ObjectiveTo explore the differences in human papillomavirus (HPV) genotype among patients with varying stages of cervical intraepithelial neoplasia (CIN). MethodsThis retrospective study included 113 patients with CIN who were treated at the Third People’s Hospital of Hangzhou from April 2020 to March 2023. The patients were divided into three groups based on the progression of CIN: CIN Ⅰ (n=30), CIN Ⅱ (n=42), and CIN Ⅲ (n=41). The influencing factors of CIN progression were analyzed, and HPV genotyping was performed logistic regression analysis was used to examine relationship between HPV genotypes and the degree of CIN progression, and the interaction between HPV genotypes and CIN progression-related factors were assessed. ResultsHPV genotype analysis revealed that HPV16, HPV58, HPV18, and HPV52 were the most common genotypes among CIN patients. The proportion of negative HPV cases in CIN I patients (53.33%) was significantly higher than that in CIN Ⅱ (33.33%) and CIN Ⅲ (0) patients. The infection rates and multiple infection rates of HPV16, HPV58, HPV18, and HPV52 (3.33%, 3.33%, 0, 0, 0.) were significantly lower in CIN Ⅰ patients compared to CIN Ⅱ (7.14%, 4.76%, 2.38%, 4.76%, 2.38%) and CIN Ⅲ patients (24.39%, 19.51%, 12.20%, 14.63%, 12.20%) (P<0.05). After adjusting for confounding factors, HPV16, HPV18, HPV52, and HPV58 remained significantly associated with the progression of CIN (P<0.05). Having ≥2 sexual partners (OR=3.529, 95%CI: 2.756~6.925, P=0.025), abnormal discharge odor (OR=5.684, 95%CI: 2.439~8.942, P=0.036), contact bleeding (OR=2.679, 95%CI: 1.627~3.192, P=0.018), and chronic cervicitis (OR=3.894, 95%CI: 1.952~4.931, P=0.031) were independent factors influencing the progression of CIN. HPV16, HPV18, HPV52, HPV58 exhibited positive interactions with the number of sexual partners, abnormal discharge odor, contact bleeding, and chronic cervicitis (P<0.05). ConclusionHPV16, HPV58, HPV18, and HPV52 are the most common genotypes of HPV infection in CIN patients, which are positively correlated with the degree of CIN progression. These genotypes are also closely related to factors influencing CIN progression.

Objective To express recombinant Burkholderia pseudomallei(B.pseudomallei)type Ⅲ secretion system BipD protein,prepare its polyclonal antibodies and verify their immunological traits.Methods The recombinant pET-28a-BipD plasmid was generated,and the pET-28a-BipD-carried E.coli BL21(DE3)bacteria were induced with isopropyl-β-d-thiogalactoside(IPTG)to express recombinant BipD(rBipD)protein.The rBipD was obtained by affinity chromatography using His Trap column,then mixed with Fredrick's adjuvant to immunize BALB/c mice by intraperitoneal injection in order to obtain anti-rBipD polyclonal antibodies.The immunoreactivity of rBipD was detected by Western blot assay using rabbit anti-melioidosis serum and the serum from melioidosis patients.The immunogenicity of rBipD was evaluated using Western blotting and immunofluorescence staining.Finally,rBipD was used to establish an indirect ELISA to detect serum antibodies of clinical melioidosis patients.Results The recombinant plasmid pET-28a-BipD was successfully constructed and transformed into E.coli BL21(DE3)to induce rBipD expression with IPTG treatment.The obtained rBipD had a relative molecular weight of 36×103 and a purity of 95.4％,and had good immunogenicity and immunoreactivity.It could induce the production of specific antibodies after immunizing mice,and mouse polyclonal antibodies against rBipD were prepared with the titer of 1∶512 000.rBipD of 5.0 μg/mL produced specific immune response with the serum of melioidosis patients,but had no specific reaction with the serum of tuberculosis patients,with statistical difference(P＜0.01).Conclusion rBipD with immunological activity is successfully prepared and purified,and its polyclonal antibodies are also developed,which provide a good tool for clinical immunological diagnosis and study of immune mechanism of B.pseudomallei infection.

