ObjectiveTo explore the procedures and methods for genetic diagnosis in one nonsyndromic variants of congenital neutropenia (NSVCN) patient and its pathogenic mutation.Methods Genomic DNA was prepared from one NSVCN patient who had progressed to chronic myelomonocytic leukemia and ELA2,HAX1,WASp and GFI1 genes were amplified and sequenced.Results A novel compound heterogeneous mutation consisting of two frame-shift mutations (c.430-1insG and c.655- 9del5bp) was found in HAX1 gene.ConclusionA practically genetic diagnosis procedure for NSVCN has been established,and the novel HAX1 gene mutation may contribute to the etiology of NSVCN.

Background It is well known that sclera remodeling occurs during axial elongation in myopia under the control of growth hormone or its downstream effectors.The role of transforming growth factor-β (TGF-β) in myopia has been determined in previous studies.Bone morphogenetic protein (BMP) is one of members of the TGF-β superfamily,but if it plays an important role in the genesis and development of myopia is not completely clear.Objective This study was to identify the presence of BMPs in normal guinea pigs sclera and investigate the change of BMPs in the sclera in form-deprivation myopia (FDM) of guinea pigs.Methods Thirty young guinea pigs were randomized into normal control group and experimental group using table of random number.FDM models were established by occluding unilateral eyes of guinea pigs with a translucent lens for 14 days in the experimental group,and the fellow eyes served as the controls.Diopter of all eyes was tested by retinoscopy optometry,and ocular axial length was measured by A-sonography before and after modeling.Posterior sclera tissue of the animals was obtained on 14 days,and the relative expression level of BMPs mRNA and protein were assayed by reverse transcription PCR (RT-PCR) and Western blot.The use and care of the animals complied with ARVO Statement.Results On 14 days after occluding of unilateral eyes,the refraction diopter of the experimental group was (-0.48±0.51) D,and that of the fellow eyes was (3.22 ±0.34) D,showing a significant difference between them (t =-12.814,P =0.000).Also,a significant difference in the diopter was seen between the experimental group and normal control group ([-0.48±0.51]D vs.[2.97±0.70]D,t =-11.878,P=0.000).Axial length was (8.30 ± 0.05) mm in the experimental group,(8.11 ±0.06) mm in the fellow eyes and (8.06±0.06) mm in the normal control group,showing a significant increase in the experimental group compared with the fellow eyes and normal control group (t =7.230,P =0.000 ; t =9.084,P=0.000).The expressions of BMP-2 mRNA,BMP-4 mRNA,BMP-5 mRNA in posterior sclera were detected in the normal guinea pigs.Fourteen days after the induction of myopia,the relative levels of BMP-2 mRNA and BMP-5 mRNA in sclera were 0.41 ± 0.11 and 0.65 ± 0.06 in the experimental eyes,which were significantly lower than 0.62 ± 0.07 and 0.84 ± 0.03 in the fellow eyes with the descent range of 34.48％ and 23.67％ respectively (t=2.838,P=0.017; t=2.524,P=0.028).The relative values of BMP-2 protein and BMP-5 protein were 0.44±0.06 and 0.70±0.05 in the experimental eyes,and those of the fellow eyes were 0.61±0.05 and 0.82±0.03,showing significant decline in the experimental eyes with the lowing range of 23.42％ and 15.21％,respectively (t =2.465,P =0.030;t =2.445,P=0.031).No significant differences were found in the expression of BMP-4 mRNA and protein in posterior sclera between the experimental eyes and the normal control eyes (mRNA:t =0.704,P=0.460;protein:t=0.987,P=0.365).Conclusions The expressions of the BMP-2 and BMP-5 in sclera down-regulate significantly in FDM eyes,which suggest that BMP-2 and BMP-5 participate in sclera remodeling during myopia induction.

OBJECTIVETo explore the clinical effects of colonic dripping with Taihuang liquid (THL) in treating neonatal hyperbilirubinemia (HBE).

METHODSOne hundred and thirty-eight neonates with HBE were randomly assigned to two groups. Conventional treatment and nursing were given to both groups, and THL was given additionally to the observation group by colonic dripping.

