OBJECTIVESince glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary hemolytic erythrocyte enzyme deficiency, most cases have single nucleotide mutations in the coding region, and current test methods for gene mutation have some missed detections, this study aimed to investigate the feasibility of RT-PCR sequencing in the detection of gene mutation in G6PD deficiency.

METHODSAccording to the G6PD/6GPD ratio, 195 children with anemia of unknown cause or who underwent physical examination between August 2013 and July 2014 were classified into G6PD-deficiency group with 130 children (G6PD/6GPD ratio <1.00) and control group with 65 children (G6PD/6GPD ratio≥1.00). The primer design and PCR amplification conditions were optimized, and RT-PCR sequencing was used to analyze the complete coding sequence and verify the genomic DNA sequence in the two groups.

RESULTSIn the G6PD-deficiency group, the detection rate of gene mutation was 100% and 13 missense mutations were detected, including one new mutation. In the control group, no missense mutation was detected in 28 boys; 13 heterozygous missense mutations, 1 homozygous same-sense mutation (C1191T) which had not been reported in China and abroad, and 14 single nucleotide polymorphisms of C1311T were detected in 37 girls. The control group showed a high rate of missed detection of G6PD deficiency (carriers) in the specimens from girls (35%, 13/37).

CONCLUSIONSRT-PCR sequencing has a high detection rate of G6PD gene mutation and a certain value in clinical diagnosis of G6PD deficiency.

Adolescent ; Child ; Child, Preschool ; Female ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; genetics ; Humans ; Infant ; Male ; Mutation ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

Allergic diseases such as allergic asthma, allergic der-matitis, allergic rhinitis, are polygenic diseases, involving the interaction between the environment, genes and immunity. In the past few decades, the incidence rate of allergic diseases in-creased predominantly and influenced the quality of people's lives seriously, so looking for new targets for the prevention and treat-ment of allergic diseases and drugs with less adverse reaction be-comes a hot topic for researchers. MicroRNAs(miRNAs)are a class of endogenous non-coding small RNAs that mediate nega-tively posttranscriptional regulation of gene expression by targe-ting specific mRNA sequences to inhibit the translation of mR-NAs. They are widely involved in the biological processes of cell differentiation, immune response and tumor development. The study shows that miRNAs can control the occurrence and devel-opment of allergic diseases. Studying the regulatory role of miR-NAs in allergic diseases has important implications for exploring the immunopathological mechanisms and discovering new thera-peutic targets of drugs.

Objective:To discuss the type, treatment and results of different therapies of biliary fistula after orthotopic liver tansplantation(OLT).Methods:Data of 24 patients who developed biliary fistula after OLT in the First Affiliated Hospital of Xi′an Jiaotong University from January 2000 to March 2019 were retrospectively analyzed. Patients with biliary fistula were classified into 4 types according to presence or absence of stricture. All patients were treated by endoscopic retrograde cholangiopancreatography (ERCP) or interventional therapy, including endoscopic nasobiliary drainage (ENBD), endoscopic retrograde biliary drainage (ERBD) or percuteneous transhepatic cholangial drainage (PTCD). Main outcome measurements were the onset time of biliary fistula, the site of biliary fistula, the complications of ERCP or PTCD, the time of removing abdominal or biliary drainage tube, and the onset of new biliary stricture.Results:Biliary fistula was found in (46.5±36.6) days (6-122 days) after OLT. The numbers of patients in four types of biliary fistula were 6, 14, 2 and 2, respectively. Biliary fistula was cured in 22 patients, with clinical cure rate of 91.7%. All patients underwent ERCP first, and the technical success rate and clinical cure rate were 87.5% (21/24) and 85.7% (18/21), respectively. The clinical cure rates of ERCP forⅠ-Ⅳ biliary fistula were 6/6, 84.6%(11/13), 1/2, and 0, respectively. The clinical cure rates of ENBD and ERBD were 8/10 and 6/8, respectively. Five cases in whom ERCP failed, underwent PTCD, with technical success and clinical cure rates of 4/5 and 3/4 respectively. Eight patients(33.3%)developed cholangitis after treatment, and the incidence rate seemed higher in type Ⅱ biliary fistula than that in type Ⅰ [35.7% (5/14) VS 16.7% (1/6)]. Incidence of cholangitis was higher in patients with non-anastomotic stricture than those with anastomotic stricture [83.3%(5/6) VS 16.7%(3/18)].Conclusion:The first line treatment for biliary fistula after OLT is ERCP, followed by PTCD. The best procedures of biliary fistula typeⅠ-Ⅳ were ENBD, ENBD combined with ERBD, ENBD and PTCD, respectively.

