Objective:To explore changes in immune status in patients with cervical intraepithelial neoplasia(CIN)after cer-vical conization and its relationship with prognosis of high-risk human papilloma virus(HR-HPV).Methods:A total of 227 patients with CINⅡ～Ⅲ admitted to Jinjiang Municipal Hospital(Shanghai Sixth People's Hospital Fujian Campus)from January 2019 to November 2022 were included to analyze HPV typing situation of different patients.Patients were divided into conversion group and persistent infection group according to postoperative conversion status.Different patients'postoperative immune indexes and regression were observed,and relationship between HPV types and immune indexes and postoperative regression was analyzed.Results:HPV types 16,33,52 and 58 were predominant in CINⅡ patients;HPV types 16 and 58 were predominant in CINⅢ patients.Differences between conversion and persistent infection groups were statistically significant regarding time to surgery,IL-10,TGF-β,TNF-α,CD4+T and CD4+T/CD8+T(P<0.05).After surgery,patients'CD8+T level decreased with time,CD4+T and CD4+T/CD8+T levels showed an increasing trend with time,and HPV types 16,33,52 and 58 were significantly correlated with immune indicators.Generalized linear mixed-effects models showed that CD8+T was a risk factor for patient regression,CD4+T and CD4+T/CD8+T were protective factors for patient regression.Generalized multifactor downscaling model interaction analysis showed that CD4+T,CD8+T,CD4+T/CD8+T and HPV typing were interactive on postoperative regression.Conclusion:Postoperative patients of cervical conization have decreased level of CD8+T and increased levels of CD4+T and CD4+T/CD8+T,CD8+T is risk factor for patient regression,while CD4+T and CD4+T/CD8+T are protective factors for patient regression.

Objective:To investigate the regulatory role and mechanism of synaptic vesicle protein 2A (SV2A) in apoptosis of drug-resistant epilepsy neurons.Methods:One hundred and fifty specific pathogen-free (SPF) Sprague-Dawley (SD) rats were randomly numbered; 20 rats were selected as a normal control group (NC group), and the remaining 130 rats were subjected to chronic epilepsy models of lithium-pilocarpine. Phenobarbital and phenytoin sodium for 2 weeks were given to these rats after modeling; fanally, 94 rats with chronic epilepsy were divided into a drug-resistant group (phenobarbital and phenytoin sodium-resistant epilepsy [PRE] group, reduction in episodes<50%, n= 55) and a drug-sensitive group (phenobarbital and phenytoin sodium-sensitive epilepsy [PSE] group, reduction in episodes by≥50%, n=38). Rats in the PRE group were further randomly divided into 5 groups, namely a up-regulated-SV2A PRE group (UPRE group), a down-regulated-SV2A PRE group (DPRE group), a up-regulated-SV2A control group (UPRC group), a down-regulated-SV2A control group (DPRC group), and a non-transfected PRE group; different types of lentivirus were given to the first 4 groups via stereotactic brain injection. Ten days after lentiviral transfection, virus detection was performed; and 2 weeks after lentiviral transfection, occurrence of epileptic seizures was observed, and after that, rats were sacrificed and the hippocampal tissues were collected for subsequent experiments. Western blotting was used to detect the expressions of SV2A, cyclophilin A (CyPA), apoptosis-inducing factor (AIF), and superoxide dismutase 2 (SOD2) in the cell nucleus or mitochondria; coimmunoprecipitation experiment was performed to observe the interaction among SV2A, CyPA and AIF proteins; immunofluorescent co-staining was used to observe the CyPA/AIF localization; mitochondrial damage was detected by electron microscopy; ATP content in the hippocampal tissues was detected by luciferase method, 8-hydroxy-2-deoxyguanosine (8-OHDG) expression was detected by immunofluorescent staining, and neuronal apoptosis rate was calculated by double staining with neuron-specific nuclear protein (NeuN) and TUNEL. Results:(1) Confocal microscopy revealed that the hippocampal tissues in the 4 transfected groups showed green fluorescence inherent to the lentivirus, indicating successful viral infection. Compared with the UPRC group, the UPRE group had significantly reduced frequency of epileptic