Objective To explore the predictive value of serum interleukin-17A(IL-17A),pentraxin-3(PTX3)and serum amyloid A(SAA)expression in Kawasaki disease(KD)children for non-response to in-travenous immunoglobulin(IVIG).Methods A total of 120 KD children who received IVIG treatment in the hospital from January 2022 to December 2024 were selected as the research objects.According to the response to IVIG treatment,they were divided into the sensitive group(n=90)and the non-response group(n=30).The clinical data of all children were collected.The predictive value of serum IL-17A,PTX3 and SAA expres-sion alone and in combination for non-response to IVIG treatment were explored by receiver operating charac-teristic(ROC)curve.The influencing factors of non-response to IVIG treatment in KD children were explored by multivariate Logistic regression.Results The levels of serum IL-17A,PTX3 and SAA in the non-response group were higher than those in the sensitive group(P＜0.05).The area under the curve(AUC)of serum IL-17A,PTX3 and SAA levels and their combined detection for predicting non-response to IVIG treatment were 0.704(95％CI:0.659-0.749),0.769(95％CI:0.719-0.819),0.813(95％CI:0.768-0.863),and 0.922(95％CI:0.877-0.967),respectively.The AUC of the combined detection of the three was larger than those of the individual detection of serum IL-17A,PTX3 and SAA(Zcombination-IL-17A=8.465,P＜0.001,Zcombination-PTX3=12.791,P＜0.001,Zcombination-SAA=9.984,P＜0.001).There were no statistically significant differences in age,body mass index(BMI),gender,KD type,fever duration before initial IVIG treatment,time from onset to ini-tial treatment,conjunctival congestion,changes in fingers and toes,rash,lymph node enlargement,platelet(PLT)count,hemoglobin(Hb)between the two groups(P＞0.05).The white blood cell(WBC)count,neu-trophil count(NEU),alanine aminotransferase(ALT),aspartate aminotransferase(AST)in the non-re-sponse group were higher than those in the sensitive group(P＜0.05).Multivariate Logistic regression analy-sis showed that serum IL-17A(OR=2.555,95％CI:1.529-4.270),serum PTX3(OR=3.473,95％CI:1.940-6.216),and serum SAA(OR=3.022,95％CI:1.823-5.011)were the risk factors of non-response to IVIG treatment(P＜0.05).Conclusion The combined detection of serum IL-17A,PTX3 and SAA levels can be used as important biological markers for predicting non-response to IVIG in KD children,providing a theoretical basis for early identification of high-risk children.

OBJECTIVE: To assess the feasibility of first polar body transfer (PB1T) combined with preimplantation mitochondrial genetic testing for blocking the transmission of a pathogenic mitochondrial DNA 8993T>G mutation. METHODS: A Chinese family affected with Leigh syndrome which had attended the Reproductive Medicine Centre of the First Affiliated Hospital of Anhui Medical University in September 2021 was selected as the study subject. Controlled ovarian hyperstimulation was carried out for the proband after completing the detection of the mitochondrial DNA 8993T>G mutation load among the pedigree members. Mature MII oocytes were inseminated by intracytoplasmic sperm injection (ICSI), cultured in vitro for 5 to 6 days to the blastocyst stage, and trophoblastocytes were obtained by microbiopsy. Mitochondrial DNA testing (PGT-MT) and chromosomal aneuploidy (PGT-A) analyses were carried out after whole-genome amplification, and the embryos with zero mutation load were selected for transfer. Amniotic fluid and umbilical cord blood samples were collected during middle pregnancy and after birth respectively for mitochondrial DNA testing to verify the reliability of embryo screening. As an attempt, PB1 with good morphology of MII oocytes was selected for transfer into the enucleated oocytoplasm from healthy donors, followed by ICSI fertilization, blastocyst culture and PGT of embryos using the same procedure. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (No. 2021zhyx-B12). RESULTS: An antagonist protocol was used for ovarian stimulation, and a total of 19 oocytes were obtained, of which 14 MII were fertilized by ICSI, and 2 had developed into blastocysts. PGT-MT was carried out on biopsied trophoblastocytes, in which the mitochondrial DNA 8993T>G mutation load was not detected in one embryo, the other was 100% mutated, and the mutation loads of the remaining unfertilized eggs and developmentally arrested embryos ranged from 0% ~ 100%, presenting a clear biased distribution. With fully informed consent, one PGT-MT zero mutation load blastocyst was transferred and clinical pregnancy was achieved. Mitochondrial DNA and chromosomal testing of amniotic fluid cells during middle pregnancy had revealed no abnormalities. The proband had delivered a healthy boy through Caesarean section at 39⁺⁵ weeks of gestation, and no mutation was detected in the cord blood sample. Five well-formed PBs from 14 eggs were selected for PB1 transfer, followed by ICSI and culture, and two of the reconstituted embryos had formed blastocysts, with none of the above mutations detected in the biopsied samples. CONCLUSION The PGT-MT technology can help families affected with mitochondrial diseases to have healthy offspring. PB1 transfer in combination with ICSI and PGT-MT holds the promise of turning waste into treasure and providing an alternative means of fertility for such families.
Humans ; Preimplantation Diagnosis/methods* ; Female ; DNA, Mitochondrial/genetics* ; Genetic Testing/methods* ; Pregnancy ; Mitochondrial Diseases/genetics* ; Polar Bodies ; Adult ; Feasibility Studies ; Sperm Injections, Intracytoplasmic/methods* ; Embryo Transfer/methods* ; Mutation ; Male ; Blastocyst/metabolism* ; Pedigree