Objective To analyze the mechanism that Hcp1 protein in type Ⅵ secretion system of Burkholderia pseudomallei(B.pseudomallei)mediates the formation of multinucleated giant cells(MNGCs)when host cells are infected by the bacterium.Methods The mutant strain(BPC006 Δhcp1)and complementation strain(BPC006 Δhcp1::hcp1)were constructed by homologous recombination and plasmid complement technology,respectively.After RAW264.7 cells were infected with B.pseudomallei,the localization of Hcp1 in host cells was analyzed by immunofluorescence staining.The localization was further verified by cytoplasmic-membrane isolation in 293T cells after transfecting pCDNA4.1-Hcp1.The biological significance and effect of Hcp1 were explored by the anti-Hcp1 polyclonal antibody blocking and the formation of MNGC was detected by Giemsa staining.Results Western blotting showed that BPC006 Δhcp1 could not express Hcp1,while BPC006 Δhcp1::hcp1 restored Hcp1 expression.The above results proved that the mutant and complement strains were successfully constructed.Both cellular immunofluorescence co-localization and cytoplasmic-membrane isolation experiments showed that Hcp1 localized to host cell membranes.Last but not least,compared with the control group,anti-Hcp1 polyclonal antibodies inhibited the formation of MNGC(P＜0.01).Conclusion Hcp1 protein in type Ⅵ secretion system of B.pseudomallei is able to translocate to the RAW264.7 cell membranes and plays an important role in the formation of MNGCs.

Objective:To improve the understanding of primary pulmonary lymphoepithelioma-like carcinoma (PPLELC).Methods:A retrospective case series study was conducted. The clinical data of 31 patients with PPLELC who were admitted to the First Affiliated Hospital of Guangxi Medical University from January 2012 to June 2023 were retrospectively analyzed, and their clinical features were summarized. The correlations of organ metastasis, tumor stage, serum tumor markers, lactate dehydrogenase, and albumin with survival time were analyzed.Results:Among the 31 patients, 13 (41.9%) were male and 18 (58.1%) were female, aged (50±9) years old, with no smoking history in 24 cases (77.4%). The common clinical manifestations were cough(24 cases, 77.4%) and sputum (19 cases, 61.3%), and 7 patients (22.6%) were detected by physical examination; 24 cases (77.4%) had elevated levels of serum tumor markers, and the rest of the 7 cases (22.6%) had normal levels of various tumor markers. All of the patients had a single lesion, with a predominance of the right middle lung (8 cases, 25.8%), and 23 cases (74.2%) had lymph node metastasis. Immunohistochemical detection showed that the positive rate of CK was 67.7% (21/31), and the positive rates of squamous cell carcinoma markers CK5/6, p63 and p40 were 90.3% (28/31), 80.6% (25/31) and 77.4% (24/31), respectively. The positive rate of EBER in situ hybridization detection was 85.2% (23/27). Genetic testing showed 6 cases had epidermal growth factor receptor (EGFR) mutation. The median survival time [ M ( Q1, Q3)] of the groups without lymph node metastasis and with lymph node metastasis was 33.0 months (7.3 months, 9.3 months) and 19.0 months (7.0 months, 27.0 months), and the difference was statistically significant ( P < 0.001). The median survival time of patients with stage Ⅰ-Ⅱ and with stage Ⅲ-Ⅳ was 20.0 months (12.5 months, 42.0 months) and 18.5 months (6.5 months, 38.5 months), and the difference was statistically significant ( P = 0.002). One stage Ⅰ A patient was treated with surgery alone and survived at 92 months of follow-up. Ten cases were treated with immunotherapy and had a good outcome. Conclusions:PPLELC is prevalent in non-smokers, the lesions are mostly in the right middle lung, and it is easily misdiagnosed as squamous cell carcinoma. The positive EBER in situ hybridization detection can help the diagnosis; lymph node metastasis is common. Tumor stage, lymph node metastasis and CYFRA21-1 level may be correlated with the survival of patients. The patients can benefit from immunotherapy, and anti-angiogenic therapy combined with chemotherapy is an optional treatment regimen.

