Objective To investigate the availability of early gestation termination with low frequency ultrasound.Methods The 10th day rabbit embryos were irradiated with low frequency focused ultrasound of 6 different levels of sound intensity.The rates of embryonic mortality were observed and the expression of p53 was detected by using immunohistochemistry 10 days after the irradiating.Results The rates of embryonic mortalities were 25.00%,72.88 % and 100% with the intensity of 30 W/cm~2,35 W/cm~2 and 40 W/cm~2 for 60 seconds,respectively.The expression of p53 in placenta cells increased after irradiating,peaked at 96 hours,and recovered to control level at 192 hours.Conclusion It is feasible for early pregnancy termination with low frequency ultrasound.The low frequency ultrasound may induce the increased expression of p53 in placenta cells,but the expression may be recovered.

Objective To develop a rapid molecular biological method for detection of the asymptomatic infection of Leish?mania. Methods Two pairs of primers named RV1?RV2 and K13A?K13B were selected to be the fast diagnosis primers since they were designed according to the conserved region of Leishmania kinetoplast DNA(kDNA)minicircles. The PCR amplifica?tion products of Leishmania donovani promastigote from Shandong Province were sequenced to compare their conservatism. The method was applied to detect 105 venous blood samples from healthy home canine and 7 venous blood samples from home canine suffered from Kala?azar in Heishui County of Sichuan Province,and 75 venous blood samples from susceptible population(no leishmaniasis symptoms)and 7 venous blood samples from patients in Xinjiang Kashi area in order to verify the feasibility and accuracy of the method. Results The size of PCR products was consistent with the expected fragments with high conservative among Leishmania species. The positive rates of 105 home canine samples and 75 susceptible population samples were 37.14%(39/105)and 82.67%(62/75)rspectively,and the positive rates of home canine suffered from Kala?azar and patients were all 100%(7/7). Conclusion This rapid diagnosis method is suitable for detection of asymptomatic infection of Leishmania in Kala?azar endemic areas of China with high sensitive and specific,thus it has bright perspective to be used.

Objective To establish the primary cat intestinal epithelial cells(IECs)culture methods and construct the cD-NA library for the following yeast two-hybrid experiment,so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection ,by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMARTTM technology,and then the double-strand cDNAs were acquired by LD-PCR,which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recom-bination. Matchmaker?Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calcula-tion of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of colla-genase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1× 106 independent clones. The titer was 2.8 × 109 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research,and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

Objective:To investigate the azole susceptibility of Candida albicans (C. albicans) from vulvovaginal candidosis patients and to analyze the relationship between ERG11 gene mutations in these isolates and azole resistance.
Methods:Three hundred and two clinical isolates of Candida species were collected. Azole susceptibility was tested in vitro in microdilution studies. The ERG11 genes of 17 isolates of C. albicans (2 susceptibles, 5 dose-dependent resistants and 10 resistants) were amplified and sequenced.
Results:Of the 302 isolates collected, 70.2%were C. albicans, of which 8.5%, 3.8%and 4.2%were resistant to fluconazole, itraconazole and voriconazole, respectively. In total, 27 missense mutations were detected in ERG11 genes from resistant/susceptible dose-dependent isolates. Among them, Y132H, A114S, and Y257H substitutions were most prevalent and were known to cause fluconazole resistance. G464S and F72S also has been proved to cause fluconazole resistance. Two novel substitutions (T285A, S457P) in hotspot regions were identified.
Conclusions:Twenty seven mutations in the ERG11 gene were identified in azole-resistant C. albicans isolates, which indicated a possible relation with the increase in resistance to azole drugs and the recurrence of vulvovaginal candidosis. The relationship of two novel substitutions (T285A, S457P) with fluconazole resistance needs to be further verified by site-directed mutagenesis.

OBJECTIVETo investigate the effects of Osterix (Osx) overexpression on the osteogenic differentiation of human periodontal ligament cells in response to mechanical force.

METHODSHuman periodontal ligament cells were isolated and cultured in vitro with explant method. Cells were transfected with either an Osx expression vector pcDNA3.1 flag-Osx or the mock control vector pcDNA3.1 flag. Then, cells were centrifuged for 6 h. After transfection and centrification, the expression of Osx mRNA and protein in untransfected cells, mock-transfected cells and Osx-transfected cells were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. Furthermore, the changes of mRNA expressions of core-binding factor cal (Cbfal), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OC), bone sialoprotein(BSP) and collagen protein al (Col I ) genes were measured to assess the differentiation of human periodontal ligament cells.

