Objective To investigate the mechanisms underlying the protection by punicalagin against ultraviolet B (UVB)-induced damage to keratinocytes.Methods Cultured human HaCaT keratinocytes were divided into several groups:blank control group receiving no treatment,punicalagin groups treated with various concentrations of punicalagin,UVB group irradiated with UVB at 30 mJ/cm2,combination groups pretreated with different concentrations of punicalagin followed by UVB radiation at 30 mJ/cm2.The concentrations of punicalagin were 5,10,20,40 and 80 μmol/L in the cell proliferation assay,10,20 and 40 μmol/L in the other assays.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of HaCaT cells,Hoechst and propidium iodide (PI) staining as well as flow cytometry to detect the apoptosis in cells,reverse transcription-PCR to quantify the mRNA expressions of matrix metalloproteinase-1 (MMP1) and tissue inhibitor of metalloproteinase-1 (TIMP1) in HaCaT cells,Western blot to determine the phosphorylation levels of the mitogen-activated protein kinase (MAPK) pathway-related proteins including P38,JNK and ERK.Statistical analysis was carried out by t test,one-way analysis of variance,and Dunnett's t-test.Results As the MTT assay showed,punicalagin at 10-40 μmol/L showed stronger pre-protective effects against UVB-induced damage to HaCaT cells compared with punicalagin at the other concentrations.The number of cells highly positive for both Hoechst and PI staining was larger in the UVB group than that in the blank control group,but smaller in the combination groups than in the UVB group.The percentage of apoptotic cells increased significantly in the UVB group compared with the blank control group (9.82％ ± 0.11％ vs.1.24％ ± 0.91％,P ＜ 0.01),but decreased significantly in the three combination groups (punicalagin (10,20 and 40 μmol/L) + UVB) compared with the UVB group (6.38％ ± 0.14％,5.24％ ± 0.17％ and 3.77％ ± 0.11％ vs.9.82％ ± 0.11％,all P＜ 0.01).Theexpression of MMP1 mRNA was significantly higher,but that of TIMP1 mRNA was significantly lower in the UVB group than in the blank control group (both P ＜ 0.01),whereas no statistically significant difference was observed in the expression of MMP1 or TIMP1 mRNA between the punicalagin groups and blank control group (all P ＞ 0.05).The pretreatment with punicalagin significantly reduced the expression level of MMP1 mRNA (P ＜ 0.01),but elevated that of TIMP1 mRNA (P ＜ 0.01) in the combination groups compared with the UVB group.As Western blot showed,the phosphorylation levels of P38,JNK and ERK were markedly increased in the UVB group (all P ＜0.01),but experienced no significant changes in the punicalagin groups (all P ＞ 0.05) compared with the blank control group,and decreased to different degrees in the combination groups compared with the UVB group (all P ＜0.01).Conclusion Punicalagin has a pre-protective effect on UVB-induced damage to HaCaT cells.

Background Exfoliation syndrome (XFS) is a systemic disease with abnormal accumulation of extracellular matrix.Researches showed that the single nucleotide polymorphisms (SNPs) of lysyl oxidase-like 1 (LOXL1) gene is associated with the pathogenesis of XFS in global population.However,the results are varied among different ethnicity and regions.Objective This study aimed to assess the association between LOXL1 gene polymorphisms and XFS in Uygur population.Methods One-hundred and fifty-two Uygur XFS patients without relativeness were enrolled from January to August in 2014,and 228 ethnicity-and gender-matched normal controls were recruited at the same period from the same region.Each individual underwent comprehensive eye examinations and 5 ml peripheral blood was collected.Genomic DNA was extracted from peripheral blood.PCR-ligase detection response (LDR) was used to determine the allele and genotype frequencies of the six SNPs rs12914489,rs4886467,rs4558370,rs4461027,rs4886761 and rs16958477 in the promoter region of LOXL1 gene.The distribution frequency between the patients and normal controls was compared by x2 test.Logistic regression analysis was used for age adjustment.This study was approved by Ethic Committe of Xinjiang Medical University,and informed consent was obtained from the subjects.Results rs12914489 site in the normal control group diverged from Hardy-Weinberg equilibrium (HWE) (P =0.033),and the rs4886467,rs4558370,rs4461027,rs4886761 and rs16958477 sites followed HWE.The frequencies of G allele and GG genotype of rs4886467 in the XFS group were lower than those in the control group (both at P =0.00) and were protective factors of XFS (OR =0.54,95 ％ CI:0.40-0.74,P =0.000;OR=0.51,95％ CI:0.33-0.78,P=0.001);the frequencies of T allele and TT genotype of rs4558370 in the XFS group were significantly higher than those in the control group (both at P=0.00) and were the risk factors of XFS (OR=1.96,95％ CI:1.23-3.11,P =0.004;OR =2.18,95％ CI:1.31-3.64,P =0.002);the frequencies of C allele and CC genotype of rs4461027 in the XFS group were significantly higher than those in the control group (both at P=0.00) and were the risk factors of XFS (OR=2.25,95％ CI:1.67-3.04,P=0.000;OR=3.06,95％ CI:1.89-4.96,P=0.000);the frequencies of T allele and TT genotype of rs4886761 in the XFS group were significantly higher than those in the control group (both at P=0.00) and were the risk factors of XFS (OR=2.44,95％ CI:1.79-3.33,P =0.000;OR =3.02,95％ CI:1.63-5.60,P =0.000);the frequencies of C allele and CC genotype of rs16958477 in the XFS group were significantly higher than those in the control group (both at P=0.00) and were the risk factors of XFS (OR =2.00,95 ％ CI:1.47-2.71,P =0.000;OR =2.37,95 ％ CI:1.31-4.27,P =0.004).Conclusions The SNPs of promoter region of LOXL1 gene are associated with hereditary susceptibility of XFS individually in Uygur population.The SNPs of rs4886467 locus are protective factor,while the SNPs of rs4558370,rs4461027,rs4886761 and rs16958477 locus are risk factors for pathogenesis of XFS.

