Objective:The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored.Methods:The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate, collagen, epinephrine, arachidonic acid, and ristocetin. The expression levels of CD41 (αⅡb), CD61 (β3), and CD42b (GPⅠb) on the platelet surface was detected by flow cytometry. Gene sequencing technology was used for the genetic identification of the family. RT-PCR was used in the detection of mRNA splicing, and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. The expressions of total αⅡb and β3 in platelets were analyzed by Western blot.Results:Except ristocetin, the other four inducers could not induce platelet aggregation in the proband. Flow cytometry showed that the expression levels of αⅡb and β3 were only 0.25% and 9.76%, respectively, on the platelet surface of the proband, whereas GPⅠb expression was relatively normal. The expression levels of glycoproteins in the other family members were almost normal. c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing. The c.480C>G mutation was inherited from his mother, and the c.2929C>T mutation was inherited from his father. The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother, resulting in the deletion of 99 bases in c.476G-574A (p.S160-S192). qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father. Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5′SS splice site. The three-dimensional structural model of the αⅡb subunit showed that the β-propeller domain of the p.S160-S192 deletion lost two β-strands and one α-helix in blade 2. The c.2929C>T nonsense mutation caused premature translation termination and produced a truncated protein with the deletion of p.R977-E1039, including the cytoplasmic domain, transmembrane domain, and a β chain of the extracellular Calf-2 domain. The total αⅡb expression of the proband was absent, and the relative expression of β3 was 11.36% of the normal level.Conclusion:The compound heterozygous mutation c.480C>G in exon 4 and c.2929C>T in exon 28 of the ITGA2B gene probably underlies Glanzmann thrombasthenia in this pedigree.

Objective:To analyze the clinical features and etiological results of neonatal central nervous system(CNS) infection and provide basis for optimization of pathogen detection strategy for CNS infection.Methods:We collected the clinical and laboratory data of hospitalized neonates with clinical diagnosis of CNS infection in the neonatal department at Hebei Provincial Children′s Hospital, from January 1, 2020 to August 31, 2021.The clinical manifestations of the enrolled neonates, as well as the cerebrospinal fluid(CSF)pathogens detected by conventional and molecular biological detection techniques were analyzed.Laboratory characteristics of different kinds of pathogen were compared.Results:A total of 101 eligible neonates were enrolled.The median gestational age was 38.8(36.2, 39.6)weeks, with a prematurity rate 26.7%.There were 68 boys.The median age of onset was 9(2, 14)days.Blood culture was positive in 19(18.8%) cases, including 17 cases of bacteria and two cases of fungus.Positive findings were found in CSF specimens of 33(32.7%)cases by various methods including 13 bacteria, 19 viruses and one fungi.Streptococcus group B and Escherichia coli were the first two bacteria in CSF.Enterovirus was the most common virus in CSF.In terms of detection methods of CSF pathogens, seven cases(7/101, 6.9%) were detected by CSF culture, two cases(2/21, 9.5%)by smear, 22 cases(22/45, 48.9%)by single-virus targeted/multiplex polymerase chain reaction and four cases(4/7, 57.1%)by metagenomic next-generation sequencing.The CSF white blood cell counts, protein levels and blood C-reactive protein levels were higher in the cases with bacteria/fungi detection from CNS infection than in those with virus detection( P<0.05). Almost all neonates(98/101, 97.0%)were clinically cured or significantly improved before discharge.Two neonates were discharged against medical advice and one neonate was transferred to the other hospital after clinical improvement. Conclusion:Combined use of conventional and molecular biological detection techniques can significantly improve the etiological positive rate of neonatal CNS infection.Viral infection is not rare in the neonatal population.Our study demonstrated the spectrum of organism causing neonatal CNS infection, which provided a basis for the optimization of pathogen detection strategy.