RESULTSAt 24 h after transfection, Osx mRNA and protein level increased significantly in Osx-transfected cells (P < 0.01), while there were no significant difference in Osx mRNA and protein levels between mock-transfected cells and untransfected cells(P > 0.05). Simultaneously, the upregulated mRNA expressions of all the five osteogenic genes were observed (P < 0.05, P < 0.01). After 6 h of mechanical stimulation, a significant increase in Osx expression was shown in all three groups. However, compared to mock-transfected and untransfected cells, Osx-transfected cells further showed the highest Osx mRNA and protein expression level. Furthermore, the mRNA expressions of all five osteogenic markers in Osx-transfected cells also exhibited the greater increase and showed the highest levels.

CONCLUSIONThe overexpression of Osx promotes the mechanical stress-induced osteogenic differentiation of human periodontal ligament cells. Osx may be essential for mechanical stress-induced differentiation of human periodontal ligament cells to osteoblas tic-like cells and be involved in orthodontic osteogenic remodeling.

Alkaline Phosphatase ; Cell Differentiation ; Cells, Cultured ; Humans ; Integrin-Binding Sialoprotein ; Osteocalcin ; Osteogenesis ; Osteopontin ; Periodontal Ligament ; RNA, Messenger ; Stress, Mechanical ; Transfection

OBJECTIVETo investigate miR-20a expression in human glioma and normal brain tissues and its effect on the proliferation of glioma cells in vitro.

METHODSThe expression of miR-20a was detected in human normal brain tissues and glioma tissues by real-time RT-PCR. miR-20a mimics were synthesized and transfected into U251 cells via liposome, and the cell proliferation were detected using MTT assay and flow cytometry.

RESULTSThe glioma tissues showed significantly up-regulated expression of miR-20a compared with normal brain tissues (P=0.035). The expression level of miR-20a was higher in high-grade than in low-grade gliomas. miR-20a mimics significantly enhanced the proliferation of U251 cells and the percentage of S-phase cells.

CONCLUSIONmiR-20a shows potent effect in promoting the growth of glioma cells, suggesting its important role in the pathogenesis of human glioma.

Adult ; Brain Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; Glioma ; genetics ; metabolism ; pathology ; Humans ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Real-Time Polymerase Chain Reaction ; Young Adult

Objective To study the protective effect of ROP2 nuclei acid vaccine in mice.Methods Forty-two BALB/c mice were divided into three groups.Each mouse in experiment group was injected with 50 ?g recombinant plasmid pc-DNA3-ROP2 through musculus quadriceps fexoris.In control groups,each mouse was injected with 50 ?g blank plasmid pc-DNA3 and with 50 ?l PBS respectively.All mice were immunized for three times with an interval of three weeks.The volume was doubled for the final injection in the two plasmid groups.Blood,spleens and lymph nodes of 4 mice in each group were taken for the detection of CD4+,CD8+ T cells and cytokines 2 weeks after the final immunization.The rest mice in 3 groups were challenged with 500 tachyzoites of Toxoplasm gondii RH strain for further observation.Results The vaccine induced strong cellular and humoral immune response.The titer of antibody in serum was high after inoculation and recognized ROP2 protein antigen expressed in vitro.The lymphocyte phenotype was analyzed.CD4+ T cells proliferated sharply(69.5?3.4)%,and the ratio of CD4+/CD8+ increased considerably by(4.69?1.32)%(P

Objective To explore the biological function of rhoptry protein 38(ROP38)of Toxoplasma gondii,and to iden?tify the reactogenicity of the recombinant protein(rROP38). Methods The ROP38 was amplified by RT?PCR from T. gondii RH strain,and was cloned into prokaryotic expression vector pET?28a(+). The recombinant plasmid was transformed into E. co?li BL21(DE3)competent cells. Then the rROP38 was analyzed by SDS?PAGE and identified by Western blot. Results SDS?PAGE showed that rROP38 was efficient expression with a molecular weight of about 43 kD. Western blot showed that rROP38 reacted with antibody of His tag or human positive antibody,which indicated that ROP38 had good reactogenicity and could be a serological diagnostic antigen. Conclusion The study successfully obtains the rROP38 of T. gondii with good reactogenicity.

