BACKGROUND:The hyperpoladzation-activated cyclic nucleotide-gated cation channel (HCN) gene is not increase the risk of induced cardiac arrhythmia,and can accept the modulation function of autonomic nervous system.Therefore,it is the first candidate gene for biological pacemakers.OBJECTIVE:To construct recombinant adenovirus vector containing human HCN4 gene and evaluate its transfection efficency in rat bone marrow mesenchymal stem cells (MSCs).DESIGN,TIME AND SETTING:The in vitro cytology-gene experiment was performed at the Lin Bai-xin Experimental Center,Second Hospital of Sun Yat-sen University from February to September 2008.MATERIALS:Ten SD rats were supplied by the experimental animal center of Sun Yat-sen University.Rlasmid pcDNA3.1-HCN4 containing target gene human HCN4,human embryo kidney 293 cells and Escherichia coll DH5α were preserved by our experimental center;Shuttle plasmid pShuttle-CMV containing green fluorescent protein gene and adenoviral backbone plasmid pAdxsi were bought from SinoGenoMax Co.,Ltd.METHODS:HCN4 cDNA segment was liberated from the cloning vector of pcDNA3.1-HCN4 via Hind Ⅲ+Xba Ⅰ,and subcloned into pShuttle-CMV,which was digested by I-Ceu Ⅰ +I-Sce Ⅰ double enzyme and subcloned into adenoviral plasmid to form recombinant adenovirus plasmid.Recombinant adenovirus plasmid was transfected into 293 cell lines by liposome,and the recombinant adenovirus AdHCN4 was packaged and transfected into rat MSCs.MAIN OUTCOME MEASURES:The identification of recombinant adenovirus plasmid vector,identification of recombinant adenovirus and its titration test;the transfection efficiency of recombinant adenovirus.RESULTS:Cloned sequence about 3.6kbp was obtained by Hind Ⅲ+Xho Ⅰ digestion after HCN4 cDNA segment was cloned into pShuttle-CMV.DNA sequencing results indicated that the clone location was correct.Recombinant adenovirus plasmid was cut into seven fragments while empty vector gained only six fragments after digested by Xho Ⅰ.The recombinant adenovirus was pathogenic to 293 cells after recombinant adenovirus plasmid was packaged in it.HCN4 cDNA (657bp) was amplified by PCR with virus titer of 2.5×10~(11) PFU/mL after transfected 293 cells with supematant.The efficiency of recombinant adenovirus infecting rat MSCs was about 90% when multiplicity of infection was 800.Rat MSCs expressed green fluorescence after transfection.CONCLUSION:The adenovirus vector with human HCN4 cDNA was established successfully,which can effectively transfect rat MSCs.

National traditional Chinese medicine (TCM) master professor Ban Xiuwen had rich experiences on treatment of gynecological diseases with unique insights on diagnosis and treatment for disorders of menstruation, leukorrhea, pregnancy and delivery. FemaleYin-Wei, which was a gynecological disorder, was recorded in the book ofHuang-Di Nei-Jing. However, due to different reasons, TCM study on femaleYin-Wei was far less than the study on male impotence. ProfessorBan Xiuwen had specific considerations for this disease on the book of Treatment of Miscellaneous Gynecological Diseases. In his understanding, femaleYin-Wei was due to four aspects, which were the innate deficiency, yang deficiency and coldness of the uterus; seven emotion damage, liver-qi soothing disorder; spleen-stomach deficiency,qi-blood deficiency; and phlegm-dampness stasis,qi stagnation. Through the analysis and study on case records of femaleYin-Wei treatment by national TCM master professorBan Xiuwen, this article briefly elaborated professorBan Xiuwen’s understandings on femaleYin-Wei treatment from etiology, pathogenesis, treatment according to syndrome differentiation, and clinical experiences. Two femaleYin-Wei cases treated by professorBan Xiuwen were given as examples in order to analyze the etiology, pathogenesis, treatment according to syndrome differentiation and treatment experiences in details. The therapeutic effects of both cases were obvious, which were also important study documents.

Objective Implantable cardioverter defibrillator(ICD)can effectively treat lifethreatening ventricular arrhythmias.The most common side effect is inappropriate discharge.This study analyzes the incidence and causes of inappropriate discharges of ICD in our hospital.Methods Forty.threepatients implanted with ICD in our hospital from November 2001 to October 2007 were involved in our study.Patients were followed-up regularly.All episodes recorded and stored in the ICD were analyzed.Results Seven of the 43 patients underwent ninety-six inappropriate discharges.Inappropriate discharges in six patients were caused by supraventricular tachyarrhythmias(SVT).In one patient the discharge was caused by noise.Most inappropriate discharges occurred in the first year after implantation.The history of atrial fibrillation before implantation is an independent predictor of inappropriate discharges.Conclusions The incidence of inappropriate discharge is 16.3%in our study and the most common cause is SVT.Most inappropriate discharges occur in the first year after implantation.Patients with atrial fibrillation history have a higher risk of inappropriate discharges.