Objective To investigate the effect of plasma microRNA(miR)-93-5p on ovarian granulosa cell proliferation in patients with polycystic ovary syndrome(PCOS)and its possible mechanism.Methods A total of 120 PCOS patients of childbearing period who were treated at Jiaozuo People's Hospital from January 2022 to January 2023 were selected as the PCOS group,and 18 healthy women of childbearing period who were physically examined at the same hospital during the same period were selected as the control group.The age,body weight and height of the subjects in the two groups were recorded,and the body mass index(BMI)was calculated.Fasting venous blood was collected from the subjects during the follicular phase of the natural menstrual cycle or the progesterone-induced withdrawal bleeding phase,and serum levels of luteinizing hormone(LH),follicle-stimulating hormone(FSH),total testosterone(TT),anti-Mullerian hormone(AMH),aspartate aminotransferase(AST),alanine aminotransferase(ALT),fasting insulin(FINS),and fasting blood glucose(FBG)were measured by using the electrochemical method;and the homeostasis model assessment of insulin resistance(HOMA-IR)was calculated.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression level of miR-93-5p in plasma of patients in the two groups.Human ovarian granulosa cells(KGN cells)in logarithmic growth phase were cultured in 6-well plates with 3 × 105 cells per well,and they were randomly divided into the miR-93-5p mimics group,LY294002+miR-93-5p group,AZD5363+miR-93-5p group,negative control(NC)group,and blank control group.The KGN cells in the miR-93-5p group were transfected with miR-93-5p mimics;the KGN cells in the LY294002+miR-93-5p group were transfected with miR-93-5p mimics and treated with 50 mmol·L-1 LY294002 one hour before transfection;the KGN cells in the AZD5363+miR-93-5p group were transfected with miR-93-5p mimics and treated with 50 μmol·L-1 AZD5363 one hour before transfection;the KGN cells in the NC group were transfected with the negative control plasmids;and the KGN cells in the blank control group were not treated at all.The expression of miR-93-5p in the KGN cells in each group was detected by qRT-PCR,and the proliferation of the KGN cells in each group was detected by cell counting kit-8.Results There was no significant differences in age,FSH,ALT,and AST levels of patients between the PCOS group and the blank control group(P＞0.05).The BMI,TT,AMH,LH,FINS,FBG,and HOMA-IR of patients in the PCOS group were significantly higher than those in the blank control group(P＜0.05).The relative expression of miR-93-5p in plasma of patients in the PCOS group was significantly higher than that in the blank control group(t=-5.549,P＜0.001).miR-93-5p was moderately positively correlated with TT,FINS and HOMA-IR(r=0.434,0.622,0.586;P＜0.001)and was mildly positively correlated with FBG and LH(r=0.398,0.398;P＜0.001).The receiver operating characteristic curve showed that the optimal cut-off value for plasma miR-93-5p in diagnosing PCOS was 1.380,the area under the curve was 0.906(95％confidence interval:0.839-0.973,P＜0.001),the sensitivity was 0.858,the specificity was 0.833,and the Youden index was 0.691.The relative expression of miR-93-5p in the KGN cells in the miR-93-5p mimics group,LY294002+miR-93-5p group and AZD5363+miR-93-5p group was significantly higher than that in the blank control group and NC group(P＜0.05).After 24,48 and 72 hours of culture,the proliferation of the KGN cells in the miR-93-5p mimics group,LY294002+miR-93-5p group and AZD5363+miR-93-5p group was significantly higher than that in the blank control group and the NC group(P＜0.05);the proliferation of the KGN cells in the LY294002+miR-93-5p group and AZD5363+miR-93-5p group was significantly lower than that in the miR-93-5p mimics group(P＜0.05).Conclusion miR-93-5p in plasma is overexpressed in PCOS patients,and it may be involved in the occurrence and development of PCOS by mediating the proliferation of ovarian granulosa cells through the phosphoinositide 3 kinase/protein kinase B signaling pathway.The miR-93-5p level in plasma has a certain diagnostic value for PCOS.