Objective:To evaluate the effect of esketamine on postoperative delirium (POD) in elderly patients undergoing general anesthesia.Methods:Two hundred and twenty-four elderly patients, aged ≥ 65 yr, with American Society of Anesthesiologists Physical Status classification Ⅰ-Ⅲ, undergoing elective surgery under general anesthesia, were divided into 2 groups ( n=112 each) using a random number table method: esketamine group (S group) and control group (C group). Esketamine 0.5 mg/kg was intravenously injected before anesthesia induction in S group, while the equal volume of normal saline was given instead in C group.The Fuzzy Consciousness Assessment Scale (3D-CAM) was used to assess the occurrence of POD within 7 days after surgery.The consumption of propofol, remifentanil and sufentanil and use of vasoactive drugs were recorded during operation.The rescue analgesia within 48 h after operation and occurrence of postoperative complications were recorded. Results:Compared with C group, the incidence of POD was significantly decreased, the intraoperative consumption of remifentanil was reduced, and the utilization rate of vasoactive drugs, rate of rescue analgesia and incidence of postoperative vertigo, nausea and vomiting within 48 h after surgery were decreased in S group ( P<0.05). Conclusions:Esketamine can reduce the development of POD in elderly patients.

Objective:To evaluate the effective dose, efficacy and safety of remimazolam for induction of general anesthesia in super-elderly patients.Methods:Trial Ⅰ American Society of Anesthesiologists Physical Status classification Ⅰ-Ⅲ patients of either sex, aged ≥80 yr, with body mass index of 18-25 kg/m 2, undergoing elective surgery with general anesthesia in the Zhejiang Provincial People′s Hospital from January to March 2022, were selected. Remimazolam 0.12 mg/kg or propofol 0.8 mg/kg was intravenously injected in the first patient, and the dose of remimazolam or propofol in the next patient was determined by using the modified Dixon′s up-and-down method. The difference between the two successive doses was 0.01 mg/kg for remimazolam and 0.05 mg/kg for propofol. A positive response was defined as achieving an anesthesia depth (BIS value ≤ 65) within 5 min of administration. If the response was positive, the next patient received a lower dose, or conversely if negative, a higher dose was given in the next patient. The 50% effective dose (ED 50) and 90% effective dose (ED 90) of remifentanil and propofol and their 95% confidence intervals ( CIs) were calculated by Probit method. Trial Ⅱ One hundred and forty-six American Society of Anesthesiologists Physical Status classification Ⅰ-Ⅲ elderly patients of either sex, aged ≥80 yr, with a body mass index of 18-25 mg/kg, scheduled for elective surgery with general anesthesia from April to October 2023 in Zhejiang Provincial People′s Hospital, were selected and divided into 2 groups ( n=73 each) by using a random number table method: remimazolam group (R group) and propofol group (P group). R group was induced with intravenous remimazolam ED 90 and P group was induced with intravenous propofol ED 90, and the injection time was both 30 s. If the BIS value was still greater than 65 at 5 min after administration, remimazolam 0.05 mg/kg was intravenously added each time in R group and propofol 0.5 mg/kg was intravenously added each time in P group until the patient′s BIS value ≤65. The success of anesthesia induction, time for successful induction of anesthesia and rescue sedation were recorded. The occurrence of intraoperative injection pain, hypertension, hypotension, bradycardia, hypoxemia, intraoperative awareness and postoperative delirium, nausea and vomiting was also recorded. Results:Trial Ⅰ The ED 50 (95% CI) of remimazolam was 0.148 (0.139-0.157) mg/kg, and the ED 90 (95% CI) was 0.160 (0.153-0.202) mg/kg; the ED 50 (95% CI) of propofol was 0.824 (0.726-0.983) mg/kg, and the ED 90 (95% CI) was 0.916 (0.860-2.472) mg/kg. Trial Ⅱ Compared with group P, the time for successful induction of anesthesia was significantly prolonged, the incidence of intraoperative injection pain and hypotension was decreased ( P<0.05), and no significant change was found in the success rate of anesthesia induction, rate of rescue sedation, intraoperative hypertension, bradycardia, hypoxemia, postoperative delirium, and nausea and vomiting in group R ( P>0.05). Conclusions:The ED 50 and ED 90 of remimazolam for induction of general anesthesia are 0.148 and 0.160 mg/kg, respectively, in super-elderly patients. Compared to propofol, remimazolam has a slightly longer onset time, but it is safer when used for induction of general anesthesia in super-elderly patients.