Bronchial asthma is a kind of respiratory disease which affects people 's life quality seriously. Many factors in-volved in the occurrence and development of such disease, of which the aberrant expression of E-cad plays a critical role in it. Research found that E-cad is an important cell adhesion molecu-lar, and its main function is to maintain the structural integrity of cells and participate in the improvement of airway remodeling as well as restoration of immune function. Further study showed that the role of mucosal barrier of airway epithelial cells in bronchial asthma patients was often damaged. Moreover, the protein ex-pression of E-cad decreased significantly in mucosal molecular, which suggested that the abnormal expression of E-cad was in-volved in the development of bronchial asthma. A review on the relations between the abnormal expression of E-cad protein and bronchial asthma has been discussed in this paper, also it in-cludes the discussion about the mechanisms of E-cad’ s disorder-induced bronchial asthma as well as explores the strategies of bronchial asthma treatment, which may provide references for the follow-up research and clinical treatment.

Objective To observe the effect oftert-Butylhydroquinone (tBHQ) on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2),heme oxygenase (HO)-1 and phosphatidylinositol 3-kinase (PI3K) in high glucose cultured retinal Müller cells;and to investigate the anti-oxidative stress and anti-apoptotic effects oftBHQ.Methods Retinal Müller cells were divided into normal glucose group (5.5 mmol/L,N group),high glucose group (45 mmol/L,HG group) and tBHQ intervention group (HG+tBHQ group).After retinal Müller cells were cultured with high glucose for 48 hours,the pretreatment with tBHQ (20 μmol/L) induced the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1.The Müller cells were identified by immunofluorescence staining.The expressions of Nrf2,HO-1,PI3K,B-cell lymphoma-2 (Bcl-2) and Bax were detected by Western blot and real-time fluorescence quantitative PCR.Flow cytometry was used to detect the apoptosis of retinal Müller cells in rats.Results Müller cytoplasm and nucleus GS showed strong positive,large cell body,abundant cytoplasm,uniform green fluorescence;nuclear DAPI staining round or oval,clear boundary.The expression of Nrf2 protein (t=4.114,P=0.006),HO-1 protein (t=9.275,P=0.000),Nrf2 mRNA (t=7.292,P=0.000) and HO-1 mRNA (t=15.014,P=0.000) in the HG group were higher than those in the N group.The expressions of Nrf2 protein (t=7.847,P=0.000),HO-1 protein (t=7.947,P=0.000),PI3K protein (t=5.397,P=0.002),Bcl-2 protein (t=6.825,P=0.000),Nrf2 mRNA (t=18.046,P=0.000),HO-1 mRNA (t=39.458,P=0.000),PI3K mRNA (t=4.979,P=0.003) and Bcl-2 mRNA (t=9.535,P=0.000) in the HG+tBHQ group were significantly higher than those in the HG group.The protein and mRNA expressions of Bax protein in the HG+tBHQ group were significantly lower than those in the HG group (t=14.998,16.520;P=0.000,0.000).Flow cytometry showed that the apoptosis rate of Müiller cells in the HG group was significantly higher than that in the N group (t=39.905,P=0.000).The apoptosis rate of Müller cells in the HG+tBHQ group was significantly lower than that in the HG group (t=21.083,P=0.000).Conclusion tBHQ can inhibit the apoptosis of retinal Müller cells by up-regulating the expression ofNrf2,HO-1 and PI3K.