Objectives:To explore the influencing factors of inter-arm systolic blood pressure difference(sIAD)in young hypertensive population. Methods:A total of 12 895 young Kailuan employees aged≤40 years,who participated in the physical examination from 2010 to 2020,were enrolled in this study.All of them underwent blood pressure measurements of four limbs in supine position.Young hypertensive group(n=3 584)and young non-hypertensive group(n=3 584)were 1∶1 matched by sex and age(±1 year),and participants were further divided into sIAD＜10 mmHg(1 mmHg=0.133 kPa)and sIAD≥10 mmHg subgroups.A stepwise multivariate logistic regression model was established to analyze the determinants of sIAD≥10 mmHg. Results:The detection rate of sIAD≥10 mmHg was significantly higher in the young hypertensive group than in the young non-hypertensive group(31.72%vs.27.76%,P＜0.001).Stepwise multivariate logistic regression analysis showed that in young hypertensive population,ankle-brachial index(ABI)＜0.9,male,obesity,overweight,elevated low density lipoprotein cholesterol(LDL-C)level,and systolic blood pressure were positively associated with sIAD≥10 mmHg,while college education or above,physical exercise were negatively correlated with sIAD≥10 mmHg(all P＜0.05).In the young non-hypertensive population,ABI＜0.9,systolic blood pressure were positively correlated with sIAD≥10 mmHg,while age was negatively associated with sIAD≥10 mmHg(all P＜0.05). Conclusions:The detection rate of sIAD≥10 mmHg is higher in young hypertensive population than in young non-hypertensive population.Decreased ABI,male sex,obesity,overweight,increased LDL-C level,systolic blood pressure,college education and above,and physical exercise are the influencing factors of sIAD≥10 mmHg in young hypertensive population.

Objective:To construct a recombinant herpes simplex virus 2 (HSV-2) expressing enhanced green fluorescent protein (EGFP) using clustered, regularly interspaced, short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology.Methods:Four strategies for inserting exogenous EGFP gene into HSV-2 genome using CRISPR/Cas9 technology were designed: (1) conventional homology-directed repair: circular two homology arm donor-mediated gene knock-in; (2) linearized single homology arm donor-mediated gene knock-in; (3) homology-independent targeted integration; (4) conventional homology-directed repair-mediated by cell lines stably expressing Cas9 and sgRNA.Results:The recombinant virus HSV-2-EGFP was successfully constructed based on the second, the third and the fourth strategies. The second strategy was the most efficient, followed by the third and the fourth strategies. The purified recombinant virus could stably express green fluorescent protein in seven passages and shared similar growth characteristics in Vero cells to the parental virus.Conclusions:Linearized single homology arm donor could increase the efficiency of gene knock-in, and cell lines stably expressing Cas9 and sgRNA could increase the efficiency of gene knock-in mediated by homology-directed repair.

