[Correlation of extracellular enzymes activity of Candida glabrata clinical isolates with in vivo pathogenicity in Galleria mellonella larvae].

Objective: To explore the relationship between extracellular enzymes activity and virulence of Candida glabrata clinical isolates based on the infection model of Galleria mellonella larvae. Methods: Using experimental research methods, 71 strains of non-repetitive Candida glabrata were collected from Qinghai Provincial People's Hospital from June 2021 to January 2022. Bovine serum protein agar medium, egg yolk agar medium, sheep blood agar medium, Tween-80 agar medium and triglyceride agar medium were used to detect the aspartyl protease activity, phospholipase activity, hemolysis activity, esterase activity and lipase activity of Candida glabrata. Median lethal concentration (LC50) was calculated by using 1.25×108 CFU/ml,2.50×108 CFU/ml,3.75×108 CFU/ml,5.00×108 CFU/ml suspension of Candida glabrata ATCC2001 to infect Galleria mellonella larvae. Histopathological and etiological analysis was performed to determine whether the infection model was successfully established. The clinical isolates of Candida glabrata were configured to infect Galleria mellonella larvae with LC50 concentration to detect the pathogenicity of Galleria mellonella larvae.Spearman test or Pearson test were used to analyze the correlation between the extracellular enzyme activity of Candida glabrata clinical isolates and the pathogenicity of Galleria mellonella larvae. Results: 71 strains of Candida glabrata isolated clinically were detected to have low hemolytic activity after 2 days of culture. Aspartyl protease was detected after 4 days of culture, among which 7 strains (9.86%), 19 strains (26.76%) and 45 strains (63.38%) showed low, medium and high aspartyl protease activity. After 7 days of culture, 71 strains did not detect phospholipase, esterase and lipase activities. Candida glabrata on Galleria mellonella larvae of LC50=2.5×108 CFU/ml Fungal spore were found in the intestinal tissue pathological section of Galleria mellonella larvae in the experimental group, and Candida glabrata was identified by the microbial Mass Spectrometry after culture, while no fungi were found in the pathological section and culture of the control group. Spearman test shows that, there was a linear positive correlation between aspartyl protease activity and the survival rate of Galleria mellonella larvae (r = 0.73, P<0.01), the difference was statistically significant.Pearson test shows that, there was no significant linear relationship between hemolytic activity and survival rate of Galleria mellonella larvae (r = 0.16, P = 0.34), the difference was not statistically significant. Conclusion: The clinical isolates of Candida glabrata in this study had aspartyl protease activity and low hemolytic activity, but no phospholipase, esterase and lipase activity. The activity of aspartyl aspartyl protease of Candida glabrata was positively correlated with the pathogenicity of Galleria mellonella larvae.

[Epidemiologic characteristics and drug resistance of isolated from blood culture escherichia coli in a hospital in Qinghai Province from 2016 to 2022].