seizures and seizure duration ( P<0.05). (2) Western blotting showed that, in mitochondria, the UPRE group had significantly higher expressions of SV2A (0.475± 0.105 vs. 0.136±0.043), CyPA (0.473±0.041 vs. 0.175±0.047), AIF (0.443±0.058 vs. 0.131±0.037), and SOD2 (0.457±0.037 vs. 0.152±0.038) compared with the UPRC group ( P<0.05); the DPRE group had significantly decreased expressions of SV2A (0.038±0.013 vs. 0.184±0.047), CyPA (0.041±0.010 vs. 0.214±0.040), AIF (0.040±0.019 vs. 0.175±0.046), and SOD2 (0.043±0.017 vs. 0.187±0.039) compared with the DPRC group ( P<0.05). In the cell nucleus, the UPRE group had significantly lower expressions of AIF (0.336±0.084 vs. 0.649±0.209) and CyPA (0.331±0.086 vs. 0.620±0.162) compared with the UPRC group ( P<0.05); the DPRE group had statistically higher expressions of AIF (0.771± 0.180 vs. 0.519±0.144) and CyPA (0.738±0.223 vs. 0.488±0.091) compared with the DPRC group ( P< 0.05). (3) Co-immunoprecipitation experiment results showed that the bidirectional precipitation results of SV2A and AIF, SV2A and CyPA, and AIF and CyPA were all positive, suggesting existence of interactions. (4) Immunofluorescent co-staining showed that the fluorescence changes of CyPA and AIF were consistent. (5) Electron microscopy showed that mitochondria in the NC group had intact structure; mitochondria in the UPRE group, UPRC group, DPRC group and DPRE group showed swelling and cristae fragmentation; among them, injury in the UPRE group was relatively less severe than that in the UPRC group, while that in the DPRC group was more severe than that in the DPRE group. (6) Compared with the UPRC group, the UPRE group had statistically higher ATP content ( P<0.05); compared with the DPRC group, the DPRE group had significantly lower ATP content ( P<0.05).(7) Immunofluorescent staining results showed that the UPRE group had significantly lower 8-OHDG expression than the UPRC group ( P<0.05); compared with the DPRC group, the DPRE group had statistically higher 8-OHDG expression ( P<0.05). (8) The UPRE group had significantly lower NeuN-TUNEL double staining positive rate than the UPRC group ( P<0.05); compared with the DPRC group, the DPRE group had significantly higher NeuN-TUNEL double staining positive rate ( P<0.05). Conclusion:SV2A can play a role in the apoptosis of hippocampal neurons in rats with drug-resistant epilepsy by regulating the nuclear translocation of AIF/CyPA and mitochondrial damage.

Objective:To explore changes in immune status in patients with cervical intraepithelial neoplasia(CIN)after cer-vical conization and its relationship with prognosis of high-risk human papilloma virus(HR-HPV).Methods:A total of 227 patients with CINⅡ～Ⅲ admitted to Jinjiang Municipal Hospital(Shanghai Sixth People's Hospital Fujian Campus)from January 2019 to November 2022 were included to analyze HPV typing situation of different patients.Patients were divided into conversion group and persistent infection group according to postoperative conversion status.Different patients'postoperative immune indexes and regression were observed,and relationship between HPV types and immune indexes and postoperative regression was analyzed.Results:HPV types 16,33,52 and 58 were predominant in CINⅡ patients;HPV types 16 and 58 were predominant in CINⅢ patients.Differences between conversion and persistent infection groups were statistically significant regarding time to surgery,IL-10,TGF-β,TNF-α,CD4+T and CD4+T/CD8+T(P<0.05).After surgery,patients'CD8+T level decreased with time,CD4+T and CD4+T/CD8+T levels showed an increasing trend with time,and HPV types 16,33,52 and 58 were significantly correlated with immune indicators.Generalized linear mixed-effects models showed that CD8+T was a risk factor for patient regression,CD4+T and CD4+T/CD8+T were protective factors for patient regression.Generalized multifactor downscaling model interaction analysis showed that CD4+T,CD8+T,CD4+T/CD8+T and HPV typing were interactive on postoperative regression.Conclusion:Postoperative patients of cervical conization have decreased level of CD8+T and increased levels of CD4+T and CD4+T/CD8+T,CD8+T is risk factor for patient regression,while CD4+T and CD4+T/CD8+T are protective factors for patient regression.