Objective To investigate the mutations of epigenetic regulation factor ASXL1 gene in myelodysplastic syndrome(MDS).Methods Mutation analysis of ASXL1 gene in 53 de novo MDS patients and 20 healthy persons was performed by using polymerase chain reaction(PCR)followed by sequence analysis at DNA level.The clinical and laboratory characteristics were compared in MDS patients with ASXL1 gene mutation and ASXL1 wild type.ASXL1 mutation in mRNA level was detected by using reverse transcription PCR(RT-PCR)followed by sequence analysis.Results ASXL1 gene mutations were observed in 9 cases(16.9%)of 53 MDS patients.6 mutation types were detected,including 4 frameshift mutations types(2 cases with p.Glu635ArgfsX15,3 cases with p.Gly646TrpfsX12,1 case with p.Ala640GlyfsX14 and 1 case with p.Gly790TrpfsX10)and 2 nonsense mutation types(1 case with p.Gln1063X and 1 case with p.Gln695X).All the mutations were heterozygous,and p.Gly790TrpfsX10 and p.Gln695X were new mutation types.In addition,a single nucletide polymorphism(SNP)p.Gly652Ser was also detected in 4 cases with MDS.5 cases of p.G652S SNP and 1 case of p.Leu1173Leu SNP were detected in 20 healthy people.Frameshift mutation(p.Gly646TrpfsX12)could be detected at mRNA level by using RT-PCR.Differences were not observed in red blood cell counts,white blood cell counts,platelet counts,hemoglobin levels,reticulocyte,neutrophil granulocyte,the peripheral blood lymphocytes percentage,T-cell subsets in the peripheral blood,the proportion of primitive cell in the marrow and MDS types between the patients with ASXL1 gene mutation and ASXL1 wild type patients(P >0.05).Conclusion There is a high frequency of ASXL1 gene mutation in MDS patients,which can be detected at mRNA level.

Objective: To investigate the effect of biology and mTOR pathway activity of down-regulated TSC2 gene expression on U937 leukemia cells. Methods: Gene expression was down-regulated by lentivirus induced RNA interference on TSC2 high expressed U937 cell line; the proliferation, apoptosis and differentiation were detected by CCK-8 assay, colony formation assay and flow cytometry; the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot. Results: Down-regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability (P<0.05) . The proportion in G₀/G₁ phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ (52.53±3.75) % vs (75.10±4.33) %, t=6.829, P=0.002], the S phase [ (22.43±1.00) % vs (15.47±1.20) %, t=-5.581, P=0.019] and G₂/M phase [ (25.03±4.34) % vs (14.33±0.91) %, t=-5.413, P=0.013] was remarkably higher than that of the control cells (P<0.05) . There were no statistically significant differences in cell apoptosis and differentiation (P>0.05) . Down-regulation of TSC2 led to the increased activity of mTOR, 4EBP1 and S6K1, but did not influence the activity of AKT. The expressions of proliferation related cyclinD1, c-myc and PTEN were also up-regulated after TSC2 silenced, but the expressions of P27KIP and BCL-XL were not changed. Conclusion Downregulation of TSC2 could promote the proliferation of U937 cells through up-regulation of mTOR activity.

Objective To analyze the impact of uterine septum on pregnancy and influencing factors on postopera-tive pregnancy. Methods 125 patients with septate uterus and bearing requirement who underwent TCRS were fol-lowed up to assess fertility outcome. The clinical data was retrospectively analyzed. Results Spontaneous abortion rate was 70.40%and 19.39%, live birth rates was 10.40%and 72.45 %in preoperation and postoperation respec-tively. The difference was statistically significant ( < 0.05). Spontaneous abortion rate in older than 35 years old group was significantly higher than that in younger than 35 years old group, but live birth rate was lower. There was no significant difference in different times of operation in uterine cavity, number of abortion and septum length and so on. Conclusions TCRS can significantly improve pregnancy outcome. The age has influence on postoperative pregnancy outcome. Abortion numbers, septum length, septal base width, intrauterine device (IUD) and hormone re-placement therapy (HRT) may have no effects.