Objective To explore the effects of Fractalkine (FKN) on the biological functions of human pancreatic cancer cell lines SW-1990 and PNAC-1.Methods Adenovirus mediated FKN-small interfering RNA (siRNA) was transfected into human pancreatic cancer cell lines SW 1990 and PNAC-1.The differences in proliferation and invasion ability between before and after FKN-siRNA transfection were determined by clone formation assay,MTT assay and cells invasion assay.After FKN-siRNA transfection,the expression of FKN,tumor necrosis factor (TNF)-α and interleukin (IL)-6 at protein and mRNA level in human pancreatic cancer cell were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR).The data were analyzed by one way analysis of variance.Results After human pancreatic cancer cell lines SW-1990 and PNAC-1 transfected with FKN-siRNA,the clone numbers (5.27 ％ ± 0.35 ％ and 4.60 ％ ± 0.30％ ) increased compared with those of control group ( 1.97％ ±0.25％ and 1.77％ ± 0.25％ ) and negative FKN-siRNA group (2.10％±0.30％ and 1.97％±0.25％),and the difference was statistically significant (F=113.51,103.86; both P＜0.05).The clone size was also enlarged.After human pancreatic cancer cell lines SW-1990 and PNAC-1 transfected with FKN-siRNA for 48 hours and 72 hours,the MTT test results showed the absorbance value (48 h:1.28±0.07 and 1.19±0.14; 72 h:1.49±0.11 and 1.52±0.16) was higher than that of control group (48 h:0.80±0.03 and 0.74±0.11;72 h:0.89±0.03 and 0.93±0.04) and negative FKN-siRNA group (48 h:0.85±0.02 and 0.76±0.05; 72 h:0.89±0.02 and 1.07±0.09),and the difference was statistically significant (F=83.80,71.99,17.19,23.51; all P＜0.05).The invasion ability assay showed that the invasion ability of FKN-siRNA transfected cells was stronger than that of control group and negative FKN-siRNA group,and the difference was statistically significant (F=37.37,9.08; both P＜0.05).After FKN-siRNA transfection,the expression of FKN at protein and mRNA level in SW-1990 and PNAC-1 cell line decreased (protein:F=118.93 and 88.62,mRNA:F=47.91 and 72.59),at the same time the expression of TNF-α and IL-6 at protein and mRNA level increased (protein:FTNF-α =112.90 and 77.88,FIL-6 =165.27 and 286.49,mRNA:FTNF-α ==47.93 and 45.19,FIL-6 =36.41 and 23.67),and the differences were statistically significant (all P values＜0.05).Conclusion With siRNA technology to silent FKN function,the proliferation and invasion ability of pancreatic cancer cell lines increased,which indicated FKN might inhibit certain biological functions of pancreatic cancer cells.

Objective To obtain novel vaccine candidate antigens against Schistosoma japonicum. Methods S. japonicum schistosomula cDNA library was screened by using sera of Microtus fortis that was naturally resistant to schistosomiasis. The positive clones were transformated into Escherichia coli BM25.8, E. coli clones containing the plasmid cultured in LB, and then selected for plasmid extraction, the plasmid DNA was digested by EcoRⅠand Hind Ⅲ, and analysed by agarose gel electrophoresis. The positive clones were also sequenced and the data were analysed through the internet Nucleotide BLAST software of NCBI and Expert Protein Analysis system of GeneRunner and HNN. Results Twelve positive clones were obtained after repeatedly immunoscreening the library and their sizes ranged from 300 bp to 1100 bp. Two novel genes (named as Sj-sMf1 and Sj-sMf2) with complete ORF were obtained. The deduced protein of Sj-sMf1 consisted of 93 amino acids while Sj-sMf2 consisted of 61 amino acids. Sj-sMf1 protein predicted containing one cAMP phosphorylation site and Casein kinase C phosphorylation site, respectively. Sj-sMf1 protein predicted containing one Casein kinase C phosphorylation site and two Protein kinase C phosphorylation sites. Conclusion Two novel genes predictably encoding unknown proteins are obtained from immunoscreening of Schistosoma japonicum schistosomula cDNA library by M. fortis sera.

