E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.

Objective To observe the effect of DNA methyltransferase 1 (DNMT1 ) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells. Methods Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3.1-HI and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8 ) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry(FCM ) method. Results Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector PShRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMTI-B was the better choice. While no effect of pshRNA-DNMTI-C was seen. BT-PCR results showed that the relative mRNA expression of DNMT1 gene in Helm cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406±0.057,0.191±0.036 and 0.104±0.015, which were significantly lower than that in Helm cells transfected by empty vector and non-transfected cells (0.520±0.020, 0.537±0.041, respectively, P ＜ 0.05 ). The western blotting analysis manifested that the relative expression of DNMT1 protein of Helm cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197±0.024, 0.075±0.015, 0.040± 0.013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273±0.010, 0.283±0.016, respectively, P ＜0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P ＜ 0.01 ). The results of FCM indicated that the apoptesis rate of HeLa cells trandected with pshRNA-DNMTI for 24, 48 and 72 hours were (17.7± 1.3 ) %, (35.3±1.3 ) %, (47.6±1.6 ) %, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9±0.5 ) %, (5.1±0.7 ) %, respectively, P ＜ 0.05]. Conclusions DNMT1 can be successfully silenced by RNA interfering in cervical Helm cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.

OBJECTIVE To investigate the prevalence of nasal colonization among health care workers(HCWs).METHODS Nasal swabs from 93 ICU workers and 98 other clinical workers were cultured and isolated and the tests of antibiotic susceptibility were performed by using paper diffusion method.RESULTS In total,214 isolates of 8 species from 191 health care workers were recovered,of which 187 isolates were coagulase-negative Staphylococcus(CNS)(the carriage rate of 93.71%) and 8 isolates were Staphylococcus aureus(the carriage rate of 4.19%).While the total Gram-negative bacteria carriage rate was 14.14%(27 isolates).The most frequent CNS species were S.epidermidis and S.haemolyticus.The antibiotic susceptibility profiles of S.aureus and CNS differed sharply: all 8 S.aureus strains were resistant to penicillin but were fully susceptible to oxacillin,in contrast,most of CNS were resistant to both penicillin and oxacillin.The carriage rate of CNS(60.2%)and Gram-negative bacteria(26.9%)in HCWs of ICU were higher than other HCWs(P

Objective To investigate the effect of telerehabilitation on memory disorders. Methods From August, 2010 to April, 2015, 81 patients with memory disorders were randomized into control group (n=26), computer-assisted training group (n=33) and telerehabilita-tion training group (n=22). All the patients accepted medicine to facilitate the recovery of memory. Besides, the computer-assisted training group and the telerehabilitation training group accepted memory-based training programs with cognitive rehabilitation system locally or on network respectively, for six weeks. They were evaluated with Wechsler Memory Scale, Rivermead Behavioural Memory Test-2nd Edition and Rey Auditory Verbal Learning Test before and after training. Results Both computer-assisted and telerehabilitation training groups im-proved in all the assessment after training (t>4.059, P<0.001). There was no significant difference between them (P>0.05). There was no sig-nificant improvement in the control group after training (t<0.771, P>0.05). Conclusion Memory rehabilitation training can significantly im-prove memory abilities, similar with locally or telerehabilitation system.

Objective To investigate the possible mechanism of aggregation and activation of neu-trophils(polymorphonuclear neutrophils,PMN)in mice with chlamydial pneumonitis. Methods C57BL/ 6 mice were inoculated intranasally with 3×103 inclusion-forming units(IFU)of Chlamydia muridarum(Cm) to induce the murine model of chlamydial pneumonitis. Samples of lung tissues collected at different time points after infection were stained by hematoxylin and eosin for histopathological assessment of inflammation. The levels of myelo-peroxidase(MPO)were detected for the evaluation of PMN aggregation. The mononu-clear cells were isolated from lung tissues. The inflammatory cells were counted with Giemsaˊs staining. CD11b+Gr1+ cell population and CD11b expression in lung mononuclear cells were analyzed by flow cytome-try. The expression of chemokines(MIP-2,LIX,KC and MCP-1)in lung tissues at mRNA level was meas-ured by RT-PCR. Results Chlamydial pneumonitis was induced in mice by intranasal inoculation of 3×103 IFU of Cm. Compared with the mice from control group,large amounts of inflammatory cells including PMN, monocytes and lymphocytes were induced in lung tissues of mice with Cm infection. PMN responded earlier than monocytes to the infection. The levels of MPO were significantly increased in mice with Cm infection and reached the highest level on the 7th day after infection. A decline in MPO levels was observed on the 14th day but the levels were still higher than those on day 0. The percentages and total numbers of CD11b+Gr1+ cells were significantly increased after Cm infection. Moreover,an increased expression of PMN CD11b was also detected by flow cytometry. The expression of chemokines(MIP-2,LIX,KC and MCP-1)was in-creased in lung tissues of mice after Cm infection. The results of the study indicated that Cm infection in-duced the expression of PMN chemoattractants,resulting in the recruitment of PMN. Conclusion The infil-tration and activation of PMN in lung tissues of mice were induced by Cm infection through increasing the ex-pression of chemokines. PMN played an important role in immune responses against Cm infection.