Objective:To investigate the regulatory role and mechanism of synaptic vesicle protein 2A (SV2A) in apoptosis of drug-resistant epilepsy neurons.Methods:One hundred and fifty specific pathogen-free (SPF) Sprague-Dawley (SD) rats were randomly numbered; 20 rats were selected as a normal control group (NC group), and the remaining 130 rats were subjected to chronic epilepsy models of lithium-pilocarpine. Phenobarbital and phenytoin sodium for 2 weeks were given to these rats after modeling; fanally, 94 rats with chronic epilepsy were divided into a drug-resistant group (phenobarbital and phenytoin sodium-resistant epilepsy [PRE] group, reduction in episodes<50%, n= 55) and a drug-sensitive group (phenobarbital and phenytoin sodium-sensitive epilepsy [PSE] group, reduction in episodes by≥50%, n=38). Rats in the PRE group were further randomly divided into 5 groups, namely a up-regulated-SV2A PRE group (UPRE group), a down-regulated-SV2A PRE group (DPRE group), a up-regulated-SV2A control group (UPRC group), a down-regulated-SV2A control group (DPRC group), and a non-transfected PRE group; different types of lentivirus were given to the first 4 groups via stereotactic brain injection. Ten days after lentiviral transfection, virus detection was performed; and 2 weeks after lentiviral transfection, occurrence of epileptic seizures was observed, and after that, rats were sacrificed and the hippocampal tissues were collected for subsequent experiments. Western blotting was used to detect the expressions of SV2A, cyclophilin A (CyPA), apoptosis-inducing factor (AIF), and superoxide dismutase 2 (SOD2) in the cell nucleus or mitochondria; coimmunoprecipitation experiment was performed to observe the interaction among SV2A, CyPA and AIF proteins; immunofluorescent co-staining was used to observe the CyPA/AIF localization; mitochondrial damage was detected by electron microscopy; ATP content in the hippocampal tissues was detected by luciferase method, 8-hydroxy-2-deoxyguanosine (8-OHDG) expression was detected by immunofluorescent staining, and neuronal apoptosis rate was calculated by double staining with neuron-specific nuclear protein (NeuN) and TUNEL. Results:(1) Confocal microscopy revealed that the hippocampal tissues in the 4 transfected groups showed green fluorescence inherent to the lentivirus, indicating successful viral infection. Compared with the UPRC group, the UPRE group had significantly reduced frequency of epileptic seizures and seizure duration ( P<0.05). (2) Western blotting showed that, in mitochondria, the UPRE group had significantly higher expressions of SV2A (0.475± 0.105 vs. 0.136±0.043), CyPA (0.473±0.041 vs. 0.175±0.047), AIF (0.443±0.058 vs. 0.131±0.037), and SOD2 (0.457±0.037 vs. 0.152±0.038) compared with the UPRC group ( P<0.05); the DPRE group had significantly decreased expressions of SV2A (0.038±0.013 vs. 0.184±0.047), CyPA (0.041±0.010 vs. 0.214±0.040), AIF (0.040±0.019 vs. 0.175±0.046), and SOD2 (0.043±0.017 vs. 0.187±0.039) compared with the DPRC group ( P<0.05). In the cell nucleus, the UPRE group had significantly lower expressions of AIF (0.336±0.084 vs. 0.649±0.209) and CyPA (0.331±0.086 vs. 0.620±0.162) compared with the UPRC group ( P<0.05); the DPRE group had statistically higher expressions of AIF (0.771± 0.180 vs. 0.519±0.144) and CyPA (0.738±0.223 vs. 0.488±0.091) compared with the DPRC group ( P< 0.05). (3) Co-immunoprecipitation experiment results showed that the bidirectional precipitation results of SV2A and AIF, SV2A and CyPA, and AIF and CyPA were all positive, suggesting existence of interactions. (4) Immunofluorescent co-staining showed that the fluorescence changes of CyPA and AIF were consistent. (5) Electron microscopy showed that mitochondria in the NC group had intact structure; mitochondria in the UPRE group, UPRC group, DPRC group and DPRE group showed swelling and cristae fragmentation; among them, injury in the UPRE group was relatively less severe than that in the UPRC group, while that in the DPRC group was more severe than that in the DPRE group. (6) Compared with the UPRC group, the UPRE group had statistically higher ATP content ( P<0.05); compared with the DPRC group, the DPRE group had significantly lower ATP content ( P<0.05).(7) Immunofluorescent staining results showed that the UPRE group had significantly lower 8-OHDG expression than the UPRC group ( P<0.05); compared with the DPRC group, the DPRE group had statistically higher 8-OHDG expression ( P<0.05). (8) The UPRE group had significantly lower NeuN-TUNEL double staining positive rate than the UPRC group ( P<0.05); compared with the DPRC group, the DPRE group had significantly higher NeuN-TUNEL double staining positive rate ( P<0.05). Conclusion:SV2A can play a role in the apoptosis of hippocampal neurons in rats with drug-resistant epilepsy by regulating the nuclear translocation of AIF/CyPA and mitochondrial damage.

