Objective To perform PCR site-directed mutagenesis of nine novel PAH gene mutations (Y154H, R157I, Y206C, G247R, D282G, G346R, S349A, A389G, R400K) identified in northern Chinese and construct mutant recombinant plasmids of PAH gene. Methods 1) Every mutant recombinant plasmid was constructed according to the site of the mutation localized in functional domain of PAH gene and the related clinic phenotype of patients with the gene mutation. 2) Using the wild-type PAH expression vector as a templet, the mutant recombinant plasmids were directly amplified by PCR with Platinium Taq DNA polymerase and nine pairs of primers which were designed according to the human PAH cDNA sequence and the requirement for site-directed mutagenesis technology. 3) The positive strains were selected by Amp resistant test, PCR and restriction endonuclease analysis. The Mva Ⅰ, Mva Ⅰ, Hind Ⅲ, Rsa Ⅰ, Rsa Ⅰ sites exist in the sequences near the mutant sites of S349A, D282G, G247R, Y206C, Y154H, respectively, but not in the related sequence of wild-type PAH expression vector. Restriction endonuclease digestion could be directly used in identifying the mutant sites. However, the amplification created restriction site (ACRS) analysis was supplied in the followed identification of R157I, G346R, A389G, R400K. Finally the sequences of mutant recombinant plasmids of PAH gene were confirmed by DNA sequence analysis. Results Every sequence analysis showed that the mutant nucleic acids were introduced at the expected sites of PAH gene, suggesting that the mutant recombinant plasmids of PAH gene were constructed successfully. Conclusion PCR site-directed mutagenesis is accurate and highly efficient. The successfully mutagenized plasmids of PAH gene lay the foundation for the functional analysis of phenylalanine hydroxylase in mammalian cell system.

Index Medicus/MEDLINE/PubMed published by U. S. National Library of Medicine (NLM) is the most important and commonly used biomedical literature retrieval system in the world. According to the"List of Journals Indexed in Index Medicus (2004)", 4,098 journals are indexed for Index Medicus, including 70 journals from mainland China and Hong Kong and 9 journals from Taiwan. Journal of Chinese Integrative Medicine established in May, 2003 is indexed in Index Medicus in 2004. This article outlines the critical elements of journal selection for Index Medicus/MEDLINE and the journal selection process for indexing at NLM, and introduces some measures for the Journal of Chinese Integrative Medicine being indexed in Index Medicus/MEDLINE.

Objective To investigate the expression of DNA methyltransferase (DNMT) in HL-60 cells induced by tetrandrine (Tet).Methods HL-60 cells were treated with different concentrations of Tet and decitabine (DAC) alone and in combination with both.Methyl thiazolyl tetrazolium (MTT) assay was used to assess cytotoxic effect.Flow cytometry (FCM) was used to determine apoptosis rate.Real-time quantitative polymerase chain reaction (PCR) assay was used to quantify mRNA levels of DNMT.Western blot was used to quantify the expression of DNMT protein.Results Tet inhibited the growth and proliferation of HL-60 cells in a time-and dose-dependent manners (both P ＜0.01).Tet treated HL-60 cells after 48 h at the concentration of 2 μmol/L,and 4 μmol/L,the levels of DNMT gene and protein in the drug administration group decreased compared to the control group.After incubation for 48 h with Tet 2 μmol/L combined with DAC 4 μmol/L,the combination group was significantly depressed.Conclusions Tet could potently inhibit the growth and proliferation of HL-60 cells,reduce the expression levels of DNMT mRNA and protein,and have a more obvious effect in the combination group.