RESULTSSignificant differences between the observation group and the control group were shown in frequency of defecation (4.6 +/- 1.3 times/d vs 2.0 +/- 1.1 times/d), daily serum bilirubin reduction (31.5 +/- 10.1 micromol/L vs 23.3 +/- 8.3 micromol/L), and days for normalizing serum bilirubin level (5.6 +/- 3.5 d vs 7.8 +/- 4.1 d, all P < 0.01).

CONCLUSIONColonic dripping of THL could promote the excretion of bilirubin, so as to decrease the level of serum bilirubin in neonates with HBE.

Bilirubin ; blood ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Hyperbilirubinemia, Neonatal ; blood ; drug therapy ; Infant ; Infant, Newborn ; Male

OBJECTIVETo explore the effect of Qiling Decoction (QD) combined highly active antiretroviral treatment (HAART) on expression levels of peripheral blood Th17 and Treg cells in HIV/AIDS patients.

METHODSTotally 55 HIV/AIDS patients were randomly assigned to the treatment group (28 cases) and the combination group (27 cases). Besides, 21 HIV negative patients were recruited as the healthy control group. Those in the treatment group received HARRT alone, while those in the combination group received HAART combined QD. The observation lasted for 24 weeks. Meanwhile, according to peripheral blood CD4+ T cell counts before treatment, HIV/AIDS patients were assigned to three subgroups. For patients in subgroup 1, 1 cells/microL < CD4+ T cell counts < or = 100 cells/microL; For patients in subgroup 2, 101 cells/microL < CD4+ T cell counts < or = 200 cells/lL; For patients in subgroup 3, 201 cells/microL < CD4+ T cell counts < or = 350 cells/microL. Expression of peripheral blood Th17 and Treg cells, and number of CD4+ T cell counts were detected using flow cytometry (FCM)in HIV/AIDS patients at the pre-treatment baseline, week 4, 12, and 24, as well as those in the healthy control group.

RESULTSCompared with the healthy control group, CD4+ T cell counts and the baseline expression level of Th17 cells in the peripheral blood of HIV/AIDS patients significantly decreased, the expression level of Treg cells significantly increased P < 0.01). Compared with before treatment in the same group, CD4+ T cell counts all increased at week 4, 12, and 24 in the two treatment groups, showing statistical difference (P < 0.05, P < 0.01). There was no statistical difference in the effective rate at various CD4+ T cell levels between the two groups (P > 0.05). There was no statistical difference in expression levels of Th17 and Treg cells between the combination group and the treatment group at any time point (all P >0.05). The Th17/Treg ration significantly increased in the combination group after 24 weeks of treatment, showing statistical difference when compared with the treatment group (U = 2.135, P = 0.038).

CONCLUSIONQD could improve the immune balance of Th17/Treg cells, which might be one of its mechanisms for improving HIV/AIDS patients' immunity.

Acquired Immunodeficiency Syndrome ; drug therapy ; immunology ; Adult ; Antiretroviral Therapy, Highly Active ; CD4 Lymphocyte Count ; Case-Control Studies ; Drugs, Chinese Herbal ; therapeutic use ; Female ; HIV Infections ; drug therapy ; immunology ; Humans ; Male ; Middle Aged ; Phytotherapy ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

Objective To assess the effect of ursodeoxycholic acid on postoperative outcomes in biliary atresia (BA) patient undergoing Kasai procedure.Methods Correlative english articles published until Jun.2013 were searched by computer in Medline (PubMed),EMBASE,Cochrane library,Wanfang Database,China Knowledge Resource Integrated Database and China Biology Medicine Database,the output articles were screened according to inclusion and exclusion criteria.The selected articles were evaluated with the of Review Manager 5.2 software.Results Nine articles were obtained,but only 4 articles were included,including 284 patients with BA were conformed to the inclusion and exclusion criteria.The sensitivity analysis indicated that the total effective rate in ursodeoxycholic acid group was superior to that in the control group,and the difference between the 2 groups was notable significant (OR =7.24,95 ％ CI:2.42-21.68,Z =3.55,P =0.0004).There was no significant difference between ursodeoxycholic acid group and control group in the effect of ursodeoxycholic acid on outcome of patients who had BA(OR =5.50,95％ CI:0.96-31.64 ; Z =1.91,P =0.06).Conclusions Ursodeoxycholic acid has certain effect on alleviating jaundice after Kasai procedure,but it has no advantage on improving the long outcomes as comparison with control group.

