological features of 1 2 children with MELAS diagnosed through muscle biopsy or gene sequencing in the Fifth Affiliated Hospital of Zhengzhou University from January 201 1 to December 201 5 were retrospectively analyzed.Results (1 )Clinical features:the main manifestations included headache and vomiting in 1 1 cases,epileptic seizures in 9 cases,short stature in 8 cases,hairy in 7 cases,intolerance fatigue in 7 cases,cogni-tive decline in 7 cases,visual disturbance in 6 cases,hearing disturbance in 6 cases,and 5 cases had positive family history.In addition,7 cases had the serum lactic acid level increase in a rest for 1 0 min after exercise.(2)Imaging fea-tures:4 cases showed bilateral basal ganglia calcification symmetry in 8 patients who underwent head CT scan.The most frequently involved parts of the lesion were occipital in 1 0 cases,temporal in 9 cases and parietal lobe in 7 cases in stroke -like episodes.The lesions were lamellar necrosis.The abnormal areas by MRI showed low signal intensity on T1 weighted imaging,high signal intensity on T2 weighted imaging and fluid attenuated inversion recovery,high or equal signal intensity on diffusion weighted imaging,high or low signal intensity on apparent diffusion coefficient;the lactate peak significantly increased on magnetic resonance spectroscopy.The distribution was not in accordance with the control region of the cerebral vessels.Dynamic observation revealed that the lesions were reversible and migratory.(3)Myo-pathological features:muscle biopsy was performed in all children,and ragged -red fibers were found in 1 0 cases by im-proved Gomori staining,strongly succinate dehydrogenase -reactive were found in 9 cases,and the lipid droplets slight-ly increased in 8 cases by oil red O staining.Besides,the crystalline inclusion bodies in mitochondria were arranged in a parking lotpattern in 9 cases by electromicroscope.(4)Molecular biological characteristics:the mitochondrial gene mutations were analyzed in peripheral blood of 9 children and their parents,including 8 cases with A3243G muta-tion and 1 case with G13513A mutation.Five mothers had the same A3243G mutation site in 8 cases.Conclusions Children with MELAS have complex and varied clinical manifestations and certain characteristic of neuroimaging.More-over,muscle pathology and gene sequencing have important diagnostic value.Fully understanding the clinical,muscle pathology,imaging and molecular biological characteristics of children with MELAS can be helpful to the early diagnosis and treatment,also reduce misdiagnosis.

Objective To investigate the neuroprotective effect of thyroid hormones T3 on cerebral ischemia-reperfusion injury in rats and its mechanism.Methods SD rats were divided into four groups:sham+saline group,sham+T3 group,MCAO+saline group,MCAO+T3 group.The cerebral ischemia-reperfusion injury rat models were established by right middle cerebral artery occlusion.Thyroid hormones(10 μg/100 g)or normal saline were given respectively by intraperitoneal injection twice at 1 h after the onset of ischemia and 6 h after reperfusion.Neurobehavioral score was evaluated at 24 h after reperfusion;TTC staining was used to label infarction area;RT-PCR was used to detect the mRNA level of nerve growth factor(NGF)and brain derived neurotrophic factor(BDNF)in brain tissue;Western blot was employed to determine alterations in protein levels of NGF and BDNF.Results Compared with MCAO+saline group,the neurological deficit and the volume of cerebral infarction of MCAO+T3 group was decreased,and the mRNA and protein expression of NGF and BDNF of MCAO+T3 group were increased(P<0.05).Conclusion Thyroid Hormones could promote the nerve repair,stimulate the nerve regeneration and improve the nervous behavioral function by up-regulating the expression of NGF and BDNF.