Objective To investigate the possible mechanism of mitochondrial in chronic myeloid leukemia cells K562/G01 cells apoptosis induced by triptolide.Methods K562/G01 cells were treated with different concentrations of triptolide.MTT assay was used to assess cytotoxic effect.FCM was used to determine apoptosis rate,mitochondrial membrane potential and the activity of Caspase-9 of each experimental group.Real-time quantitative PCR assay was used to quantify mRNA levels of Caspase-9 and cytochrome C and Western blot assay was used to determine protein levels of cytochrome C.Results Triptolide inhibited the growth and proliferation of K562/G01 cells in a time-and dose-dependent manner (both P ＜ 0.001).Meantime,triptolide could make the mitochondria membrane potential fade away and enhance the activity of Caspase-9 (F =566.431,2 555.485,P ＜ 0.001).In addition,triptolide could dose-dependently up-regulated the transcription of Caspase-9 and cytochrome C (F =61 007.702,452 121.760,P ＜ 0.001),and the protein expression of cytochrome C,whose gray value in each experimental group was 21.54±0.59,39.63±0.58,53.29± 1.47 and 75.68±1.87 (F =5 677.928,P ＜ 0.001) respectively.Conclusion Triptolide could potently inhibit the growth and proliferation of K562/G01 cells,and the mitochondria apoptosis pathway might be one of the important apoptosis mechanisms in chronic myeloid leukemia cells induced by triptolide.

Objective To perform mutation analysis of survival motor neuron gene 1 (SMN1 in two spinal muscular atrophy (SMA) patients and their parents to evaluate the effects of the two SMN1 gene mutations on the transcript levels of the gene and preliminarily predict their effects on the structure and function of SMN protein.Methods Mutation analysis of SMN1 gene was carried out by multiplex ligationdependent probe amplification,reverse transcript-polymerase chain reaction (RT-PCR) and cloning sequencing.Transmission of the mutations was confirmed by the mutation analysis in patients' parents.The full-length SMN1 (SMN1-fl) transcript levels of the patients carrying these subtle mutations were detected using quantitative RT-PCR.Results The two patients were diagnosed as SMA Ⅱ and SMA Ⅲ.They carried p.Val19GlyfsX21 and p.Ala2Gly SMN1 mutations in SMN1 gene,respectively.Both of the two mutations were originated from their fathers.Compared with the healthy individuals (23.5 ± 4.9),the two patients had a significant reduction in the level of SMN1-fl transcripts (t =3.322,P =0.011 (p.Ala2Gly) ;t =6.964,P =0.000 (p.Val19GlyfsX21)).However,compared with the healthy carriers (14.1 ±4.5),the patient with p.Ala2Gly mutation had no significant reduction in the level of SMN1-fl transcripts (13.9 ±3.6,t =0.058,P =0.955) ; however,the patient with p.Val19GlyfsX21 mutation had a significant reduction (4.9± 2.4,t =3.725,P =0.004).Conclusions Two SMN1 gene mutations are identified in our study.The mutation p.Val19GlyfsX21 is a novel mutation and p.Ala2Gly is firstly reported in Chinese SMA patients.p.Val19GlyfsX21 may possibly lead to decreased SMN1-fl mRNA by nonsense-mediated messenger RNA decay,however,p.Ala2Gly has no obvious effects on the amount of the SMN1-fl transcripts,indicating that its deleterious effect may be occurring at SMN protein level or the function of SMN protein.

Objective To investigate the value of susceptibility weighted imaging (SWI) in the diagnosis of hemorrhagic diffuse axonal injury (DAI). Methods A retrospective study was conducted on 20 patients with DAI who received MRI examination at day 3 post-injury.MRI sequences included T1WI,T2WI,fluid attenuated inversion recovery ( FLAIR),diffusion weighted imaging (DWI) and SWI.There were 15 patients with the Glasgow Coma Scale (GCS) score≤8,three with GCS score of 9-12 and two with GCS of 13-15.The location and quantity of hemorrhage focus were counted.The area of hemorrhage focus was measured on each MR sequence.Differences of detection rate of hemorrhage focus on each sequence were compared by using X2 test.The correlation between DAI related bleeding area and GCS score was analyzed. Results DAI related hemorrhage focus showed a larger number in superficial cerebrum than that in posterior cranial fossa and in deep cerebrum.The detection rate of hemorrhage focus on SWI was the highest,as compared with other sequences ( P ＜ 0.05 ).Bleeding area and GCS score showed a negative correlation (r =-0.921,P ＜ 0.01 ). Conclusion SWI is very sensitive in detection of the intracerebral hemorrhage focus in the acute period of traumatic DAI.

