Objective:To construct a bait vector for HSPC238, and to screen the target proteins which interact with the HSPC238.Methods:Gene synthesis method was used to synthetic gene HSPC238, then connected with the pGBKT7 vector after digesting by the sfiIA and sfiIB,to obtain the bait plasmid pGBKT7-HSPC238,then transferred into the yeast strains AH109 with the empty plasmid pGBKT7after sequencing,to observe its self-activating effect in the nutrient deficiencies medium,and further to screen the target proteins which interact with HSPC238 from the human fetal liver cDNA library.Results:The bait vector pGBKT7-HSPC238 was successfully constructed,and it had no self-activating effect through the phenotypic screening,after the yeast two-hybrid technology with literature analysis,we preliminary screened and found that the ribosomal protein L5(RPL5) may be the one of the target proteins which interacted with HSPC238 from the human fetal liver cDNA library.Conclusion: We successfully constructed the bait plasmid vector PGBKT7-HSPC238,and after the yeast two-hybrid technology with literature analysis,we preliminary screened and found that the ribosomal protein L5( RPL5) may be the one of the target proteins which interacted with HSPC238.

Objective To explore the clinical signiifcance of combined detection of thyroid stimulating hormone (TSH) and free thyroxin (FT4) in dried blood spots in screening for congenital hypothyroidism (CH) in neonates. Methods The TSH and FT4 levels in dried blood spot were measured by time-resolved lfuorescence immunity in live born neonates from June to December 2013. If the screening was positive, the blood was drawn and the serum TSH and FT4 were measured and compared with the results from dried blood spots. Results In a total of 31 199 neonates screened, 12 cases were diagnosed with CH and the prevalence rate of CH was 1/2 600;4 cases were hyperthyropinemia and no pituitary CH was detected. There was no signiifcant difference between TSH or FT4 levels in dried blood spot and those in serum in neonates diagnosed with CH (P>0.05). Conclusions Combined detection of TSH and FT4 in dried blood spot can be used for neonatal screening of CH. It can be applied for early distinguishing CH from hyperthyropinemia, and also helpful for early diagnosis and treatment of central CH.

Objective:To construct the recombinant eukaryotic expression vector pCDNA3.1-MT2A and to investigate the cellular localization of MT2A protein in 293T and SMCC7721cell lines.Methods: Gene synthesis method was used to synthetic gene MT2A,added a Kozak sequence and His tag sequence at the N-terminus,the amplified target gene was connected to the pcDNA3.1(+) vector which was double digested between the BamH Ⅰ and Not Ⅰ.After transformation to E.coli DH5α, the positive clones were picked for plasmid extraction then Electrophoretic and sequenced.The pCDNA3.1-MT2A plasmids which passed through electrophoretic and sequenced were transfected 293T and SMMC7721 cell lines by liposome method,and then observed their expression and localization in eukaryotic cells by laser confocal microscopy.Results: The recombinant plasmid pCDNA3.1-MT2A was confirmed by restriction analysis and DNA sequencing,the sequence of the target gene MT2A was entirely correct,eukaryotic expression vector was successfully constructed and cell lines which had transfected recombinants could see the expression of green fluorescent protein in the cytoplasm.Conclusion:Successfully constructed fusion gene of pCDNA3.1-MT2A and expressed in eukaryotic cells,we found that the MT2A was mainly localized in the cytoplasm of 293T and SMMC7721 cell lines.The findings can help us to lay the foundation for the functions of MT2A in hepatoma cells.