Objective To investigate ACEI ( benazepril ) and tranilast exert renoprotective properties in diabetic nephropathy( DN) through the inhibition of thioredoxin( Trx) . Methods Forty male SD rats were randomly divided into normal control group,model control group,tranilast group and benazepril group (n=10 each).Normal control group was fed with normal diet. Other groups were fed with high-glucose high-fat diet to make DN models. Rats in model control, tranilast, and benazepril groups were fed with normal diet,400 mg??kg-1??d-1 tranilast plus normal diet,and 10 mg??kg-1??d-1 benazepril plus normal diet,respectively,via oral gavage for 12 weeks.The 24-hour proteinuria,blood glucose(BG),blood urea nitrogen (BUN),serum creatinine ( Scr) and renal pathology changes were detected. Expression of Trx was measured by Western-blot. Results The 24 h urine protein, BG, BUN, Scr, kidney/body weight, and glomerular sclerosis index were significantly decreased in tranilast group and benazepril group,as compaired with model control group ( P<0.05) ,but there was no statistical difference between the two drug groups (P>0.05).Both tranilast and benazepril can reduce renal pathological changes,and can increase the expression of Trx of DN rats, but benazepril had a more significant effect on increasing Trx expression. Conclusion Both tranilast and benazepril have renoprotective function in DN, and benazepril is more effective than tranilast in delaying the progression of diabetic nephropathy by increasing Trx expression and decreasomg oxidative stress.

To investigate the effect of Kozak sequence (+4A or +4G) on expression of green fluorescent protein (GFP) gene in HEK293 cells. The eukaryotic expression vectors containing GFP gene with different Kozak sequence (+4A or +4G) were constructed by classic DNA recombination methods, including PCR, enzyme digestion, ligation, transformation, identification, et al. Two different Kozak sequences (+4A or +4G) were obtained through PCR with different mutagenic primers. The right recombinant plasmids pHGFP-A and pHGFP-G were transfected into HEK293 cells by liposome-mediated gene transfer method. The expression level of GFP was observed by fluorescent microscope, flow cytometry and Western blot. The flow cytometry revealed that the expression levels of GFP fluorescence in pHGFP-A and pHGFP-G transfected cells were about 15% and 45%, respectively. Western blot showed the specific bands of about 27 kD (GFP) both in pHGFP-G and pHGFP-A sample lanes; and the GFP expression density of pHGFP-G was about 3.87-fold as that of pHGFP-A by ImageJ software analysis. These results indicated that the +4G in Kozak sequence (when -3 site is purine base pair) plays an important role in GFP protein translation, which enhances the GFP expression up to 4-fold in HEK293 cells.
Actins ; biosynthesis ; genetics ; Cell Line ; Cloning, Molecular ; Gene Expression Regulation ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Protein Processing, Post-Translational ; RNA, Messenger ; metabolism ; Recombination, Genetic ; Transfection