Objective: To explore the drug resistance of Isolated From Blood Culture Escherichia coli (E. coli) in a hospital in Qinghai over the past seven years, to evaluate the ability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the homologous origin of E. coli, and to establish a protein fingerprint library to match with it, adjuvant clinical experience medication so as to provide the basis for the prevention and control of hospital-acquired infections. Methods: Retrospective analysis of blood cultures sent to hospitals from January 2016 to December 2022. Drug resistance and resistance changes in E. coli.A total of 1 841 E. coli strains were isolated from Qinghai Provincial People's Hospital from January 2016 to December 2022; all strains were identified by MALDI-TOF MS, and the VITEK2.0 drug sensitivity analyzer was applied for drug sensitivity analysis of the strains, and the mass spectrometry homology analysis and self-constructed protein fingerprint library were carried out by MALDI-Biotyper software; the protein fingerprint library was built by using WHONET5.6 software was used to statistically analyze the drug sensitivity results, SPSS23.0 software was used to analyze the relationship between fingerprint typing and drug sensitivity, and the χ2 test was used for intergroup comparisons. Results: A total of 1 841 strains of E. coli were detected in 4 582 positive blood culture specimens from January 2016 to December 2022, with a detection rate of 40.17%; the resistance rate of E. coli from blood sources to piperacillin/tazobactam and ceftriaxone was on the rise, and it was slightly decreased to cefepime, amikacin, levofloxacin, and sulfamethoxazole, and there was not much change to the rest of the drugs; After MALDI-Biotyper clustering analysis, the 1841 E. coli strains from Isolated From Blood Culture were classified into two major clusters and five subtypes, of which type Ⅰa1 accounted for about 40%, type Ⅰa2 accounted for about 2.7%, type Ⅰb accounted for about 3.8, type Ⅱa accounted for about 46%, and type Ⅱb accounted for about 7.5%. The detection rate of type Ⅰa1 E. coli was higher in general surgery (50.45%) and emergency surgery (50.92%), and the detection rate of type Ⅰb E. coli was higher in emergency medicine(10.05%)than in other departments. The drug sensitivity results of different subtypes were compared with each other, the resistance rate of type Ⅰa1 E. coli to cefepime was 21.3% higher than that of the remaining four types, and the difference was statistically significant (χ2=37.74,P=0.000); the resistance rate of type Ⅱ E. coli(>60%) to sulfamethoxazole was higher than that of type Ⅰ (<60%) as a whole, and the difference was statistically significant (χ2=15.248,P=0.004); and a preliminary database of homologous protein fingerprints of E. coli has been established E. coli homologous protein fingerprint library and validated. The drug susceptibility results of 1 288 E. coli strains in the validation set were statistically analyzed and compared with those in the training set. There was no significant difference(P>0.05). Conclusion: In recent years, the resistance rate of E. coli isolated from a hospital in Qinghai province to piperacillin/Tazobactam, cefepime, amicacin and other antibiotics has changed greatly. A fingerprint database of E. coli homologous protein was established, and it was found that the drug sensitivity data of E. coli were different among different fingerprint types. According to drug sensitivity, drug use could assist clinical experience and provide evidence for prevention and control of hospital illness.

Feedback regulation between orphan nuclear receptor TR2 and human papilloma virus type 16.

The human TR2 orphan receptor (TR2), initially isolated from testis and prostate cDNA libraries, is a member of the steroid receptor superfamily. TR2 can regulate several target genes via binding to a consensus response element (AGGTCA) in direct repeat orientation (AGGTCAX((n))AGGTCA, n = 0-6). Here we show that TR2 is able to induce the expression of human papilloma virus type 16 (HPV-16) genes via binding to a DR4 response element in the long control region of HPV-16. Additionally, one of the HPV-16 gene products, the E6 oncogene, regulates TR2 gene expression. A likely mechanism for this regulation involves E6-mediated degradation of the tumor suppressor p53, a protein known to suppress TR2 expression. Together our data provide evidence for feedback regulation between TR2 and HPV-16, which represents a novel regulatory pathway involving a member of the steroid receptor superfamily and the HPV-16 DNA tumor virus.

The p53/retinoblastoma-mediated repression of testicular orphan receptor-2 in the rhesus monkey with cryptorchidism.

Whereas the linkage of infertility to cryptorchidism, the failure of the testis to descend into the scrotum at birth, has been well documented, the detailed molecular mechanism remains unclear. Here we report that the testicular orphan receptor-2 (TR2) expression, which modulates many signal pathways, was completely repressed in the surgery-induced cryptorchidism of the rhesus monkey. Further studies link TR2 repression to the induction of p53 and results suggest that induced p53 could repress TR2 expression via the p53-->p21-->CDK-->Rb-->E2F signal pathway. In return, TR2 could also control the expression of p53 and Rb through the regulation of human papillomavirus 16 E6/E7 genes. Together, our data suggest a feedback control mechanism between TR2 and p53/Rb tumor suppressors, which might play important roles in male infertility associated with cryptorchidism.

Cloning of novel temperature-related expressed sequence tags in rat testis during spermatogenesis.