BACKGROUNDThis study was to examine the expression of total vascular endothelial growth factor (VEGF) and the anti-angiogenic VEGF 165 b isoform in the vitreous body of retinopathy of prematurity (ROP) patients, and to further study the role of the VEGF splicing in the development of ROP.

METHODSThis was a prospective clinical laboratory investigation study. All patients enrolled received standard ophthalmic examination with stage 4 ROP that required vitrectomy to collect the vitreous samples. The control samples were from congenital cataract patients. The expression of total VEGF and the anti-angiogenic VEGF 165 b were measured by enzyme-linked immunosorbent assay. Results were analyzed statistically using nonparametric tests.

RESULTSThe total VEGF level was markedly elevated in ROP samples while VEGF 165 b was markedly decreased compared to control group. The relative protein expression level of VEGF 165 b isoform was significantly decreased in ROP patients which were correlated with the ischemia-induced neovascularization.

CONCLUSIONSThere was a switch of VEGF splicing from anti-angiogenic to pro-angiogenic family in ROP patients. A specific inhibitor that more selectively targets VEGF 165 and controls the VEGF splicing between pro- and anti-angiogenic families might be a more effective therapy for ROP.

Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infant, Newborn ; Infant, Premature ; Male ; Prospective Studies ; Protein Isoforms ; metabolism ; Retinopathy of Prematurity ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Vitreous Body ; metabolism

De-identification of personal health information is essential in order not to require written patient informed consent. Previous de-identification methods were proposed using natural language processing technology in order to remove the identifiers in clinical narrative text, although these methods only focused on narrative text written in English. In this study, we propose a regular expression-based de-identification method used to address bilingual clinical records written in Korean and English. To develop and validate regular expression rules, we obtained training and validation datasets composed of 6,039 clinical notes of 20 types and 5,000 notes of 33 types, respectively. Fifteen regular expression rules were constructed using the development dataset and those rules achieved 99.87% precision and 96.25% recall for the validation dataset. Our de-identification method successfully removed the identifiers in diverse types of bilingual clinical narrative texts. This method will thus assist physicians to more easily perform retrospective research.
Algorithms ; *Data Anonymization ; *Electronic Health Records ; *Health Records, Personal ; Humans ; Multilingualism ; Natural Language Processing ; Research Design

OBJECTIVE To examine the synergistic inhibiory effect of combination of mammalian target of sirolimus (Rapamycin) (mTOR) inhibitor everolimus and AKT inhibitor MK-2206 on hepatocar-cinoma cell proliferation. METHODS HepG2 and BEL-7402 cells were treated with sirolimus and evero-limus alone for 0, 1, 3, 6, 12 and 24 h or in combination with insulin-like growth factor 1 receptor (IGF-1R) inhibitor NVP-AEW541 or AKT inhibitor MK2206 for 24 h. p70S6K and AKT kinase activityies were detected by Western blotting. Plate clone formation assay and CCK8 assay were used to detect the growth and proliferation of hepatocarcinoma cells treated with everolimus and MK2206 alone or in combi-nation. RESULTS Sirolimus and everolimus inhibited p70S6K activity while causing feedback activa-tion of AKT kinase activity at different time points (P<0.01). NVP-AEW541 and MK-2206 could inhibit AKT kinase feedback activation by everolimus (P<0.05). Colony formation of hepatocarcinoma cells treated with everolimus and MK-2206 in combination was significantly inhibited compared with everolimus or MK-2206 alone (P<0.01). Everolimus and MK-2206 in combination inhibited the proliferation rate of two types of hepatocarcinoma cancer cells by more than 45％ compared with everolimus used alone (P<0.01). CONCLUSION The resistance of sirolimus and its derivatives in hepatocellular carcinoma cells may be achieved throngh the feedback-activated PI3K/AKT pathway, and the combination therapy can synergistically inhibit the growth and proliferation of hepatocarcinoma cells.

