Objective To analyze the clinical features and pathogenic gene mutation in a Chinese family with cerebro-oculo-facio-skeletal(COFS)syndrome,in order to summarize the relationship between phenotype and genotype.Methods The clinical data of the proband and his family members were collected.Genomic DNA from the proband and his parents were extracted by using standard procedures from the peripheral blood leukocytes.Next-generation sequencing was used to detect gene mutation in the patient with COFS syndrome.Sanger sequencing was applied to confirm the results.Results The proband,male,1 year and 3 months old,presented with microcephaly nystagmus,large ears,prominent nose,high arched palate,overhanging upper lip,micrognathia,widely set nipples,flexion contractures(especially involving the elbows and knees),failure to thrive,developmental retardation and feeding difficulty.His parents were normal phenotype.Two different heterozygous mutations c.1843G>T(P.G615W)and c.1996 C> T(P.R666W)were identified in the ERCC2 gene.The proband's father had the heterozygous mutation c.1843G>T(P.G615W)and his mother had the heterozygous mutation c.1996 C> T(P.R666W).Meanwhile,this heterozygous mutation c.1996 C> T(P.R666W)had been reported as a pathogenic gene mutation.Conclusions COFS syndrome is characterized by microcephaly,prominent nose,arthrogryposis and severe developmental delay.This is the first report on COFS syndrome patient in the mainland of China.The pathogenic gene mutations and gene status were identified through genetic studies.The result has laid the foundation for accurate genetic counseling and further prenatal diagnosis.

BACKGROUND: Classic isolation method of bone marrow mesenchymal stem cells (BMSCs) is Percoll density gradient centrifugation. Blood cell component was removed. However, this method is complicated. Preparation density was needed when isolating dog bone marrow. Moreover, centrifugation was frequent, which had a great damage to cells. OBJECTIVE: To establish methods of the isolation, proliferation, culture and osteoinduction of canine BMSCs, and observe the in vitro proliferation and ability to osteoinduction differentiation. METHODS: 10 mL bone marrow was extracted from dog posterior superior iliac spine, heparin anticoagulation, diluted using Hanks juice, treated with 1.077 g/mL Ficoll solution 3 mL, and centrifuged at 2 000 r/min for 20 minutes. Karyocytes were absorbed to form white cloudlike layering interface, and then centrifuged twice using DMEM supplemented with fetal bovine serum, incubated at 12×10~4/cm~2 at 37 ℃ in a 5% CO_2 incubator. Following subculture, cells were incubated in DMEM containing dexamethasone, β-sodium phosphoglycerol and ascorbic acid 2-phosphate. Immunocytochemical staining and immunofluorescence staining were utilized to detect osteocalcin, osteopontin and type Ⅰ collagen expression in osteoblasts. Alkaline phosphatase staining and alizarin red staining were performed. RESULTS AND CONCLUSION: 1.077 g/mL Ficoll density gradient centrifugation was used to isolate karyocytes that were significant compared with Percoll solution. Obtained BMSCs had high purity, good growth and the mean doubling time was 24 hours. Following in vitro osteogenic incubation of dog BMSCs, osteocalcin, osteopontin and type Ⅰ collagen showed positive expression. Alkaline phosphatase staining demonstrated bluish-green cytoplasm. Alizarin red staining showed red nodes in extracellular matrix, and could differentiate into osteoblasts in vitro.

Objective: To investigate the current status of malaria rapid diagnostic test (RDT) strips application and malaria laboratory technicians' evaluation about them at primary healthcare provider level in Jiangsu Province. Methods: From November to December 2016, 878 medical institutions and 118 CDCs of city, county and township/community level in Jiangsu Province were selected as study samples using stratified random sampling method. Self-designed questionnaire was distributed to investigate the institution's malaria work task, RDT strips application and evaluation status in 2015. We also investigated the socio-demographic information and collected the RDT strips evaluation score from the malaria laboratory technicians selected from the institutions investigated (one technician from each institution). Rank sum test was performed to compare the RDT strips evaluation scores between medical institutions and CDCs, and among different medical institutions and CDCs. Results: In 2015, 405 cases of malaria were reported, 362 200 person-time of malaria blood testing task was conducted, and 100 000 RDT strips were procured and provided for healthcare providers in Jiangsu province for free. Of the 996 healthcare institutions investigated, 628 used RDT strips in the year 2015 and the median (P₂₅, P₇₅) of RDT strips volume used in these institutions was 10 (2, 25). The volume of RDT strips used in CDCs (15 (5, 52)) was significantly higher than that in medical institutions (10 (2, 25), (Z=3.42, P=0.001)). The investigated CDCs gave higher score on RDT strips' testing time per operation (10 (8.5, 10)) than medical institutions (9(8, 10), (Z=-2.20, P=0.028)). The employers of 614 investigated malaria laboratory technicians used RDT strips in 2015. The median of the scores given by CDC malaria laboratory technicians for RDT strips in terms of testing time per operation, testing operation and results judgement difficulties were 10 (9, 10), 10 (9, 10) and 10 (9, 10), respectively, which were significantly higher than those from technicians of medical institutions (9 (8, 10), 9 (8, 10), 9 (8, 10), (Z values were -2.55, -2.97 and -2.96, respectively; P values were all less than 0.05)). Conclusion RDT strips had been widely performed in health institutions in Jiangsu Province. The amount of RDT strips used in CDCs was significantly higher than that in medical institutions. Primary-level institutions and malaria laboratory technicians generally recognized RDT strips' advantage for application in terms of testing time and operational procedure. CDCs and malaria laboratory technicians from them gave higher regards on RDT strips in terms of testing time per operation, testing operation and results judgement difficulties compared with that of medical institutions.