Objective: To discuss the clinical value of neck circumference in screening for overweight and obesity in children and adolescents, so as to provide a theoretical basis and new ideas for overweight and obesity screening. Methods: From May to July 2023, a total of 2 493 children and adolescents aged 3 to 16 were sampled from three schools by the cluster random sampling method. General data, neck circumference, body mass index (BMI), waisttoheight ratio, and other information were collected. Mann-Whitney ranksum test, Spearman correlation analysis, and receiver operating characteristic (ROC) curve analysis were used to statistically analyze the data. Results: The prevalence of overweight and obesity in children and adolescents aged 3 to 16 was 33.05%, and the prevalence of abdominal obesity was 28.33%.The neck circumferences of boys and girls in the overweight and obese group showed statistically significant differences from the normal weight group during the preschool, schoolage, and adolescence periods (Z=-16.69--4.54, P<0.05). The neck circumferences of children of different genders were moderately positively correlated with BMI (r=0.50-0.79, P<0.01). ROC curve analysis showed that neck circumference had good accuracy in assisting the screening for the occurrence of overweight and obesity in boys and girls during the preschool, schoolage, and adolescence periods [the AUC for boys:0.80(0.71-0.89), 0.89(0.86-0.91), 0.84(0.81-0.87); the AUC for girls:0.76(0.67-0.86), 0.81(0.74-0.88), 0.73(0.68-0.77), P<0.01], the optimal neck circumference cutoff values for boys were 26.95, 28.95, 33.55 cm, and for girls were 25.05, 26.55, 28.35 cm respectively. The neck circumferences of boys and girls were moderately positively correlated with the waisttoheight ratio during the schoolage and adolescence periods (r=0.41-0.70, P<0.01). ROC curves showed that neck circumferences had good accuracy in assisting the screening for the occurrence of abdominal obesity in boys and girls during the schoolage and adolescence periods [the AUC for boys:0.89(0.86-0.92), 0.82(0.78-0.85); the AUC for girls:0.84(0.75-0.93), 0.87(0.75-0.93)], the optimal cutoff values for boys were 28.95, 32.75 cm; for girls were 26.45, 31.85 cm respectively. Conclusion The neck circumference can be applied to screen for overweight and abdominal obesity in children and adolescents, and can provide new ideas for the prevention and control of overweight and obesity in children and adolescents.

The present study was performed to investigate the seroprevalence and risk factors for Dirofilaria immitis infection in cats from Liaoning province, northeastern China. From October 2014 to September 2016, sera of 651 cats, including 364 domestic cats and 287 feral cats (332 females and 319 males) were assessed. They were tested for the presence of D. immitis antigen using SNAP Heartworm RT test kit. In this population, the average prevalence was 4.5%. Age and rearing conditions (feral or domestic) were found to be associated with the prevalence of D. immitis. The prevalence was significantly higher in feral cats compared with domestic cats (8.4% vs 1.4%, P < 0.01). There was no significant difference between males and females (4.7% vs 4.2%, P>0.05), but older cats (≥3 years old) showed a statistically higher prevalence compared with younger cats ( < 3 years old) in feral populations (16.8 vs 2.4%, P < 0.01), while the difference between the age groups was not statistically significant in domestic cats (2.4% vs 0.51%, P>0.05), all these results suggest that outdoor exposure time may be one of the most important factors for D. immitis prevalence in cats. Results reveal that D. immitis are prevalence in domestic and feral cats in northeastern China, which indicates that appropriate preventive measures should be taken to decrease the incidence of feline heartworm disease in Liaoning province, northeastern China.
Animals ; Cats* ; China* ; Dirofilaria immitis* ; Dirofilaria* ; Dirofilariasis ; Female ; Humans ; Incidence ; Male ; Prevalence ; Risk Factors ; Seroepidemiologic Studies*

Leishmaniasis is a prevalent cause of death and animal morbidity in underdeveloped countries of endemic area. However, there is few vaccine and effective drugs. Antimicrobial peptides are involved in the innate immune response in many organisms and are being developed as novel drugs against parasitic infections. In the present study, we synthesized a 5-amino acid peptide REDLK, which mutated the C-terminus of Pseudomonas exotoxin, to identify its effect on the Leishmania tarentolae. Promastigotes were incubated with different concentration of REDLK peptide, and the viability of parasite was assessed using MTT and Trypan blue dye. Morphologic damage of Leishmania was analyzed by light and electron microscopy. Cellular apoptosis was observed using the annexin V-FITC/PI apoptosis detection kit, mitochondrial membrane potential assay kit and flow cytometry. Our results showed that Leishmania tarentolae was susceptible to REDLK in a dose-dependent manner, disrupt the surface membrane integrity and caused parasite apoptosis. In our study, we demonstrated the leishmanicidal activity of an antimicrobial peptide REDLK from Pseudomonas aeruginosa against Leishmania tarentolae in vitro and present a foundation for further research of anti-leishmanial drugs.