Objective:To investigate the behavioral, electroencephalographic, and cognitive functional differences in drug-resistant epileptic rat models of cognitive impairment prepared by intraperitoneal injection of lithium chloride-pilocarpine followed by intracranial injection of pilocarpine or carbamylcholine.Methods:One hundred and sixty adult male SD rats were randomly divided into normal control group ( n=10), lithium chloride-pilocarpine group (establishing epileptic rat models by intraperitoneal injection of lithium chloride-pilocarpine, n=50), pilocarpine-pilocarpine group (intracranial injection of pilocarpine after intraperitoneal injection of lithium chloride-pilocarpine, n=50)and pilocarpine-carbamylcholine group (intracranial injection of carbamylcholine after intraperitoneal injection of lithium chloride-pilocarpine, n=50). Frequency and duration of spontaneously recurrent seizures (SRSs) were observed by video monitoring system, and 2 weeks after that, phenobarbital and phenytoin sodium were injected intraperitoneally to screen drug-resistant models. Frequency and amplitude of the epileptic waves in EEG were recorded by BL-420 Bio-signal Acquisition and Processing System. Novel object recognition experiment was used to detect the novel exploration, Y-maze free exploration experiment and new and different arm experiment were used to detect the spatial recognition and memory ability, and Morris water maze experiment was used to detect the spatial memory ability. Results:(1) Twenty-four rats (48.00%) survived in the lithium chloride-pilocarpine group, 25 (78.00%) in the pilocarpine-pilocarpine group, and 21 (65.62%) in the pilocarpine-carbamylcholine group; and ultimately 7, 9, and 8 drug-resistant epileptic rat models were identified, respectively; frequency and duration of SRSs in the pilocarpine-pilocarpine group and pilocarpine-carbamylcholine group were significantly higher/longer than those in the lithium chloride-pilocarpine group ( P<0.05). (2) The pilocarpine-pilocarpine group and pilocarpine-carbamylcholine group had significantly higher amplitude of the epileptic waves in EEG compared with the lithium chloride-pilocarpine group ( P<0.05); the frequency of the epileptic waves in EEG increased gradually in the lithium chloride-pilocarpine group, pilocarpine-pilocarpine group, and pilocarpine-carbamylcholine group ( P<0.05). (3) Discrimination index, accuracy, ratio of distance traveled in novel arm to total distance, and time of novel arm entries gradually decreased in the normal control group, lithium chloride-pilocarpine group, pilocarpine-pilocarpine group, and pilocarpine-carbamylcholine group, with significant differences ( P<0.05). (4) Compared with the normal control group, the pilocarpine-pilocarpine group and pilocarpine-carbamylcholine group had significantly decreased frequency in crossing the original platform ( P<0.05); compared with the normal control group, lithium-pilocarpine chloride group and pilocarpine-pilocarpine group, the pilocarpine-carbamylcholine group had statistically shorter distance of target quadrant activity ( P<0.05); number of entries in the target quadrant gradually decreased in the normal control group, lithium chloride-pilocarpine group, pilocarpine-pilocarpine group, and pilocarpine-carbamylcholine group, with significant differences ( P<0.05). Conclusion:Drug-resistant epileptic rat models established by intracranial injection of carbamylcholine after intraperitoneal injection of lithium chloride-pilocarpine have high survival rate, high SRSs rate, and severe cognitive impairment, which is suitable for studying drug-resistant epilepsy combined with cognitive impairment.