Objective To summarize the clinical efficacy of liver transplantation from donation after cardiac death (DCD). Methods Clinical data of both the donors and recipients (n=182) undergoing liver transplantation from DCD were retrospectively analyzed. According to the type of primary diseases, 182 recipients were divided into the benign group (n=135) and hepatocellular carcinoma (liver cancer) group (n=47). Perioperative conditions, 1- and 3-year survival rate of the recipients were statistically compared between two groups. Clinical prognosis and the incidence of postoperative complications of the recipients were summarized. Postoperative complications mainly included early allograft dysfunction (EAD), vascular complications, acute kidney injury (AKI), pulmonary infection, acute rejection, cytomegalovirus (CMV) infection and billiary tract complication. Results No statistical significance was identified in the anhepatic phase, operation time and length of intensive care unit (ICU) stay between two groups (all P>0.05). The 1-year survival rates of the 182 recipients and grafts were 93.1%, and 84.9% for the 3-year survival rates. In the benign group, the 1- and 3-year survival rates of the recipients were 92.5% and 88.1%. In the liver cancer group, the 1-year survival rate of the recipients was 95%, 91% for the disease-free survival rate, and 78% for the 3-year survival rate, respectively. No statistical significance was noted in the overall survival rate of the recipients between two groups (P=0.879). In terms of postoperative complications, billiary tract complications occurred in 26 patients, vascular complications in 14, AKI in 34, pulmonary infection in 22, acute rejection in 11, EAD in 11 and CMV infection in 10. The incidence of postoperative billiary tract complications in patients with T-tube insertion was significantly lower than that in their counterparts without T-tube insertion (8% vs. 19%, P<0.05). Conclusions Liver transplantation from DCD is an efficacious treatment for end-stage liver diseases and liver cancer, which yields relatively high short-term clinical efficacy.

Objective:To evaluate the effect of hepatic ischemia-reperfusion (I/R) rats-derived exosomes on microglial pyroptosis.Methods:Twenty clean-grade healthy male Sprague-Dawley rats, aged 2-3 weeks, weighing 20-50 g, were divided into 2 groups ( n=10 each) using a random number table method: sham operation group (group S) and hepatic I/R group (group I/R). The serum of rats in S group and I/R group was collected, and exosomes were isolated from the sera using differential centrifugations.Microglial cells were co-cultured with PKH26-labeled exosomes for 6 h. The intake of exosomes in microglial cells was determined using immunofluorescence staining.Primary microglial cells were seeded onto 6-well culture plates at a density of 5×10 5 cells/ml and were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), 10 7 cells/ml I/R-exosomes treated group (group 10 7), 10 8 cells/ml I/R-exosomes treated group (group 10 8), and 10 9 cells/ml I/R-exosomes treated group (group 10 9). Microglia in each group were co-cultured with the corresponding concentration of I/R-exosomes for 6 h. The expression of NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cleaved-caspase-1 and gasdermin-D (GSDMD) was detected using Western blot.Primary microglial cells were divided into 3 groups ( n=24 each) by a random number table method: control group (group C), sham operation-exosomes treated group (group S-exosome) and I/R-exosomes treated group (group I/R-exosome). In S-exosome group and I/R-exosome group, exosomes 10 8 cells/ml in S group and I/R group were given, respectively, to incubate cells for 6 h. The expression of NLRP3, ASC and GSDMD mRNA was determined by real-time polymerase chain reaction, and the levels of interleukin-18 (IL-18), IL-1β and tumor necrosis factor-alpha (TNF-α) in the cell culture supernatant were measured by enzyme-linked immunosorbent assay. Results:The results from immunofluorescence staining showed that I/R-exosomes colocalized with microglia.The 10 8 cells/ml I/R-exosomes and 10 9 cells/ml I/R-exosomes up-regulated the expression of NLRP3, ASC, GSDMD and cleaved-caspase-1 in microglial cells ( P<0.01). Compared with group C and group S-exosome, the expression of NLRP3, ASC and GSDMD mRNA in microglial cells was up-regulated, and the levels of IL-18, IL-1β and TNF-α in the supernatant were elevated in group I/R-exosome ( P<0.01). Conclusions:Hepatic I/R rats-derived exosomes can promote microglial pyroptosis.