Objective:To investigate the effects of genomic location of a foreign gene in Shanghai-191 strain measles virus (MV) vector on gene expression and virus replication.Methods:The nucleotide sequence encoding S1 protein of SARS-CoV-2 was inserted at different positions in MV antigenome (the upstream of the N gene, between P and M genes, between H and L genes), and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and N, P, and L proteins, respectively. The transfected cells were lysed and the supernatants were used to infected Vero cells to harvest recombinant viruses. S1 proteins expressed by the recombinant viruses were identified by RT-PCR, indirect immunofluorescence assay, Western blot and ELISA. Growth kinetics of the recombinant viruses were analyzed.Results:Recombinant viruses were failed to be rescued when the S1 protein-coding sequence was cloned into the upstream of N gene. Two recombinant viruses, MV-M-S1 and MV-L-S1, were successfully rescued when cloning the S1 protein-coding sequence into the intergenic region between P and M genes, or H and L genes, and could express S1 protein. MV-M-S1 expressed more S1 protein than MV-L-S1, but the titer of MV-M-S1 was lower.Conclusions:Inserting a foreign gene at different positions in the MV genome might have different effects on gene expression and virus replication. This study provided reference for the subsequent construction of MV vector.

Objective:To evaluate the safety of two inactivated COVID-19 vaccines in a large-scale emergency use.Methods:Based on the "Vaccination Information Collection System", the incidence data of adverse reactions in the population vaccinated with the inactivated COVID-19 vaccines developed by Beijing Institute of Biological Products Co., Ltd and Wuhan Institute of Biological Products Co., Ltd, respectively, in emergency use were collected, and the relevant information were analyzed with descriptive epidemiological and statistical methods.Results:By December 1, 2020, the vaccination information of 519 543 individuals had been collected. The overall incidence rate of adverse reactions was 1.06%, the incidence rate of systemic adverse reactions was 0.69% and the incidence rate of local adverse reactions was 0.37%. The main systemic adverse reactions included fatigue, headache, fever, cough and loss of appetite with the incidence rates of 0.21%, 0.14%, 0.06%, 0.05% and 0.05%, respectively; the main local adverse reactions were injection site pain and injection site swelling with the incidence rates of 0.24% and 0.05%, respectively.Conclusion:The two inactivated COVID-19 vaccines by Beijing Institute of Biological Products Co., Ltd and Wuhan Institute of Biological Products Co., Ltd showed that in the large-scale emergency use, the incidence rate of general reactions was low and no serious adverse reactions were observed after the vaccinations, demonstrating that the vaccines have good safety.

Objective:To establish a new bile duct injury and repair model in mice by generating bile duct distal stricture and proximal dilatation.Methods:The mice were randomly divided into sham operation group, bile duct stricture (BDS) group and bile duct ligation (BDL) group. The dilated bile duct of BDS mice was injured and then repaired 14 days after the modeling operation. Biochemical markers were detected and histopathological changes were analyzed.Results:14 days after the establishment of the model, the body mass in BDL group was significantly lower than that of the sham group ( P<0.05), while the body mass in BDS group was similar to sham group. Compared with the sham group, the bile duct and gallbladder of the BDS group and BDL group were both prominently dilated, but the sum of the diameters of bile duct and gallbladder in BDS group was significantly smaller than that in the BDL group ( P<0.05). Indocyanine green fluorescence imaging confirmed that biliary tract of BDS group could still drain bile. Serum ALT, AST and TBil levels in the BDS group were slightly higher than those in the sham group (all P<0.05), but significantly lower than those in the BDL group ( P<0.05). Bile ducts of BDS mice were injured by notching and repaired with bile duct path. 30 days after the repairing, HE staining showed that the bile duct epithelium around the patch was arranged in orderliness. Immunohistochemistry confirmed the positive staining of green fluorescent protein (EGFP) and CK19 in those groups. Conclusion:This model of bile duct injury and repair in mice can provide a new model for the study of the mechanism of bile duct injury and repair and the evaluation of tissue engineering bile duct.