Objective To explore the expression level of microRNA(miR)-320a-3p in peripheral blood in patients with polycystic ovary syndrome(PCOS)and its possible mechanism for regulating PCOS.Methods A total of 149 PCOS patients admitted to the Jiaozuo People's Hospital from July 2021 to July 2022 were selected as the research subjects(PCOS group),and 18 healthy volunteers with a regular menstrual cycle(28-35 d),no clinical or biochemical manifestations of hyperandrogenism,and no polycystic ovary changes detected by ultrasonography were selected as the control group.Clinical data of the subjects in the two groups were collected and compared.The expression level of miR-320a-3p in plasma of subjects in the two groups was detected by using the real-time quantitative polymerase chain reaction.The correlation between plasma miR-320a-3p expression and clinical indexes was evaluated by using the Pearson partial correlation analysis.The predictive value of miR-320a-3p for PCOS was analyzed by using the receiver operating characteristic curve.The relationship between miR-320a-3p and androgen receptor was evaluated by using the dual luciferase reporter gene assay.Results There was no significant difference in age,hip circumference,follicle stimulating hormone(FSH),prolactin(PRL),and high-density lipoprotein cholesterol(HDL-C)between the two groups(P＞0.05).The body mass index,waist circumference,total testosterone(TT),anti-Miillerian hormone(AMH),luteinizing hormone(LH),fasting insulin(FINS),fasting blood glucose(FBG),2-hour postprandial blood glucose(2 h BG),homeostasis model assessment of insulin resistance(HOMA-IR),total cholesterol,triglycerides(TG),and low-density lipoprotein cholesterol(LDL-C)of patients in the PCOS group were significantly higher than those in the control group,while the level of estradiol(E2)was significantly lower than that in the control group(P＜0.05).The relative expression level of miR-320a-3p in plasma of patients in the PCOS group was significantly lower than that in the control group(P＜0.05).The expression level of miR-320a-3p was moderately negatively correlated with TT(r=-0.594,P＜0.05)and slightly negatively correlated with waist circumference,FINS,2 h BG,HOMA-IR,and LDL-C(r=-0.293,-0.208,-0.227,-0.208,-0.208;P＜0.05),and showed no significant correlation with hip circumference,AMH,FSH,E2,PRL,FBG,TG,and HDL-C(r=-0.079,0.020,-0.042,0.089,0.005,-0.141,-0.116,0.059;P＞0.05).The area under the curve of miR-320a-3p for predicting PCOS was 0.968(95％confidence interval:0.922-1.000,P＜0.05),with a cut-off value of 0.515,a sensitivity of 0.917,a specificity of 0.789,and a Jordon index of 0.706.miR-320a-3p negatively regulated androgen receptors.Conclusion miR-320a-3p is abnormally low expressed in peripheral blood of PCOS patients and may participate in the occurrence and development of PCOS by negatively regulating androgen receptors to increase TT levels.It has high predictive value for diagnosing PCOS.

Objective To evaluate the efficacy of MOOC combined with flipped classroom teaching in clinical train-ing of urology.Methods A total of 100 clinical medical students from Peking Union Medical College were randomly assigned to either an experimental group or a control group.The experimental group adopted the teaching mode of MOOC combined with flipped classroom,while the control group adopted classic teaching method.The two groups were compared in terms of their theoretical exam scores,case analysis skills and teaching satisfaction level.Results There were no significant difference in theoretical examination scores between experimental group and con-trol group(the mean value of scores are 45.12 and 44.50,respectively).However,the interview scores from ex-perimental group was significant higher than that of control group(the mean value of scores are 42.28 and 40.10,respectively,P＜0.001).The MOOC combined with flipped classroom mode significantly improved students'capacity building of clinical reasoning for diagnosis and communication skills.Students were more willing to continue receiving this teaching mode.Conclusions The integration of MOOC with the flipped classroom model sig-nificantly enhances the quality and efficacy of urology teaching.