Objective To investigate the difference in intestinal flora among patients with esophageal squamous cell carcinoma and normal population and to provide a basis for the early diagnosis of esophageal squamous cell carcinoma as a marker. Methods DNA was extracted from biopsy tissue samples of 30 patients with esophageal squamous cell carcinoma (observation group) and 25 healthy people (control group) by microbial amplification sequencing. The integrity and quality of DNA were detected. The composition and abundance of intestinal flora in the samples of the two groups were determined. Results A great similarity in beta diversity was found between the two groups, but some differences were also observed. The relative abundance of Proteobacteria and Verrucomicrobia in the observation group was higher than that in the control group (P<0.05). The relative abundance of Megamonas in the observation group was lower than that in the control group (P=0.025). Conclusion Strengthening the study on the changes in intestinal flora among patients with esophageal squamous cell carcinoma may be of great significance for its prevention and treatment.

Objective To explore the safety and efficacy of intrathecal administration of adipose stem cells de-rived from bioactive secretome (ASCBS)in treatment of whiter matter injury (WMI)in the preterm infants. Methods Sixty - three cases of WMI were recruited according to the uniform standards from multiple medical centers and they were divided into 3 gestational age (GA)subgroups,which were 21 cases in group A (GA 24 - 28 + 6 ),20 cases in group B (GA 29 - 32 + 6 ),and 22 cases in group C (GA 33 - 36 + 6 ). The patients were randomly divided into treatment groups and control groups by tossing coins. The treatment groups received lumbar puncture followed with ASCBS intra-thecal injection once daily for 3 consecutive days. Follow - up study included Neonatal Behavioral Neurological Assess-ment (NBNA)at term - equivalent age and neurodevelopment at corrected age of 6 - month. Neurodevelopment was assessed by using the Bayley Scales of Infant Development and Peabody Developmental Motor Scale. The survival rates, NBNA scores,mental development index (MDI),psychomotor develop index (PDI),total motor development quotient, gross motor development quotient and fine motor development among each subgroup were compared. Results Sixty -three cases were recruited,including 31 in the treatment group and 32 in the control group. Only 1 case in the treatment groups lost in the follow - up. No clinical side effects were found in the treatment groups. There was no significant diffe-rence in the survival rate and complication in the preterms in all subgroups of the treatment group and control group (all P > 0. 05). The gross and total motor development quotient in the treatment group A was higher than that in the control group A(gross motor development quotient:98. 330 ± 6. 282 in treatment group A,90. 330 ± 3. 777 in control group A, P = 0. 040;total motor development quotient:97. 330 ± 4. 803 in treatment group A,91. 000 ± 4. 472 in control group A,P = 0. 023). The rest findings showed no significant difference between groups. Conclusion The treatment of WMI in preterm infants with ASCBS is safe and can promote the motor development of preterm infants with GA in 24 - 28 weeks.

Objective:To investigate the clinical and genetic characteristics of developmental and epileptic encephalopathy (DEE) caused by syntaxin-binding protein 1 (STXBP1) gene mutation.Methods:The clinical data, gene variation and treatment outcome of 15 children with STXBP1 encephalopathy admitted to Children′s Hospital Affiliated to Capital Institute of Pediatrics from January 2014 to June 2019 were analyzed retrospectively.Results:Among 15 patients, 11 were male and 4 were female, age ranged from 2 months to 69 months. The clinical manifestations of 14 children were epilepsy and developmental delay (DD) and the remaining one showed developmental delay without seizure. The onset age of epilepsy ranged from two days to 19 months and 11 of them experienced the first attack before 1 year of age. The common seizure types were epileptic spasms and tonic seizures. Seven patients were diagnosed with Ohtahara syndrome or West syndrome. Epileptic form discharges were observed in the interictal electroencephalograms (EEG) of 11 patients, including multifocal discharges, suppression-burst and hypsarrhythmia. The brain magnetic resonance imaging of 7 children were abnormal, including myelin dysplasia, less white matter, lack of corpus callosum or hypoplasia. The follow-up time ranged from 2 months to 57 months, after the last follow-up, 3 cases were seizure free, 6 children showed partial response and the other 5 patients had no response on multitherapy. Six of 8 patients showed good responses to levetiracetam (LEV) monotherapy or in combination with other antiepileptic drugs (AEDs). Vigabatrin (VGB) was applied to 5 patients with epileptic spasms and 4 of them showed response. All patients showed different degrees of developmental delay while four of them showed autistic features. STXBP1 gene mutations were identified in all cases and there were 15 types of gene variations, including 8 missense mutations, 1 nonsense mutation, 5 frame shift mutations and 1 complex mutation. Five novel mutations were unreported before, including c.1193A>G, c.172delG, c.1769C>T, c.1038_1039delCC, c.348_351dupTGAA.Conclusions:Development delay and epilepsy are the major and independent clinical phenotypes in children with STXBP1 encephalopathy. The variation of STXBP1 gene is mainly de novo. Levetiracetam and vigabatrin may be more effective in epilepsy control than other AEDs.