Objective To analyze the distribution of common chromosomal karyotypes of patients with Turner syndrome (TS), and to explore the correlation between the age and height standard deviation scores (HSDS) on diagnosis.Methods Retrospective investigation was performed for the data of age and HSDS on diagnosis in 273 TS girls(≤ 18.0 years old)diagnosed by chromosomal karyotypes.The main statistical methods were analyzed with t-test and Pearson correlation test by using the SPSS 18.0 statistical software.Results (1) There were 4 kinds of common chromosomal karyotypes in the TS :45, X (87/273 cases,31.9％),46, X, i (Xq) (43/273 cases, 15.7％) ,45, X/46, X, i (Xq) (36/273 cases, 13.2％) and 45, X/46, XX (23/273 cases, 8.4％), respectively, the adolescent TS all had delayed puberty.For the cases with 45, X karyotypes ,3 cases presented mental retardation and 2 cases with organs deformity.(2)The patients with 45 ,X/46,X,i(Xq) karyotypes or with 46,X,i(Xq) karyotypes had the maximum(12.56 age) or the minimum(9.70 age) mean age on diagnosis, respectively, there was a significant difference between 2 groups (t =3.019, P =0.004).The maximum deviation from normal height was found in the patients with karyotypes of 46, X,i (Xq) (HSDS =-4.04), and the minimum deviation was in the patients with karyotypes of 45,X/46, XX (HSDS =-3.16), and there was a significant difference between 2 groups (t =-2.95, P =0.004).(3) More than 75.7％ of TS patients was diagnosed when their heights deviated above 3 SD,and their mean age on diagnosis was 12.10 age,which was 3 years later than those patients within 2 SD.(4) There was a significant negative correlation between the age and HSDS on diagnosis in the groups of common chromosomal karyotypes[45,X、46,X,i(Xq) and 45,X/46,XX] (r =-0.551,-0.560,-0.622,all P ＜ 0.01), except for the group with the 45, X/46, X, i (Xq).Conclusions (1) In this study, the consti-tuent ratios of these 4 common chromosomal karyotypes were different from those in Europe and America's.(2)Patients with 45 ,X may have more severe symptoms than others.(3)The mean age on diagnosis was at least 3.0 years earlier when considered HSDS below-2.00 as an indicator for chromosomal karyotype screening,which would facilitate earlier diagnosis.

OBJECTIVETo establish a hyperphenylalaninemia related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA).

METHODSThree children with known HPA mutations and a healthy control were used for setting up the method. Ten children with HPA with known mutations were recruited for validating the method. Ion Ampliseq PCR was used to amplify the 5' and 3' untranslated region, coding sequence, and flanking introns of PAH, GCH1, PTS, QDPR, and PCBD1 genes. After the enrichment with the Ion OneTouch system, the products were sequenced by PGM. Data from the PGM were processed with Torrent Suite v2.2 software package. All variations were confirmed by Sanger sequencing.

RESULTSFor the 4 samples, the PGM output was 94.22 Mb, with approximately 99.5% of reads mapping to the target regions. Among these samples, we detected 74 variations (28 positions) including 6 known mutations. Compared with database and results of Sanger sequencing, 55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed. For the 10 samples, all the known mutations were successfully identified.

CONCLUSIONIon Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways, which can meet the clinical demand for individualized diagnosis and treatment.