Objective:To investigate the effect of IL-17A on the differentiation and maturation of murine bone marrow-derived dendritic cells( BMDCs ) . Methods: Murine bone marrow cells were isolated and cultured in RPMI1640 complete medium in the presence of GM-CSF(20 ng/ml) for 8 days to induce differentiation of murine bone marrow cells to DC progenitors. Then these cells were treated with LPS(1 μg/ml) for 36 h which polarized immature DCs into mature DCs. Different concentrations of rmIL-17A(10 or 100 ng/ml) was added to the culture medium at different stages of BMDC differentiation and maturation. Co-stimulatory molecules expression on BMDC were analyzed by flow cytometry,and the culture supernatants were analyzed for IL-12p40 and IL-10 level by ELISA. Results:rmIL-17 could promote co-stimulatory molecules( CD40,CD80,CD86 and MHCⅡ) expression on BMDCs in a does-dependent manner,especially,the expression of CD40 and MHCⅡhad a significant increase in high concentration of rmIL-17A group;rmIL-17A was added while LPS induced maturation of BMDCs. CD40,CD80,CD86 and MHCⅡexpression on BMDC increased sharply in LPS plus rmIL-17A stimulation group,besides,CD86,MHCⅡ showed a higher level expression on BMDC with the increase of con-centration of rmIL-17A. Furthermore,secretion of IL-12p40 and IL-10 increased significantly in the group of DCs treated with LPS plus low concentration of rmIL-17 compared with the group without rmIL-17(P<0. 001). However,high concentration of rmIL-17A group showed significantly higher levels of IL-12p40(P<0. 001),but there was no difference in IL-10. Conclusion:IL-17A promotes the phe-notypic development of BMDC progenitors propagated in GM-CSF and cooperate with LPS to induce BMDC differentiation and matura-tion.

Objective:To investigate the effect of hydrogen sulfide on the neurogenic inflammation in mouse lung tissues ,and its mechanism .Methods:Eight‐week old ,male Balb/c mice were randomly divided into the following groups:control group , NaHS group ,CP‐96345 + NaHS group、capsaicin + NaHS group and capsazepine + NaHS group .The mice were pretreated with vehicle ,CP‐96345 (2 .5 mg/kg ,i .p .) ,capsaicin (50 mg/kg ,s .c .) or capsazepine (15 mg/kg ,s .c .) and then received NaHS (10 mg/kg ,i .p .) or 0 .9% NaCl solution .One hour after intervention ,the mice were sacrificed ,then the concentration of substance P in plasma and the levels of tumor necrosis factor‐alpha (TNF‐α) and interleukine‐1 beta (IL‐1β) in lung homoge‐nate were measured by ELISA assay .Myeloperoxidase (MPO) activity in lung tissues was detected by tetramethyl benzidine (TMB) .The pathological changes of lung tissues were stained with hematoxylin and eosin .On the other hand ,the prepro‐tachykinin‐A (PPT‐A) gene knockout mice (eight‐week old ,female ,PPT‐A‐/‐) and their corresponding wild‐type Balb/c mice (eight‐week old ,female ,PPT‐A+ /+ ) were randomly given 0 .9% NaCl solution or NaHS (10 mg/kg ,i .p .) .One hour after intervention ,the mice were sacrificed ,then MPO activity ,TNF‐α and IL‐1β levels in lung tissues as well as the pathological changes of lung tissues were examined .Results:Hydrogen sulfide donor drug NaHS elevated significantly plasma substance P concentration (P<0 .05) ,lung MPO activity as well as lung TNF‐αand IL‐1βlevels (all P<0 .05) ,resulting in lung inflam‐matory injury .The knockout of PPT‐A gene ,or pretreatment with CP‐96345 ,capsaicin or capsazepine abolished significantly the elevation of lung MPO activity ,lung TNF‐αand IL‐1βlevels ,as well as the lung injury induced by NaHS (all P<0 .05) . Conclusions :Hydrogen sulfide may induce lung inflammatory injury through stimulating the release of substance P in nerve end‐ing s .