Computational Biology ; Factor VII ; Factor VII Deficiency ; Genetic Testing ; Humans ; Pedigree

Objective To investigate the early diagnostic and the prognostic value of amplitude integrated e-lectroencephalography(aEEG)in neonates with hypoxic - ischemic encephalopathy( HIE). Methods The medical data subjects were admitted to the Department of Neonatology,Children's Hospital of Hebei Province from January 2012 to December 2013. Ninety term infants with HIE were divided into 3 groups(mild,moderate and severe),and they were investigated respectively by aEEG monitoring within 12 hours after birth,and all of the infants accepted cranial magnetic resonance imaging(MRI)on 3 to 7 days after birth. The outcomes by MRI were divided into 3 groups(mildly abnor-mal,moderately abnormal and severely abnormal). The findings of aEEG monitoring were divided into 3 groups(nor-mal,mildly abnormal and severely abnormal),the correlation between the findings of aEEG and the severity of HIE was analyzed. The correlation between the results of aEEG and severity of MRI were analyzed. Behavior evaluation of infants with HIE were applied by Neonatal Behavioral Neurological Assessment(NBNA)score on 7 d,14 d,28 d after birth and prognostic evaluation of children with HIE was conducted based on Children's Development Center of China infants intelligence development test at 12 months of age. Results (1)Among 90 term infants with HIE,44 cases(48. 9% ) had mild HIE,29 cases(32. 2% )moderate and 17 cases(18. 9% )severe HIE;49 cases(54. 4% )had mildly ab-normal MRI,23 cases(25. 6% )moderately abnormal MRI and 18 cases(20. 0% )severely abnormal MRI;43 cases (47. 8% )had normal aEEG,25 cases(27. 8% )mildly abnormal and 22 cases(24. 4% )severely abnormal aEEG. (2)The findings of aEEG classification were correlated with the severity of HIE(r = 0. 970 7,P ﹤ 0. 001). The findings of aEEG classification were correlated with the severity of MRI(r = 0. 933 5,P ﹤ 0. 001).(3)NBNA score with severe-ly abnormal aEEG was obviously lower than that with the mildly abnormal aEEG. NBNA score on 7 d after birth:(14. 1 ± 4. 2)scores vs(32. 2 ± 2. 3)scores,on 14 d after birth:(17. 8 ± 5. 6)scores vs(33. 4 ± 2. 1)scores,on 28 d after birth:(18. 9 ± 8. 4)scores vs(34. 6 ± 2. 6)scores,and the difference was statistically significant(all P ﹤0. 05).(4)The infants with HIE were followed at 12 months of age. The development quotient mental development in-dex(MDI)and psychomotor development index(PDI)with severely abnormal aEEG were obviously lower than that with the mildly abnormal aEEG[MDI(65. 1 ± 4. 1)scores vs(89. 1 ± 6. 7)scores,PDI(67. 5 ± 10. 1)scores vs(90. 7 ± 8. 3)scores],the difference was statistically significant(all P ﹤ 0. 05). Conclusion It is indicated that aEEG can early evaluate the severity of HIE and help predict its neurological outcome.