Objective To observe the clinical effects and safety of sodium valproate combined with decitabine for treatment of myelodysplastic syndrome (MDS). Methods Forty-two patients with MDS were enrolled in department of hematology in Shanxi Dayi Hospital from February 2012 to February 2017. According to random number table, the patients were divided into the control group (21 cases) and the experimental group (21 cases). The patients in the control group received decitabine at the dose of 20 mg·m-2·d-1, and intravenous infusion was completed in 2 hours, continuous therapy up to 5 days, 4 weeks as a course; the patients in the experimental group received combined medication, orally given sodium valproate 0.2 g once, 3 times per day. One week later, the dosage was added to 0.4 g once, 3 times per day. Both groups received at least 4 courses of treatment. The treatment was stopped when serious adverse reactions or obvious disease progression occurred. The bone marrow smear was rechecked every 4 weeks after treatment to evaluate the efficacy. The expressions of ASXL1, DNMT3A and TET2 in bone marrow cells were detected by fluorescence quantitative PCR before and after treatment. Results The total treatment response rate of the experimental group and the control group were 76.2 % (16/21) and 57.1 % (12/21) respectively, and there was statistically significant difference (P< 0.05); the total remission rate of the two groups was 47.6 % (10/21) and 38.1 %(8/21) respectively, and there was no significant difference (P> 0.05). All patients had slight adverse reactions, and the adverse reaction rate was 42.9 % (9/21) and 38.1 % (8/21), and there was no significant difference (P>0.05). The content of TET2 mRNA and DNMT3A mRNA after treatment in both groups were decreased compared with the expressions before treatment, and there were significant differences (P<0.05). However, there was no significant difference between the two groups after treatment (P> 0.05); the content of ASXL1 mRNA had no obvious change in the control group and a dramatic decrease in the experimental group compared with that before treatment (P<0.05). Conclusion Sodium valproate combined with decitabine has favorable effects and mild adverse reactions for treatment of MDS, besides, it can influence the expressions of TET2, DNMT3A and ASXL1.

Objective:To explore the distribution of the copy number of survival motor neuron gene 2 ( SMN2) and the transcript level of the full-length SMN2 ( fl-SMN2) transcript level in patients with type 1-3 spinal muscular atrophy (SMA), and to evaluate their influences on disease severity, progression, and prognosis. Methods:It was a retrospective study involving 78 therapy-naive SMA patients with SMN1 gene homozygous deletion who were diagnosed and treated in the Capital Institute of Pediatrics from January 2019 to December 2021.Cross-sectional clinical data, including age at onset, motor milestones, and complications were recorded.They were followed up for monitoring motor function degeneration and survival.The copy number of SMN2 and the transcript level of fl-SMN2 were detected.Differences between groups were compared by the Student′s t-test or One- Way ANOVA or Chi- square test.Kaplan-Meier analysis was used for survival analysis, and Kendall′ s tau- c was performed to assess the correlation of these two biomarkers with SMA phenotypes, age at onset, motor milestones, and survival. Results:Of the 78 SMA patients, there were 17 cases (21.8%) of type 1, 34 cases(43.6%) of type 2, and 27 cases(34.6%) of type 3.Seven cases(41.2%) type 1 SMA patients died, with a median survival time of 11 months, and no deaths were observed in type 2 and type 3 SMA patients.There was a significant difference in the median age at onset among SMA patients with 2, 3, and 4 copies of SMN2 (1.8, 12.0, and 24.0 months, respectively; F=4.943, P=0.01). The mean transcript level of fl-SMN2 in type 1, 2 and 3 SMA patients were 196.25±68.79, 331.21±108.79 and 455.69±122.27, respectively ( F=37.154, P<0.001). The survival rate of SMA with 2 SMN2 copies at 1, 2, and 5 years were 50.5%, 0, and 0, respectively, and their median survival age was 7 months.The survival rate of SMA with 3 and 4 SMN2 copies at 5 years were 97.4% and 100.0%, respectively.Moreover, a negative correlation was observed between the transcript level of fl-SMN2 and phenotype severity ( Kendall′ s tau- c=-0.444, P<0.001), and the transcript level of fl-SMN2 of the survival group was much higher than that of the death group (342.93±125.74 vs.212.14±92.31). More copies of SMN2 and higher transcript level of fl- SMN2 indicated more motor function acquisitions (head control, sitting and walking) ( P<0.001). In addition, there was a significant difference in the transcription level of fl-SMN2 between the undegenerated group and the degenerated group in sitting and standing ( F=5.432, P=0.023 and F=4.315, P=0.047, respectively). Conclusions:Both the copy number of SMN2 and the transcript level of fl-SMN2 are correlated with SMA severity, survival, and motor milestones, serving as valuable biomarkers for evaluating phenotypic severity of SMA.The transcript level of fl-SMN2 s may play an important role in the degeneration of sitting and standing.