Objective:To analyze and determine the clinical and genetic characteristics of children with Helsmoortel-Van der Aa syndrome(HVDAS).Methods:Clinical data of 6 children with HVDAS treated at the First Hospital of Peking University from November 2018 to October 2022 and their family members were collected and analyzed retrospectively.Whole exome sequencing was performed on children and their family members to identify the genetic variants.Genotype and phenotype correlation was analyzed.Results:(1) Clinical analysis results: among the 6 children, there were 5 boys and 1 girl, and their age at diagnosis ranged from 11 months and 17 days to 12 years and 9 months.Six patients all presented with developmental delays/intellectual disabilities; (2) Genetic analysis results: 6 de novo ADNP variants were discovered in 6 children, including 1 initial codon deletion variant c. 1_2del, 2 nonsense variants c. 1175dup, p.(Tyr392*) and c. 2213C>G, p.(Ser738*), and 3 frameshift variants c. 2632dup, p.(Ser878Lysfs*3), c.1695_1696insATGGTATGTATGTATGTATG, p.(Val566Metfs*8) and c. 2120_2123del, p.(Asn707Serfs*8).All variants were classified as pathogenic variants by the American College of Medical Genetics and Genomics.Except the c. 2213C>G, p.(Ser738*), the other 5 variants are all novel variants that have not been reported before. Conclusions:All of the 6 cases of HVDAS showed typical clinical manifestations, and expanded the phenotype spectrum of microcephaly and tall stature.Six de novo mutations were discovered, expanding the ADNP mutation spectrum and providing accurate genetic counseling and prenatal genetic diagnosis of the disease.

Objective To investigate the status and influencing factors of sleep in preterm infants at 1 month corrected age.Methods 130 preterm infants admitted to the NICU of 3 tertiary hospitals in Hubei Province were recruited as participants during May 2021 and March 2022.Sleep assessment of preterm infants was conducted at 1 month corrected age.Infants'sleep was assessed using the Brief Infant Sleep Questionnaire(BISQ).Multivariate linear regression model was used to analyze the influencing factors of sleep status(sleep latency,night awakenings,nocturnal sleep duration,daytime sleep duration,24 h sleep duration)of preterm infants.Results A total of 124 preterm infants completed the follow-up at 1 month corrected age.Multiple linear regression analysis showed that feeding patterns and sleep initiation patterns affected the sleep latency;gestational age,mechanical ventilation duration,maternal education level,and sleep initiation patterns were influencing factors of night awakenings;nocturnal sleep duration was influenced by mechanical ventilation duration and feeding patterns;daytime sleep duration and 24 h sleep duration were influenced by feeding patterns and maternal educational level.Conclusion The preterm infant sleep is influenced by gestational age,duration of mechanical ventilation,feeding patterns,maternal education level,and sleep initiation patterns.Neonatology staff should focus on the preterm infants discharged from NICU and develop targeted intervention plans based on the determined influencing factors to improve the sleep status of preterm infants.

Objective:To explore the clinical and genetic characteristics of three children with 22q13 deletion syndrome. .Methods:Clinical data were collected and copy number variations in the patients and their parents were detected by using array-based comparative genomic hybridization (aCGH) and copy number variation sequencing (CNV-seq). The DECIPHER, ClinGen, OMIM, PubMed and Gene Review databases were retrieved for pathogenicity analysis. .Results:The common phenotypes of the three children have included variable global developmental delay, among which speech delay was the most obvious. Patient 1 had abnormalities of corpus callosum shown by magnetic resonance imaging. Patient 2 had dental crowding, pale skin, thick palms, hypotonia, and other facial features. Patient 3 had the mildest symptoms including language dysfunction, which has caught up with the development and improved significantly. All of the three children had harbored de novo deletions of 22q13.33q13.33 region, which spanned 0.84 Mb, 8.70 Mb and 0.90Mb and involved 37, 126, and 34 genes, respectively.Conclusion:Above finding has enriched the clinical and genetic characteristics of 22q13 deletion syndrome and laid a foundation for genetic counseling and prenatal diagnosis.