Objective To evaluate the efficacy of nasal intermittent positive pressure ventilation (nIPPV) in preventing extubation failure in very low birth weight infants (VLBWI) compared with nasal continuous positive airway pressure (nCPAP).Methods A single-center randomized controlled trial was conducted from Jun 2012 to Jun 2013 in the NICU of Children's Hospital of Hebei Province.Eighty-four cases of VLBWI (birth weight 700 ～ 1 500 g,gestational age 27 ～ 32 weeks) with respiratory failure and subjected to mechanical ventilation were eligible for the study if they needed non-invasive,assisted ventilation at the time of first extubation attempt.They were randomly assigned to receive nIPPV (40 cases) or nCPAP (44 cases) according to random number table method,using the rate of successful extubation as primary outcome.Blood gas data were obtained and PaO2,PaCO2,FiO2,PaO2/FiO2 were monitored at 0 h,24 h,48 h and 72 h after extubation as enrollment for oxygenation and duration,the rate of successful extubation and the incidence of adverse events including frequent apnea,bronchopulmonary dysplasia,intraventricular hemorrhage,periventricular leukomalacia and retinopathy of prematurity and mortality as secondary outcomes.Results There were no significant differences in the baseline characteristics including the proportion of primary disease,sex,weight,gestational age,score for neonatal acute physiology and application of pulmonary surfactant between nIPPV group and nCPAP group (P ＞ 0.05).There were no significant differences in PaO2,PaCO2 and PaO2/FiO2 ratio between the two groups at 0 h of enrollment (P ＞ 0.05).The values of PaO2 and PaO2/FiO2 ratio in nIPPV group were significantly higher[48 h:PaO2:(63.2 ± 3.6) mmHg vs (52.3 ±6.7) mmHg,PaO2/FiO2:(243.2 ±32.8) mmHg vs (187.6 ±34.0) mmHg;72 h:PaO2:(66.4 ±5.8) mmHg vs (51.8±5.9) mmHg,PaO2/EO2:(280.6 ± 16.8) mmHg vs (245.2 ±40.5) mrnHg;1 rnmHg =0.133 kPa],whereas PaCO2 lower[48 h:(40.3 ±4.8) mmHg vs (49.2 ±6.6) mmHg,72 h:(42.2 ±5.6) mmHg vs (57.3 ± 6.9) mmHg],than nCPAP group at 48 h and 72 h after extubation (P ＜ 0.05).The oxygenation status in nIPPV group were significantly improved at 48 h and 72 h after extubation compared with the intra-group data at 0 h (P ＜ 0.05).The total ventilation time was shorter in nIPPV group than nCPAP group[(130.9 ±46.7) h vs (180.5 ±50.1) h,P ＜0.05],but the oxygen exposure time had no significant difference[(190.6 ± 45.2) h vs (216.8 ± 54.4) h,P ＞ 0.05].The rate of successful extubation in nIPPV group was significantly higher as compared with nCPAP group[92.5％ (37/40) vs 75.0％ (33/44),P ＜ 0.05].The incidence of frequent apnea and bronchopulmonary dysplasia in nIPPV was lower than nCPAP group[15.0％ (6/40) vs 34.1％ (15/44) ;2.5％ (1/40) vs 15.9％ (7/44),P ＜ 0.05].There were no significant differences in the incidence of severe intraventricular hemorrhage,perivenwicular leukomalacia,retinopathy of prematurity,late of infections,necrotizing enterocolitis,patent ductus arteriosus,patent ductus arteriosus operation and mortality before discharge between the two groups (P ＞ 0.05).Conclusion nIPPV after extubation in VLBWI has beneficial effects as compared with nCPAP in improving oxygenation,shortening noninvasive ventilation time,improving the rate of successful extubation,and can reduce the incidence of frequent apnea and bronchial pulmonary dysplasia in VLBWI.

Recently, more research about the plant bioreactor expressing genes encoding human proteins was reported. In the present study, the cDNA of the human gene keratinocyte growth factor 2 (KGF2) was replaced with plant preferred codons by PCR, and the modified full-length cDNA was cloned into the plant expression vector pCAMBIA-YO containing the oil-body promoter. The fusion construct pCAMBIA-YO-KGF2 was transformed into Brassica napus by Agrobacterium tumefacien-mediated cotyledon transformation method. The transgenic seedlings were identified by PCR, Southern and western blot analysis all showed that KGF2 gene was successfully expressed in in transgenic Brassica napus.
Brassica napus ; genetics ; metabolism ; Cloning, Molecular ; DNA, Complementary ; genetics ; Fibroblast Growth Factor 7 ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Humans ; Plants, Genetically Modified ; genetics ; Rhizobium ; genetics ; Transformation, Genetic