BACKGROUND:Urine-derived stem cels are most likely to come from the kidney tissue, and therefore, these cels are more adaptable to kidney microenvironment, providing a new option for the treatment of kidney diseases.
OBJECTIVE: To explore the therapeutic efficacy of human urine-derived stem cels on chronic nephropathy rats.
METHODS:The fresh urine samples of healthy people were colected, and then human urine-derived stem cels were extracted and cultured in vitro. Twenty Sprague-Dawley rats were used to prepare chronic nephropathy models, and given injection of human urine-derived stem cel suspension (experimental) or normal saline (control) into the renal cortex, respectively. Another 10 healthy rats were used as controls. Therapeutic effects on renal function were assessed by detection of serum creatinine level and glomerular filtration rate in the three groups. The kidney tissues of rats were taken and observed histomorphologicaly in each group.
RESULTS AND CONCLUSION: Human urine-derived stem cels were found to remarkable improve rat’s renal function as wel as reduce the histomorphological changes in the kidney tissues of rats. Compared with the control group, the serum creatinine level was decreased while the glomerular filtration rate was increased significantly in the experimental group; CD68 expression and infiltration of interstitial inflammatory cels were also markedly reduced in the experimental group. To conclude, human urine-derived stem cels can improve the renal function of chronic nephropathy rats.

Objective:To explore the initial motivation of nursing students to engage in voluntary service for the aged, and to provide scientific basis for the formulation of strategies and measures of voluntary service for the aged.Methods:Totally 25 nursing students volunteers who regularly participated in the volunteer service for the aged in Changsha First Welfare Home were selected by objective sampling method for semi-structured in-depth interviews, and the data were analyzed by Colaizzi phenomenological 7-step analysis method.Results:Four themes of nursing students' initial motivation to participate in voluntary service for the aged were extracted: self-interest motivation, altruistic motivation, affinity motivation and achievement motivation. Among them, self-interest motivation included two sub-themes: enriching college life and life experience, improving one's own ability and gaining professional experience; altruistic motivation included two sub-themes: accompanying and helping the elderly and eliminating loneliness of the elderly; affinity motivation included two sub-themes: establishing emotional sustenance and making up for the lack of emotion; achievement motivation included two sub-themes: facing challenges bravely, acquiring a sense of achievement, serving society and others, and realizing one's own value.Conclusion:Nursing students have a clear initial motivation to participate in the voluntary service for the aged of the aged care facilities, and schools can strengthen the relevant education for students to participate in voluntary service in the training of nursing talents. The voluntary service for the aged can formulate corresponding development strategies and incentives according to the initial motivation of nursing students, so that promote the expansion and stability of the voluntary service for the aged team, and promote the development and improvement of voluntary service for the aged.

Objective: To establish a rapid bacterial identification and antimicrobial susceptibility testing assay in positive blood cultures, so as to provide insights into timely diagnosis and treatment of bloodstream infections. Methods: A total of 1 154 blood culture samples were collected from inpatients in Zhejiang Hospital from February to May, 2022. The bacterial isolates were enriched and purified using improved separation gel method, and bacterial identification and antimicrobial susceptibility tests were performed using VITEK2 mass spectrometry system and VITEK2 Compact automated microbiology system. The accuracy of the new assay for bacterial identification and antimicrobial susceptibility tests was evaluated with the conventional VITEK 2 compact system as the standard. Results: Of 1 154 blood culture specimens, the conventional VITEK 2 compact system detected 174 positives and 980 negatives. The new assay and the conventional VITEK 2 compact system identified consistent bacterial isolates in 165 out of 174 positive blood culture samples, and the accuracy of bacterial identification was 94.83% for the new assay, with a 99.21% accuracy for identifying Gram-negative bacteria and 82.22% for Gram-positive bacteria. Antimicrobial susceptibility tests were performed in 158 bacterial isolates, and the new assay presented a 90.17% accuracy, with a 90.27% accuracy for Gram-negative bacteria and 89.74% for Gram-positive bacteria. The conventional VITEK 2 compact system required 30 hours and longer to complete bacterial identification and antimicrobial susceptibility tests, and the new assay required 9 to 18 hours. Conclusions The new rapid bacterial identification and antimicrobial susceptibility testing assay shortens the time of bacterial culture, achieves rapid bacterial identification and antimicrobial susceptibility testing in blood culture specimens and has a high accuracy that meets clinical needs, which facilitates rapid diagnosis and treatment of bloodstream infections.