OBJECTIVE: To explore the genetic basis for a child with Neurodevelopmental disorder with or without autistic features and/or structural brain abnormalities (NEDASB). METHODS: A child with NEDASB who presented at the Third Affiliated Hospital of Zhengzhou University in July 2021 was selected as the subject. Peripheral blood samples of the child and her parents were collected and subjected to high-throughput sequencing. Candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor a heterozygous c.820_828delinsCTTCA (p.Thr274Leufs*121) variant of the NOVA2 gene, for which both of her parents were of wild type. The variant was predicted as pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION The heterozygous c.820_828delinsCTTCA (p.Thr274Leufs*121) variant of the NOVA2 gene probably underlay the disease in this child. Above finding has enriched the spectrum of NOVA2 gene variants and provided a basis for genetic counseling and prenatal diagnosis for this family.
Child ; Female ; Humans ; Pregnancy ; Autistic Disorder/genetics* ; Brain ; Computational Biology ; Genetic Counseling ; Mutation ; Nerve Tissue Proteins/genetics* ; Neuro-Oncological Ventral Antigen ; Neurodevelopmental Disorders ; RNA-Binding Proteins

OBJECTIVE: To analyze the clinical phenotype and genetic characteristics of a child with Hereditary spastic paraplegia (HSP). METHODS: A child with HSP who was admitted to the Third Affiliated Hospital of Zhengzhou University on August 10, 2020 due to discovery of tiptoeing for 2 years was selected as the study subject, and relevant clinical data was collected. Peripheral blood samples of the child and her parents were collected for the extraction of genomic DNA. And trio-whole exome sequencing (trio-WES) was carried out. Candidate variants were verified by Sanger sequencing. Bioinformatic software was used to analyze the conservation of variant sites. RESULTS: The child was a 2-year-and-10-month-old female with clinical manifestations including increased muscle tone of lower limbs, pointed feet, and cognitive language delay. Trio-WES results showed that she had harbored compound heterozygous variants of c.865C>T (p.Gln289*) and c.1126G>A (p.Glu376Lys) of the CYP2U1 gene. And the corresponding amino acid for c.1126G>A (p.Glu376Lys) is highly conserved among various species. Based on guidelines from the American College of Medical Genetics and Genomics, the c.865C>T was predicted as a pathogenic variant (PVS1+PM2_Supporting), and c.1126G>A was rated as a variant of uncertain significance (PM2_Supporting+PM3+PP3). CONCLUSION The child was diagnosed with HSP type 56 due to compound variants of the CYP2U1 gene. Above findings have enriched the mutation spectrum of the CYP2U1 gene.
Female ; Humans ; Cytochrome P450 Family 2/genetics* ; Mutation ; Pedigree ; Phenotype ; Spastic Paraplegia, Hereditary/genetics* ; Infant

OBJECTIVE: To explore the genetic basis for a EAST/SeSAME syndrome child featuring epilepsy, ataxia, sensorineural deafness and intellectual disability. METHODS: A child with EAST/SeSAME syndrome who had presented at the Third Affiliated Hospital of Zhengzhou University in January 2021 was selected as the study object. Peripheral blood samples of the child and her parents were collected and subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing. RESULTS: Genetic testing revealed that the child has harbored compound heterozygous variants of the KCNJ10 gene, namely c.557T>C (p.Val186Ala) and c.386T>A (p.Ile129Asn), which were inherited from her mother and father, respectively. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were predicted as likely pathogenic (PM1+PM2_Supporting+PP3+PP4; PM1+PM2_Supporting+PM3+PP3+PP4). CONCLUSION The patient was diagnosed with EAST/SeSAME syndrome due to the compound heterozygous variants of the KCNJ10 gene.
Humans ; Child ; Female ; Intellectual Disability/genetics* ; Hearing Loss, Sensorineural/genetics* ; Ataxia ; Genetic Diseases, X-Linked ; Mutation

Objective:To observe the clinical efficacy and any side effects of using ultrasound-guided injection of botulinum toxin A in treating juvenile sialorrhoea.Methods:Forty children with sialorrhoea were randomly divided into group A and group B, each of 20. Under the guidance of color Doppler ultrasound, botulinum toxin type A (BoNT-A) was injected into the children′s 2 parotid glands and their submandibular glands. Each parotid gland was injected with 20u of BoNT-A, while 10u was injected into the submandibular gland in group A and 20u was injected in group B. Before and 2, 8 and 12 weeks after the injections, the children′s sialorrhoea was evaluated using teacher drooling sizing (TDS), the drooling quotient and the Saxon test (ST). Any side-effects were also observed.Results:There was no significant difference in the average TDS score, drooling quotient or ST score between the two groups before the intervention. After the intervention all of those measurements had decreased significantly, but there were still no significant differences between the two groups in any measurement at any time point.Conclusions:Botulinum toxin type A injection under the guidance of ultrasound is accurate and safe. The injection of 10u is sufficient to relieve children′s sialorrhoea without serious side effects.