OBJECTIVE: To analyze a child with 11β hydroxylase deficiency (11β-OHD) due to CYP11B2/CYP11B1 chimeric gene. METHODS: Clinical data of the child who was admitted to Henan Children's Hospital on August 24, 2020 were retrospectively analyzed. Peripheral blood samples of the child and his parents were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. RT-PCR and Long-PCR were carried out to verify the presence of chimeric gene. RESULTS: The patient, a 5-year-old male, had featured premature development of secondary sex characteristics and accelerated growth, and was diagnosed with 21 hydroxylase deficiency (21-OHD). WES revealed that he has harbored a heterozygous c.1385T>C (p.L462P) variant of the CYP11B1 gene, in addition to a 37.02 kb deletion on 8q24.3. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.1385T>C (p.L462P) was rated as a likely pathogenic variant (PM2_Supporting+PP3_Moderate+PM3+PP4). The results of RT-PCR and Long-PCR suggested that CYP11B1 and CYP11B2 genes have recombined to form a CYP11B2 exon 1~7/CYP11B1 exon 7~9 chimeric gene. The patient was diagnosed as 11β-OHD and effectively treated with hydrocortisone and triptorelin. A healthy fetus was delivered following genetic counseling and prenatal diagnosis. CONCLUSION 11β-OHD may be misdiagnosed as 21-OHD due to the potential CYP11B2/CYP11B1 chimeric gene, which will require multiple methods for the detection.
Child, Preschool ; Humans ; Male ; Adrenal Hyperplasia, Congenital/genetics* ; Cytochrome P-450 CYP11B2/genetics* ; Exons ; Retrospective Studies ; Steroid 11-beta-Hydroxylase/genetics*

Objective:To assess the value of serum tumor specific protein 70 (SP70) for prognostic stratification in acute myeloid leukemia (AML).Methods:A cohort study design was adopted. 129 newly diagnosed AML patients from September 2022 to January 2024 at the Hematology Department of the First Affiliated Hospital of Nanjing Medical University were included, as well as a control group consisted of 120 healthy individuals and 7 cases with benign hematologic diseases during the same period (total 127 cases). Clinical data were collected from Electronic Medical Records. According to the 2023 edition of the Chinese Leukemia Diagnosis and Treatment Guidelines, AML patients with good or moderate prognosis were categorized as low-to-intermediate risk, while those with poor prognosis were high-risk group. Univariate and multivariate logistic regression analyses were used to identify variables significantly associated with AML prognostic risk. ROC analysis was used to evaluate diagnostic performance. A nomogram for predicting patient prognostic risk was constructed by R 4.0.2 software, and the internal validation was performed using bootstrapping.Results:Among 129 AML patients, there were 71 males (55.0%) and 58 females (45.0%), with 42 (32.6%) classified as high-risk and 87 (67.4%) as low-intermediate risk. The high-risk group had a significantly higher median age [62 (48, 67) years] compared to the low-intermediate risk group [50 (35, 63) years, Z=-2.381, P=0.017], and a significantly higher proportion of males (30 patients, 71.4%) compared to the low-intermediate risk group (41 patients, 47.1%, χ 2=6.760, P=0.009). Multivariate logistic regression analysis indicated that serum SP70 ( OR=2.54, 95% CI: 1.68-3.84, P<0.001), hemoglobin (HB) ( OR=0.96, 95% CI: 0.93-0.99, P<0.05), and bone marrow blast (BM blast) ( OR=1.07, 95% CI: 1.02-1.13, P<0.05) were independent risk factors for high-risk prognosis in AML patients. ROC analysis showed that the area under the curve (AUC) for SP70 predicting high-risk patients was 0.908 (cut-off value of 5.74 ng/ml, 95% CI: 0.845-0.952, sensitivity 90.5%, specificity 82.8%). The combined model of serum SP70, HB, and BM blasts had an AUC of 0.931 (95% CI: 0.890-0.973); C-index=0.925 (95% CI: 0.876-0.963),with no statistically significant difference compared to serum SP70 alone ( Z=1.693, P>0.05). Conclusion:Serum SP70 may be a promising non-invasive molecular biomarker for prognostic stratification in AML.