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
Astrocytes/*cytology ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; Neurons/*cytology ; Stem Cells/*cytology ; Tretinoin/pharmacology

Objective:To investigate the association between rs1042713 polymorphisms of ADRB2 gene and the susceptibility of asthma in Chinese Population by meta-analysis.Methods: The Pubmed database,Emabase database,Web of Knowledge database, CNKI database,Wanfang database and Weipu database were searched for all publications about the susceptibility of asthma and the rs1042713 polymorphisms of ADRB2 gene in Chinese Population.The article which met the inclusion criteria were assessed by the STA-TA12.0 software.Results:12 studies were included,with 2 193 asthmatic patients and 2 033 controls.All the included articles were satisfied to the Hardy-Weinberg equilibrium.The results of Meta-analysis was showed that the risk of asthma of the mutations G carriers ( GG +GA) on ADRB2 gene rs1042713 loci in Chinese people compared with the wild-type homozygotes( AA) was not significantly in-creased overall(OR=1.08,95% CI=0.82-1.44).However,subgroup analysis showed that the risk of G carriers of children had a relatively higher incidence(OR=1.69,95% CI 0.99-2.87),while the risk of adult-onset have a relatively lower incidence(OR=0.88,95%CI 0.68-1.15).Conclusion:The ADRB2 gene rs1042713 polymorphisms have a certain correlation with the susceptibility of asthma in Chinese children,the mutant gene G carriers may relatively increase the risk of asthma in childhood.

Objective To investigate the application of capillary electrophoresis by dried filter blood paper for screening of α-thalassemia in neonates .Methods The hemoglobin (Hb) of 46 718 cases of neonatal dried heel blood spots were analyzed by the capillary electrophoresis and the content of HbA ,HbF ,HbA2 and abnormal Hb were detected ,the phenotype cases which was screened positive were recalled for genetic analysis .Results A total of 2 598 cases of Bart hemoglobin (Hb Bart′s) positive were detected in 46 718 cases of neonatal heel blood dried blood spots .The screening positive rate was 5 .56% (2 598/46 718) .A total of 477 cases of α-thalassemia gene carriers were confirmed by genetic analysis in the 544 cases which were recalled .The coincidence rate of Hb Bart′s screening and genetic diagnosis was 87 .68% (477/544) .By analyzing the relationship between the clinical pheno-types and the content of Hb Bart′s ,we found the Hb Bart′s content gradually increased with the severity of clinical phenotype ,and the difference was statistically significant (P= 0 .000) .Conclusion There is a good consistency between the capillary electrophore-sis of dried filter blood paper and the genetic analysis .It could be determined α-thalassemia clinical type according to the Hb Bart′s content .

Objective To analyze the value of conventional T2WI combining with breath-hold Cartesian VIBE sequence, and compared with Blade combining with breath-free radial VIBE sequences in pre-operative T staging of potentiallly resectable esophageal carcinoma. Methods Sixty-five cases of esophageal carcinoma were concluded prospectively. All patients had received pathological examination of gastroscope biopsy before MRI. Patients received MR examination, including T2WI, breath-hold VIBE, Blade, and breath-free radial VIBE sequences. Two radiologists with more than five years experiences in the diagnosis of chest, performed T staging in MRIby referring to the the 7th edition of UICC-AJCC TNM classification. The results of MRI T staging and the postoperative pathological T staging were analyzed byχ2 test. Results Sixty-five patients were included. Diagnostic coincidence rates of the preoperative T staging by using conventional T2WI combining with breath-hold Cartesian VIBE sequences and Blade combining with breath-free radial VIBE sequences were 51%(33/65) and 88%(57/65) ,with 32 and 8 cases overstaging or understaging respectively, and the statistical differences were significant(χ2=20.80, P<0.05). The former diagnostic accuracy of esophageal carcinoma in situ, muscularis violation and esophageal fiber membrane were 42%(8/19), 54%(14/26), 55%(11/20), and the latter were 89%(17/19), 88%(23/26), and 85%(17/20). Conclusions Diagnostic coincidence rate of the preoperative T staging by using Blade combining with breath-free radial VIBE sequences is much higher than conventional T2WI combining with breath-hold Cartesian VIBE sequences. Blade combining with breath-free radial VIBE sequences could be used as non-invasive imaging method in preoperative T staging of potentially resectable esophageal carcinoma.

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem ceils, neurons and astrocytes,including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio irthe cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5 % among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.

OBJECTIVEThe effect of allitridin on the transcription levels of immediate-early (ie), early(e) and late (1) genes of human cytomegalovirus (HCMV) was investigated in order to explore the mechanism of allitridin against HCMV.