OBJECTIVETo investigate the antithrombin (AT) activity (AT: A) and AT antigen (AT: Ag) level in a Chinese family with type I antithrombin (AT) deficiency, and to explore the molecular mechanism of AT deficiency.

METHODSImmuno-nephelometry and chromogenic assay were used to detect the plasma level of AT: A and AT: Ag, respectively. Genomic DNA was isolated from the peripheral blood, and all the seven exons and exon-intron boundaries of AT gene were amplified by PCR and direct sequencing.

RESULTSThe plasma levels of AT: A and AT: Ag of the proband were 45% and 97 mg/L, respectively, which led to a type I AT deficiency. A heterozygous T to A mutation was found at nucleotide 9833 in exon 5 resulting in a Tyr363Stop nonsense mutation. The sequencing results from the pedigree indicated that four other members also had this mutation.

CONCLUSIONThis heterozygous nonsense mutation of T9833A in exon 5 resulting in venous thrombosis is a novel genetic defect of hereditary AT deficiency, which has not been described before.

Antithrombin III Deficiency ; genetics ; Antithrombins ; genetics ; Blood Coagulation Tests ; Female ; Humans ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo investigate the antithrombotic mechanisms of holothurian glycosaminoglycan (GAG) extracted from sea cucumber.

METHODSHuman endothelial cell line EA. hy926 cells were treated with 10 mg/L GAG or 10U/mL unfractionated heparin (UFH) by short-term (15 min - 2 h) and longer-time incubation (6 h - 48 h). Different doses of GAG were used to stimulate EA. hy926. Released free tissue factor pathway inhibitor(TFPI) was determined by ELISA assay. TFPI expression was investigated by immunofluorescent method and TFPI mRNA level by real-time PCR. In a 96-wells microtitre plate, pooled normal plasma containing different concentrations of GAG was allowed to clot by addition of thrombin and calcium chloride, fibrinolysis was induced by addition of t-PA. TRR (TAFI-related retardation of clot lysis) was used to assess thrombin-activatable fibrinolysis inhibitor(TAFI) functional activity.

RESULTSGAG increased TFPI synthesis, expression and secretion in a dose- and time dependent manner. GAG at low concentrations could lengthen while at intermediate concentrations could shorten clot lysis times significantly as compared to control values. TRR was dose-dependently decreased on addition of GAG.

CONCLUSIONSGAG increases TFPI synthesis, expression and secretion of endothelial cells. GAG at intermediate concentrations significantly affects clot stability of a developing clot by means of diminishing TAFI activation.

Animals ; Carboxypeptidase B2 ; antagonists & inhibitors ; Cell Line ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Glycosaminoglycans ; pharmacology ; Heparin ; pharmacology ; Holothuria ; Humans ; Lipoproteins ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; Tissue Extracts ; pharmacology

OBJECTIVECleidocranial dysplasia (CCD) is a rare skeletal disease with autosomal dominant inheritance associated with mutation in RUNX 2. The authors report a Chinese girl with CCD in whom the mutation in RUNX 2 was identified.

METHODSClinical diagnosis was based on physical examination, radiological findings, and biochemical tests. For mutation detection, genomic DNA was extracted from peripheral blood using standard method. All 7 coding exons of RUNX 2 and their flanking intronic sequences were amplified by polymerase chain reaction (PCR), and the PCR products were then subjected to automatic DNA sequencing.

RESULTSThe affected girl showed typical clinical manifestations of CCD, including patent fontanelles, absent clavicles, short stature and dental anomalies. Direct sequencing of PCR-amplified fragments revealed a recurrent missense mutation, R190W (568 C > T), in RUNX 2. The mutation was further confirmed by Hae III restriction analysis.

CONCLUSIONA Chinese case of CCD was confirmed and the disease-causing mutation was linked to a recurrent point mutation in RUNX 2.