Objective:To study the expression of zonula occludens-1(ZO-1) in neonates with necrotizing enterocolitis (NEC) and to explore the effects of disialyllacto-N-tetraose (DSLNT), a compound extracted from human milk, on the intestinal barriers in rat model of NEC.Methods:(1) Human study: From Feb 2013 to Dec 2020, the pathological samples of ileum tissue from 21 neonates (12 patients with NEC and 9 with intestinal atresia) from Pathology Department of our hospital were collected. The expressions of ZO-1 in these samples were examined using immunohistochemistry (IHC) method. (2) Animal study: A total of 28 newborn rats were randomly assigned into control group ( n=8), NEC group ( n=10) and DSLNT+NEC group ( n=10). Experimental NEC model was established based on hypoxia (95%N 2 10 min) /cold exposure (4 ℃ 10 min) three times a day for consecutive 3 days. DSLNT+NEC group were fed with formula+DLSNT (300 μmol/L) during hypoxia/cold exposure. All the surviving rats were sacrificed at the end of the experiment (72 h) and the terminal ileum tissues were collected. Hematoxylin-Eosin (HE) staining was used to evaluate tissue damage and Western blotting was used to determine the expressions of ZO-1. (3) Cellular study: Bacterial lipopolysaccharide (LPS) was used to establish a cellular inflammation model in human intestinal epithelial cell lines (Caco-2) and DSLNT (300 μmol/L) was applied to this model. Thiazolyl blue assay was used to examine cell viabilities and immunofluorescence assay was used to detect ZO-1 expression. The effects of DSLNT on cell growth and tight junctions of Caco-2 cells were analyzed. Results:(1)Human study: The villi of mucous layer of the lesion were damaged in NEC patients. ZO-1 expressions at the epithelial junction of NEC patients were decreased compared with intestinal atresia patients and non-lesion intestines of NEC patients. (2)Animal study: Apical extrusion, necrosis and shedding of epithelial cell were seen at the lesions in NEC group. The expression of ZO-1 in NEC group was significantly lower than the control group and DSNLT+NEC group ( P<0.05).DSNLT+NEC group had higher survival rates (8/10 vs. 6/10) and lower ileum inflammatory pathological scores [2.0(1.0, 2.8) vs. 3.5(3.0, 4.0)] than NEC group. (3) Cellular study: Caco-2 cells exposed to LPS showed inhibited cell growth and decreased ZO-1 immunofluorescence staining. Caco-2 cells in the DSLNT+LPS group showed better viability than LPS group and were comparable with the control group. The expression of ZO-1 was significantly increased in the DSLNT+LPS group. Conclusions:Tight junction injury of the intestinal epithelial cell is an important characteristic of NEC. ZO-1 is a potential target for the prevention and treatment of NEC. DSLNT may protect the neonatal intestines by modulating the expression of ZO-1 and keeping tight junction integrity.

OBJECTIVES: Abdominal aortic aneurysm is a pathological condition in which the abdominal aorta is dilated beyond 3.0 cm. The surgical options include open surgical repair (OSR) and endovascular aneurysm repair (EVAR). Prediction of acute kidney injury (AKI) after OSR is helpful for decision-making during the postoperative phase. To find a more efficient method for making a prediction, this study aims to perform tests on the efficacy of different machine learning models. METHODS: Perioperative data of 80 OSR patients were retrospectively collected from January 2009 to December 2021 at Xiangya Hospital, Central South University. The vascular surgeon performed the surgical operation. Four commonly used machine learning classification models (logistic regression, linear kernel support vector machine, Gaussian kernel support vector machine, and random forest) were chosen to predict AKI. The efficacy of the models was validated by five-fold cross-validation. RESULTS: AKI was identified in 33 patients. Five-fold cross-validation showed that among the 4 classification models, random forest was the most precise model for predicting AKI, with an area under the curve of 0.90±0.12. CONCLUSIONS Machine learning models can precisely predict AKI during early stages after surgery, which allows vascular surgeons to address complications earlier and may help improve the clinical outcomes of OSR.
Humans ; Aortic Aneurysm, Abdominal/complications* ; Endovascular Procedures/methods* ; Retrospective Studies ; Blood Vessel Prosthesis Implantation/adverse effects* ; Acute Kidney Injury/etiology* ; Machine Learning ; Treatment Outcome ; Postoperative Complications/etiology* ; Risk Factors