Spermatogenesis needs the relatively cool environment of the scrotum in most mammals, it would be arrested when the testis was exposed to abdominal temperature. In this study, we have used a differential display PCR technique (DD-PCR) to screen temperature-related ESTs during spermatogenesis (TRS) in scrotal testes through a unilateral cryptorchid rat model after in situ analysis of testis cell DNA fragmentation. We reported here the cloning and sequencing of three such ESTs: TRS1, TRS3, and TRS4. Northern blot analysis confirmed that they were expressed specifically in scrotal testes. In situ hybridization showed that TRS1 was mainly expressed in the spermatocytes and the round spermatids in scrotal testis. Homology searches revealed that TRS1 and TRS3 were unknown cDNA sequences, and TRS4 was identical to a known EST whose function had not been reported. TRS1, TRS2, and TRS3 were first found to be temperature-related during spermatogenesis.

Induction of an intronic enhancer of the human ciliary neurotrophic factor receptor (CNTFRalpha) gene by the TR3 orphan receptor.

A hormone response element, CNTFRalpha-NBRE (5'-AAAGGTCA-3') has been identified in the fifth intron of the alpha component of ciliary neurotrophic factor receptor gene (CNTFR-15) for the human TR3 orphan receptor (TR3). A specific binding between in vitro expressed TR3 and CNTFRalpha-NBRE was demonstrated by using electrophoretic mobility shift assay. A reporter gene assay using chloramphenicol acetyl-transferase (CAT) showed that CNTFR-15 has an enhancer activity that could be induced by TR3 in a dose-dependent manner. This induction was significantly reduced in the absence of CNTFRalpha-NBRE. Together, these results indicate CNTFRalpha-NBRE is sufficient to mediate TR3 action in inducing the enhancer activity of CNTFR-15. Our finding may, therefore, suggest CNTFRalpha is a target gene regulated by TR3 and expand the role of TR3 in the nervous system.

[Localization and expression of TR3 orphan receptor in mouse testis].

By means of in situ hybridization and immunohistochemistry, the expression and cellular localization of orphan receptor TR3 and its mRNA were observed. The results showed that both TR3 and its mRNA were expressed in a significant amount in mouse testis, and the expression level of TR3 mRNA was different in different seminiferous tubules. TR3 mRNA was specifically expressed in germ cells, mainly in spermatogonia and less advanced primary spermatocytes, whereas TR3 receptor protein was mainly localized in germ cells. It is suggested that TR3 may play an important role in regulating the early stage of germ cell development in mice.

Temporal expression of urokinase type plasminogen activator, tissue type plasminogen activator, plasminogen activator inhibitor type 1 in rhesus monkey corpus luteum during the luteal maintenance and regression.

Proteolytic activity generated by the plasminogen activator (PA) system has been associated with many biological processes. Using a pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-induced rhesus monkey corpus luteum (CL) model, we have studied how urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor type 1 (PAI-1), are temporally expressed in CL of rhesus monkey at the luteotropic and luteolytic periods. Slot blot analysis and in situ hybridization were performed to analyze the expression and distribution of uPA and PAI-1 messenger RNA (mRNA). Fibrin overlay was used to detect uPA and tPA activities. We found that uPA is the dominating PA in luteotropic CL in the monkey. Abundant expression of PAI-1 mRNA was detected. The highest expression of uPA and PAI-1 mRNA was observed at the luteotropic period, while their expression decreased approximately 50% at early luteal regression defined by considerably decreased serum progesterone levels, and remained at very low levels at the late stage of luteal regression. We also observed an increased tPA activity at the time of luteal regression. Moreover, the exogenous tPA could inhibit the progesterone production in cultured luteal cells from 13-day-old monkey CL. We also used LH receptor mRNA expression as a mark for the luteal phases. A highly expressed, evenly distributed LH receptor mRNA was detected in CL during the luteotropic phase, while its expression decreased at day 13 coinciding with the reduction of progesterone production. We conclude that proteolysis mediated by uPA and regulated by PAI-1 may play a role in the luteal maintenance, while tPA may participate in the luteal regression in the rhesus monkey.