OBJECTIVE: To explore the genetic basis for a Chinese pedigree with 6q26q27 microduplication and 15q26.3 microdeletion. METHODS: A fetus with a 6q26q27 microduplication and a 15q26.3 microdeletion diagnosed at the First Affiliated Hospital of Wenzhou Medical University in January 2021 and members of its pedigree were selected as the study subject. Clinical data of the fetus was collected. The fetus and its parents were analyzed by G-banding karyotyping and chromosomal microarray analysis (CMA), and its maternal grandparents were also subjected to G-banding karyotype analysis. RESULTS: Prenatal ultrasound had indicated intrauterine growth retardation of the fetus, though no karyotypic abnormality was found with the amniotic fluid sample and blood samples from its pedigree members. CMA revealed that the fetus has carried a 6.6 Mb microduplication in 6q26q27 and a 1.9 Mb microdeletion in 15q26.3, and his mother also carried a 6.49 duplication and a 1.867 deletion in the same region. No anomaly was found with its father. CONCLUSION The 6q26q27 microduplication and 15q26.3 microdeletion probably underlay the intrauterine growth retardation in this fetus.
Female ; Humans ; Pregnancy ; East Asian People ; Fetal Growth Retardation/genetics* ; Karyotype ; Pedigree ; Prenatal Diagnosis ; Sequence Deletion ; Chromosome Duplication

OBJECTIVE: To explore the molecular pathogenesis of a Chinese pedigree affected with inherited protein C (PC) deficiency. METHODS: The protein C activity (PC:A) and protein C antigen (PC:Ag) of the proband and his family members were determined by a chromogenic substrate method and enzyme-linked immunosorbent assay, respectively. The proband was subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing of other members of the pedigree. RESULTS: The PC:A and PC:Ag of proband were reduced to 15% and 11%, respectively. The above parameters of his parents and elder sister were also decreased to approximately 50% of reference values. Next generation sequencing has revealed that the proband has harbored a heterozygous c.572_574delAGA (p.Glu191_Lys192delinsGlu) variant in exon 7 and a missense c.752C>T (p.Ala251Val) variant in exon 8 of the PROC gene. His father was heterozygous for the c.572_574delAGA variant, while his mother and elder sister were heterozygous for the c.752C>T variant. According to the American College of Medical Genetics and Genomics Standards and Guidelines, the c.572_574delAGA (p.Glu191_Lys192 delinsGlu) variant was predicted to be likely pathogenic (PS1+PM4+PP3). c.752 C>T (p.Ala251Val) variant was also likely pathogenic (PS1+PM1+PP3). CONCLUSION The deletional variant of c.572_574delAGA (p.Glu191_Lys192delinsGlu) in exon 7 and missense variant c.752C>T (p.Ala251Val) in exon 8 of the PROC gene probably underlay the inherited protein C (PC) deficiency in this pedigree. Above finding has enriched the spectrum of PROC gene variants and provided a basis for genetic counseling for this pedigree.
Humans ; China ; Mutation ; Pedigree ; Protein C/genetics* ; Protein C Deficiency/genetics* ; Male ; Female

Objective:To explore the genetic basis for a proband with Shprintzen-Goldberg syndrome (SGS).Methods:Whole exome sequencing was carried out to detect potential variants associated with the relevant phenotypes. Candidate variants were verified by Sanger sequencing of the patient and her family.Results:DNA sequencing revealed that that the proband has carried a de novo heterozygous missense c. 94C>G (p.Leu32Val) variant in exon 1 of the SKI gene (NM_003036), which has been reported previously. The same variant was not detected in either parent. Based on the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic (PS1+ PS2+ PM1+ PM2+ PP2+ PP3). Conclusion:The SKI c. 94C>G (p. Leu32Val) variant probably underlay the autosomal dominant SGS in this patient.

Similar to other studies of bacterial pathogens, current studies of the pathogenesis of Riemerella anatipestifer (RA) are focused mainly on in vitro culture conditions. To elucidate further the pathogenesis of RA in vivo, bacterial RNA was extracted from overnight tryptic soy broth cultures (in vitro) and from the blood of infected ducks (in vivo) for comparative RNA sequencing analysis. In total, 682 upregulated genes were identified in vivo. Among the upregulated genes, a signal transduction response regulator (ArsR) and a signal transduction histidine kinase (SthK) were predicted to be located on the same operon. A mutant was constructed by deletion of both of these genes. Duck infection tests showed that genes ArsR and SthK were related to the virulence of the pathogen in vivo. Differentially expressed genes identified by comparison of in vitro and in vivo conditions provided an insight into the physiological process of RA infection and provided an opportunity to identify additional virulence factors.
Ducks ; Genes, vif ; Histidine ; In Vitro Techniques ; Operon ; Phosphotransferases ; Physiological Processes ; Riemerella ; RNA, Bacterial ; Sequence Analysis, RNA ; Signal Transduction ; Virulence Factors ; Virulence