Objective:To explore the visual fixation patterns during facial emotion recognition and the association between fixation duration percentage (FDP) of eye region with social impairment among high-functioning autism spectrum disorders (HFASD).Methods:Children were recruited from the Child and Adolescent Behavior Research Center of Sun Yat-sen University, Guangzhou, between July and December 2019 for case control study. A total of 42 children with HFASD and 23 age-and gender-matched typical development (TD) children were enrolled. Based on their performance during the facial emotion recognition task, HFASD children were further divided into ASD-1 group (correctly recognized both happy and fear emotions) and ASD-2 group (recognized happy emotions only). During the free viewing task, an infrared eye tracker was used to record the gazing data of children in the three groups. The social responsiveness scales (SRS) was utilized to evaluate HFASD children′s social impairment, with the cutoff score of 75 to differentiate those from mild and severe social impairment. The differences of FDP among the three groups were analyzed by the Analysis of Variance (ANOVA) model. Mixed linear regression model was conducted to evaluate the associations between social impairment and FDP of eye region among HFASD children.Results:The final sample consists of 25 ASD-1 (20 males, aged (7.9±1.0) years), 17 ASD-2(14 males, aged (7.2±1.2) years), and 23 TD (12 males, aged (7.7±1.3) years) children. There were no significant differences in age and gender among three groups ( F=2.05, χ2 =10.08, P=0.14 and 0.07, respectively). For the happy emotion, there are significant differences in eye FDP among the three groups (TD: 0.37±0.20, ASD-1: 0.35±0.20, ASD-2: 0.47±0.24, F=3.97, P=0.02). Mixed linear regression model revealed that, adjusting for emotion, gender, age and intelligence quotient, ASD children′s eye FDP negatively associates with social impairment ( OR=0.15, 95% CI: 0.06-0.35, P<0.01). Conclusions:Longer FDP is associated with milder social impairment among HFASD children. HFASD children with facial emotion recognition difficulties require longer fixation to recognize happy emotions.

Objective:To investigate the current status of dietary behaviors of children with inflammatory bowel disease (IBD) , explore the types of exclusion diet, screen out unreasonable dietary behaviors, and analyze the reasons leading to their behaviors, so as to provide a basis for developing individualized dietary health education plan.Methods:From January 2018 to December 2019, convenience sampling was used to select 60 IBD children who were treated and hospitalized in the Gastroenterology Department and their main caregivers of the Children's Hospital of Nanjing Medical University as research subjects. All subjects were investigated with the self-designed IBD Patients Dietary Behaviors Questionnaire. Single factor analysis and multiple linear regression analysis were performed on the influencing factors of the dietary behavior of IBD children. A total of 60 questionnaires were issued and 50 were validly recovered with a valid recovery rate of 83.3%.Results:The score of dietary behaviors of IBD children was (34.68±2.77) , and the standard score was (86.70±6.91) . Education level and residence were the effects of the dietary behavior scores of the main caregivers of children with IBD. In terms of the attitude of the main caregivers to the exclusion diet, there were unreasonable exclusion behaviors such as lack of crude fiber food, and dairy products.Conclusions:The overall dietary behaviors of IBD children are relatively good, but there are still some unreasonable dietary behaviors and excessive reliance on the exclusion diet, and the main influencing factors include the education level and residence of their caregivers. It is recommended that medical staff emphasize individualization when formulating dietary health education plan to achieve the goal of standardizing the dietary behavior of IBD children, promoting growth and development, and reducing symptom deterioration and recurrence.

Objective : To investigate the effects of 3 types of trihalomethanes ( THMs) , the by⁃products in treatment of drinking water disinfection , on the development and reproductive capacity of germ cells of Caenorhabditis elegans. Methods : Tribromomethane ( TBM) , trichloromethane ( TCM) and triiodomethane ( TIM) , 3 types of THMs were selected and wild type Caenorhabditis elegans was used as a model to detect germ cell apoptosis , oocyte number and the final brood size after exposure to different concentrations of THMs. Results : The results of germ cell apoptosis showed that the exposure to 50 μmol/L TCM and TIM caused elevated levels of germ cell apoptosis in a dose⁃dependent manner. Oocyte counting results showed that exposure to 50 μmol/L all three THMs significantly reduced the number of oocytes. The brood size results showed that only 50 μmol/L TIM exposure significantly de⁃ creased the brood size of nematodes. Conclusion THMs showed negative impacts on the reproductive system of Caenorhabditis elegans , and the toxicity is related to the halogenated elements of THMs.