Cleidocranial Dysplasia ; genetics ; Core Binding Factor Alpha 1 Subunit ; genetics ; Female ; Humans ; Mutation

OBJECTIVETaking Chinese compound medicine Yuchangning as a research model, prepared the pH and time dependent Yuchangning tablets for colon-specific delivery (PT-YT-CSD), and evaluated the releasing property in vitro.

METHODThe coating prescription is filtered by the release extent of matrine and oxymatrine in vitro and the wicking rate of the tablet, which including the category and proportion of film forming agent and porogen, the sort and dosage of fluidizing agent, the increment of weight after coating and so on. The releasing property of the preparation is evaluated by the dissolution tests in vitro through measuring the content of matrine and oxymatrine content.

RESULTThe preparation method of the PYTCSD: After prepared plain tablets, the 95% alcoholic solution of EC and Eudragit II are mixed in a 7:3 EC: Eudragit II ratio and then added in DEP up to 10% of the coating amount, reduced the alcohol concentration to 4% by diluting with ethonal. Tablet was coated by the alcohol solution and the weight of the plain tablet was increased by 3%. The dissolution tests in vitro indicated that matrine and oxymatrine were not dissolved in the simulated gastric juice after 2 h. The accumulative quantities of matrine and oxymatrine were less than 10% in the simulated intestinal juice after 4 h. The quantities of matrine and oxymatrine are 75.7% and 76.8% in the simulated colon juice after 1 h.

CONCLUSIONThe PYTCSD was prepared and the preparation could fulfil the aim of delivering in the specific colon in vitro.

Alkaloids ; analysis ; Astragalus membranaceus ; chemistry ; Cellulose ; analogs & derivatives ; Colon ; metabolism ; Delayed-Action Preparations ; Drug Combinations ; Drug Compounding ; Drug Delivery Systems ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacokinetics ; Hydrogen-Ion Concentration ; Intestinal Absorption ; Plants, Medicinal ; chemistry ; Polymethacrylic Acids ; Quinolizines ; analysis ; Sophora ; chemistry ; Tablets, Enteric-Coated

OBJECTIVETo investigate the molecular mechanism of haemophilia B caused by the novel mutation of Arg327Ile (R327I) in FIX gene.

METHODSThe R327I, R327Ala(A), R327Lys(K), R327Asn(N) and a replacement mutant (FIXβFVII), in which FIX β strand 324-329 was replaced by that of FVII 298-303, expression plasmids were constructed with site-directed mutagenesis method based on the wild-type (WT) FIX expression plasmid. The HEK293 cell was transiently transfected, then the activity of FIX (FIX:C) was assayed by one stage method in the conditioned medium, while the FIX:Ag in both the conditioned media and the cell lysates was measured by ELISA. The molecular weight and the semi-quantity of expressed FIX were analyzed by Western blot. Fluorescent protein expression plasmid was constructed to investigate the synthesis and secretion of the FIX R327I mutation in the viable cells.

RESULTSFIX:C of the R327I mutant protein was 4.49% of the level of the WT in the conditioned medium, and the FIX:Ag of the R327I mutant protein in the conditioned medium and the cell lysates was 31.02% and 129.29% compared to that of WT, respectively. The mutation was characterized as cross-reaction material reduced (CRMR). The viable cell fluorescent assays showed that the R327I protein was more in both the viable cells and in lysosome than that of WT. The FIX:C of the R327A, R327K, R327N and FIXβFVII mutants was reduced compared to that of WT, the reduction of FIX:C of FIXβFVII was the most significantly amount among all the mutants in medium. FIX:Ag of all the mutants in the medium, except that the R327K increased, was reduced. The result of Western blot showed that the molecular weight of R327I protein was the same as that of WT, but the amount of the protein was much less compared with WT in the conditioned medium.

CONCLUSIONThe abnormal synthesis and secretion as well as the abnormal function of the R327I mutant protein causes haemophilia B. The residue of R327 as well as the β strand domain of R327 located play important roles of the specific function of FIX.

Factor IX ; genetics ; HEK293 Cells ; Hemophilia B ; genetics ; pathology ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Transfection