Objective:To analyze the characteristics of cardio-metabolic risk factors coronary artery disease(CAD)patients with in-stent restenosis(ISR)after percutaneous coronary intervention(PCI).Methods:A total of 169 patients undergone primary(T1)PCI and followed up and rechecked by coronary angiography(T2)in our hospital from 2010 to 2017 were enrolled.According to the results of coronary angiography during follow-up, patients were divided into the ISR group(n=66)and the control group(non-ISR or NISR, n=103). Patients were sub-grouped into the elderly ISR group(the E-ISR group, n=17), the elderly non-ISR group(the E-NISR group, n=19), the young ISR group(the Y-ISR group, n=49)and the young non-ISR group(the Y-NISR group, n=84). Clinical data including medical history, laboratory results, and coronary angiography records during the two hospital stays were collected.Cardio-metabolic risk factors and clinical features of coronary artery disease were analyzed for the groups.Results:The levels of systolic blood pressure, glycated hemoglobin A1c(HbA1c), fasting blood glucose(FBG), the percentage of patients with diabetes or myocardial infarction(MI)history, the coronary Gensini score and the percentage of patients with triple vessel disease were higher in the ISR group than in the NISR group(128.6±16.4) mmHg vs.(123.4±10.7) mmHg, P=0.023; (7.0±1.6)% vs.(6.2±0.9)%, P<0.001; 6.6 mmol/L vs.5.7 mmol/L, P<0.001; 53.0% vs.31.1%, P=0.004; 36.4% vs.20.4%, P=0.022; 40 vs.29, P=0.01; 57.6% vs.40.8%, P=0.033). The E-ISR group had higher systolic blood pressure and a lower glomerular filtration rate than the E-NISR group(135.9±27.2) mmHg vs.(124.1±10.8) mmHg, P=0.013; 77.5±14.4 ml·min -1·1.73 m -2vs.(84.8±9.4) ml·min -1·1.73 m -2, P=0.033), but the Y-ISR group had longer hypertension history, higher levels of low-density lipoprotein and HbA1c, and higher Gensini scores than the Y-NISR group[129.7 months vs.83.1 months, P=0.008; (2.78±1.08) mmol/L vs.(2.31±0.65) mmol/L, P=0.002; (7.2±1.7)% vs.(6.1±0.9)%, P<0.001; 41 vs.29, P=0.009]. Conclusions:Clinical features are different between elderly CAD patients and young and middle-aged CAD patients with ISR after PCI, and metabolic risk factors such as blood pressure, blood lipid and blood glucose levels should be proactively controlled

Objective:To evaluate the accuracy of pRIFLE criterion and KDIGO criterion for the diagnosis of acute kidney injury (AKI) after radical operations for tetralogy of Fallot in children from the perspective of postoperative outcomes.Methods:A total of 375 children, aged<8 yr, undergoing radical operations for tetralogy of Fallot, were selected continuously and retrospectively. According to the pRIFLE and KDIGO diagnostic criteria, postoperative AKI was diagnosed, and the children were classified into different AKI grades. The prognostic parameters (postoperative mechanical ventilation time, duration of intensive care unit (ICU) stay, postoperative length of hospital stay, total costs of hospitalization, and in-hospital mortality, etc.) were collected, and the differences among different AKI grades were compared. Logistic regression method was used to analyze the risk factors for prolonged postoperative length of hospital stay (≥14 days) when two different criteria were used to diagnose AKI. The children diagnosed as non-AKI by KDIGO criterion were further confirmed using pRIFLE criterion, and the prognostic parameters in the children who were diagnosed as AKI and non-AKI were compared.Results:When two different criteria were used to diagnose AKI after radical resection for tetralogy of Fallot, the incidence was 56.8% (pRIFLE criterion) and 40.0% (KDIGO criterion). AKI diagnosed according to the two criteria was the independent risk factor for prolonged postoperative length of hospital stay, and the levels of all the prognostic parameters (postoperative mechanical ventilation time, duration of ICU stay, postoperative length of hospital stay, total costs of hospitalization, and in-hospital mortality) were significantly higher in AKI children than in non-AKI (AKI grade 0) children ( P<0.01). Among the 225 children diagnosed as non-AKI according to the KDIGO criterion, 63 cases were diagnosed as AKI and 162 cases as non-AKI according to the pRIFLE criterion, however, there was no significant difference in each prognostic parameter between children with AKI and non-AKI ( P>0.05). Conclusion:The pRIFLE criterion has a higher sensitivity, while the KDIGO criterion produces better accuracy when used to evaluate the diagnosis of AKI after radical operation for tetralogy of Fallot in children from the perspective of postoperative outcomes.

Objective: To develop a real-time quantitative PCR (qPCR) assay for detecting Sendai virus gene copy number. Methods: The recombinant plasmid was constructed and a reference used to establish the real-time qPCR method with the TaqMan probe and verified for reproducibility and sensitivity. Results: The linear range of the developed real-time qPCR assay was 1.024 × 10³ ~5 × 10¹⁰ copies/μl, with an R² value of more than 0. 99. The standard recovery of the tested samples was 84.56%~119.48%. Conclusions Compared with traditional hernagglutination assay for particle number detection, this method has higher sensitivity, better repeatability and high quantitative accuracy. It can be used for the detection of particle number of vaccine with sendai virus as the carrier, so as to detect the specific activity of vaccine products.