METHODEstablished the models of HCMV AD169 strain infected cells and AD169 strain infected cells treated with allitridin (9.6 mg x L(-1)), and they were compared with the appropriate dose(2.3 mg x L(-1)) of ganciclovir (GCV). All groups of cells were infected at 2.5 multiplicity of infection (MOI), using SYBR Green real-time PCR method to detect the dynamic change of ul122, ul123, ul54 and ul83 mRNA expression at 0.5, 2, 4, 6, 12, 24 h post-infection.

RESULTThe mRNA levels of ul122 and ul123 in AD169 infected cells treated with allitridin at all time points were markedly lower than those of AD169 infected controls (P<0.05), but there were no significant difference of ul122 gene in AD169 infected cells treated with GCV and AD169 infected cells at 0.5-6 h post-infection. The inhibitory rates of allitridin to AD169 ul122 and ul123 mRNA reached 75.2% and 70.4% at 24 h post-infection, respectively. The expression of ul54 mRNA in two drug-treatment groups at all time points were lower than those of AD169 infected cells group (P<0.05). The inhibitory rates of alltridin and GCV to AD169 ul54 mRNA were 45.4% and 27.2% at 24 h post-infection,respectively. The expression of HCMV ul83 mRNA in all groups rapidly increased after 6 h of infection,which is most obvious in AD169 infected cells group. The inhibitory rates of alltridin and GCV to AD169 ul83 mRNA were 45.9% and 26.2% at 24 h post-infection, respectively.

CONCLUSIONAllitridin could effectively suppress the transcription of ie genes (ul122 and ul123) of HCMV AD169 strain, led the expression of mRNA significantly lowerd. It was able to supress the transcription of egene (ul54) and l gene (ul83) too, indicating that HCMV ie genes may be the key target of allitridin against HCMV.

Allyl Compounds ; pharmacology ; Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; genetics ; Genes, Immediate-Early ; genetics ; Genes, Viral ; genetics ; Humans ; Sulfides ; pharmacology ; Transcription, Genetic ; drug effects

Objective:To explore the clinical phenotype and genetic characteristics of a patient with Ataxia and vitamin E deficiency syndrome (AVED) due to a variant of TTPA gene. Methods:A patient diagnosed with AVED (proband), intended for assisted reproductive technology for pregnancy in Zhongnan Hospital of Wuhan University in July 2023, was selected as research subject. Clinical data of the proband were collected, and 2 mL of peripheral venous blood samples were collected from the proband and her father and siblings for serum vitamin E level testing. Whole exome sequencing (WES) was carried out. Pathogenic variants were selected based on American public archive of interpretations of clinically relevant variants (ClinVar). Sanger sequencing was performed to validate the candidate variants detected by WES. Pathogenicity of variants was classified based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), and the impact of variants was analyzed using multiple bioinformatics tools including SIFT, Mutation Taster, CADD, and SpliceAI. Information on the protein domains was obtained from ClinVar and dbSNP databases, and a hotspot map for the variants of protein-coding region was constructed. This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (No. 2023068K).Results:The proband has a significantly low serum level of vitamin E (5.186 μ g/mL), while her father and siblings were normal. WES revealed that she has harbored a homozygous missense c. 2T>A(p.0? ) variant of the TTPA gene, for which her father and younger sister were heterozygous carriers. Based on the guidelines from the ACMG, the missense c. 2T>A(p.0? ) variant of the TTPA gene was classified as pathogenic (PVS1+ PM2+ PM3). Multiple bioinformatics tools had predicted this variant to be located in the initiation codon region and may lead to abnormal synthesis of the TTPA protein, indicating it was deleterious. The hotspot map based on ClinVar and dbSNP databases showed an even distribution of variants across 5 structural domains of the TTPA protein, with high conservation of the first amino acid residue across various species. Conclusion:The homozygous c. 2T>A(p.0? ) variant of the TTPA gene probably underlie the AVED in the proband. Above discovery has enriched the mutational spectrum of AVED and provided a basis for the diagnosis, genetic counseling, and assisted reproductive strategies for this family.