Objective To investigate the intra- and interobserver repeatability of coronary artery disease (CAD) diagnosis based on invasive coronary angiography (ICA) and CT coronary angiography (CTCA).Methods Two readers with comparable experience ( over 10 years) independently evaluated ICA results of 42 consecutive patients with a blind method. After 30 days,one of them reviewed the same patients again.Another two comparable-experience (over 10 years) readers evaluated the results of CTCA (prospectively ECG-triggering) from the same 42 patients in the same way.The inter-reader and intra-reader repeatability of ICA and CTCA were analyzed by performing Kappa test and calculating the percentage of the segments with agreement on stenotic degree.Using ICA as reference,the accuracy of CTCA in diagnosing CAD was studied by comparing the area under ROC. Results The Kappa between readers for ICA and CTCA were 0.91 and 0.81.Intra-reader Kappa were 0.92 and 0.83 respectively (x2 =509.4 and 432.5,all P ＜0.01 ).The percentage of the segments with agreement between readers on the degree of stenosis were 80.8％ (494/611) in ICA and 75.2％ (469/624) in CTCA ( x2 =2.75,P =0.10),and within the same reader,86.9％ (531/611)in ICA and 81.9％ (511/624) in CTCA(x2 =3.76,P =0.053).With≥ 50％narrowing as a CAD diagnosis criterion,the agreement rates for two readers were 96.6％ (590/611 ) in ICA and 94.4％ (589/624) in CTCA( x2 =3.36,P =0.07),and for the same reader,97.4％ (595/611) in ICA,95.4％ (595/624) in CTCA ( x2 =3.62,P =0.06).Using ICA as reference,two readers of CTCA results achieved a sensitivity and specificity of 84.9％ (530/624)and 98.1％ (612/624).The area under ROC was 0.94 (95％ CI 0.91-0.97).Conclusions Both ICA and CTCA demonstrate good repeatability in diagnosing CAD.The repeatability of ICA is superior to that of CTCA.A certain discrepancy exists in two readings from the same reader or two readers.

Objective To investigate the effect of rehabilitation on memory deficits after acquired brain injury, to compare different training models of memory rehabilitation and to analyze the possible factors affecting memory rehabilitation. Methods 144 patients with acquired brain injury following memory deficits were randomly assigned to computer-assisted training group, face-to-face training group and control group. Both training groups were given memory-based cognitive training program once a day which sustained 30 minutes for 6 or 12 weeks. The instantaneous memory, short-term memory and long-term memory were evaluated and compared before and after training. The effect of gender, age, education, course, site of injury and coma time on training efficacy were analyszed as well. Results 6 weeks and 12 weeks at training, both computer-assisted and face-to-face training groups showed a significant improvement in memory abilities when compared to controls (P<0.01), with the former making more progress (P<0.01). Negative correlation was found between age and memory performance. Conclusion Effectiveness of memory rehabilitation is proven. 12 weeks training can significantly improve memory. Cognitive training using professional equipment is significantly more effective than the face-to-face training and should be recommended.

Objective: To investigate the environmental contamination of norovirus in nurseries and primary/secondary schools, so as to provide a scientific basis for effective prevention and control measures. Methods: A total of 483 external environmental samples were collected from 34 cluster outbreaks of norovirus gastroenteritis in kindergartens and primary/secondary schools in Linhai City from 2021 to 2024. Pathogen detection was conducted using a rapid nucleic acid extraction kit and realtime fluorescence RT-PCR, and the results were analyzed using the χ2 test or Fishers exact test. Results: Among the collected external environmental samples, the total positive rate of surface contamination was 13.66%. The positive rates in kindergartens and primary/secondary schools were 12.20% and 15.82%, respectively. In kindergartens, the five surfaces with the highest detection rates were desks/chairs (23.33%), toilet stool troughs (20.69%), urinal troughs (12.00%), washbasins/sinks (11.11%), and toilet mops (9.38%). In primary/secondary schools, the top five were toilet stool troughs (38.30%), urinal troughs (23.53%), toilet door handles (13.04%), toilet mops (12.50%), and drinking cups (11.11%). The difference in positive detection rates among different external environments in primary/secondary schools was statistically significant (Fishers exact probability test, P<0.01). The positive detection rate in sanitary toilets was higher than that in classroom environments (χ2=17.38), while the positive detection rate in classroom environments of kindergartens was higher than that in primary/secondary schools (χ2=5.42)(P<0.05). Conclusions Norovirus exhibits a high contamination rate in nurseries and schools, particularly in restroom areas. Strengthening sanitation and disinfection in highrisk environments, and improving hygiene awareness among children and staff, are essential for the effective prevent and control of norovirus.