Objective To develop and validate a method for detecting factor 8 gene (F8) mutations in hemophilia A patients by Ion Torrent semiconductor sequencing .Methods Intron 22 and intron 1 inversions of F8 gene were identified by long distance PCR (LD-PCR), other mutations in the F8 gene were identified by Ion Torrent sequencing.Candidate variants were validated by Sanger sequencing .Sanger sequencing was applied to screen HA carriers from 11 female family members in the 8 pedigrees.One pregnant woman was offered prenatal diagnosis via analyzing the fetal DNA obtained through amniocentesis . Results Four missense mutations ( c.1331A >C, 1648C >T, c.6506G >A, c.6544C >T), two frameshift mutations ( c.2393 _2394insT, c.6320delG), one splicing mutation ( IVS5 +5G >A), one nonsense mutation (c.43C >T) and one Inv22 mutation were identified in all nine probands respectively . Among 11 female family members, 10 females were identified to be HA carriers, and one didn′t carry the maternal pathogenic mutation.Prenatal diagnosis result showed that the fetus inherited the wild -type maternal allele and was predicted to be unaffected by HA .Conclusion The targeted Ion Torrent sequencing is a reliable and efficient method to detect F8 mutations in patients with Hemophilia A disease .

Sodium hyaluronate is one of the natural components of articular cartilage and synovial fluid,which plays important roles in maintaining the structure and physiological functions of joints.Abnormal change of the content and physicochemical properties of sodium hyaluronate in the joints is one of the common pathological causes of osteoarthritis.Supplementation of exogenous sodium hyaluronate,which has similar physical and chemical properties as those in normal joints,has been considered as an effective strategy for clinical treatment of osteoarthritis.However,the benefits of intra-articular injection of sodium hyaluronate are still under debates.Moreover,different recommendations for clinical use were developed in several clinical guidelines.Several guidelines suggested that molecular weight of sodium hyaluronate was an important factor influencing the clinical benefits in osteoarthritis.Diverse products of sodium hyaluronate present different physicochemical and biological characteristics,which may lead to differences in clinical efficacy and safety.High-molecular-weight sodium hyaluronate,with highly modified and cross-linked sugar chains,potentially differ from those with low molecular weight in physicochemical properties,rheological characteristics,and physiological activities.In general,high-molecular-weight sodium hyaluronate have higher viscosity and elasticity.The exogenous hyaluronate,of which the molecular weight is similar to those in normal joints,probably have the similar rheological characteristics.A large number of clinical studies demonstrated that sodium hyaluronate products with high-molecular weight significantly relieved joint pain and improved joint functions in patients with osteoarthritis.In contrast,the clinical efficacy of the low-molecular-weight hyaluronate is still controversial,because several studies could not establish the superiority in osteoarthritis when comparing with the placebo.Studies on molecular mechanisms revealed that some physiological functions of sodium hyaluronate were molecular-weight dependent.High-molecular-weight sodium hyaluronate may have more pronounced impacts on the regulation of inflammation and maintaining the homeostasis of extracellular matrix.This review focused on the effects of sodium hyaluronate with different molecular weight in treating osteoarthritis.Evidence based on clinical studies related to the molecular-weight differences of sodium hyaluronate were presented.Furthermore,the optimal use of various products of sodium hyaluronate with different molecular weight was discussed.

Objective:To prepare biotin-poloxamer(BP) conjugate micelles for epirubicin through biotin conjugated on poloxam-er. Methods:Epirubicin(EPI) was encapsulated in BP micelles. The EPI-loaded BP micelles were characterized by its particle size, zeta potential,surface morphology,as well as the efficiency of drug loading and drug encapsulation and drug release. Marrow leukemia HL-60 cells were used to evaluate the cell cytotoxicity of EPI-loaded BP micelles in vitro. The tumor model in nude mice was estab-lished through the subcutaneous injection of HL-60 cells, and then the inhibitory effect of EPI-loaded BP micelles on tumor volume growth was investigated.Results:It was found that the average particle size of EPI-loaded BP micelles was about 100 nm. In addition, the enhanced cellular uptake ability of EPI-loaded BP micelles was proved by fluorescence microscope observation. The efficiency order of the tumor volume growth inhibition was:EPI-loaded BP micelles > EPI-loaded MATP micelles> EPI-loaded poloxamer micelles>EPI. BP micelles showed significant antitumor activity and low toxicity when compared with the non-targeted micelles. Conclusion:With the advantages of EPR effect and tumor-targeting potential,BP conjugate micelles might be developed as a new system for chemo-therapeutics. However,the tumor targeting technique should be demonstrated further by the other cell experiments and large animal ex-periments.

Objective To survey the current status of community pharmacy care, analyze the needs of both community residents and medical staff for community pharmacy care, and provide references for improving the quality of communi-ty pharmacy care and policy making. Methods The form of questionnaire was taken for community residents and com-munity pharmacists from community health service centers, a random survey was conducted to collect information for statistical analysis. Results Sixty-six point eight percent of community residents selected community health service center as the main choice for obtaining drugs. The residents' home-stored drugs were mainly for cold/fever, abdominal pain/diarrhea, common chronic diseases, external wounds treating and antibacterial. Only 16.8%of community residents stored the drugs according to instructions, 70.6% would use antibacterial drug by themselves under various circum-stances, 82.8% thought there were errors in drug usage, over 98.0% of community pharmacists recognized the impor-tance of pharmaceutical care and the needs for further development. 89.1% of them were eager to improve their own capability of serving and have the needs of collaboration with large hospitals. Conclusion Improper drug usage and storage are common among community residents. Their expectations for pharmaceutical service are increasing. And community pharmacists expected to improve their own pharmacy service capability.

Objective To approach the efficiency of second-trimester prenatal screening using two serum markers for Down's syndrome (DS).Methods Retrospective analysis was conducted on the results of prenatal screening using two serum markers,alpha fetoprotein (AFP) and free beta subunit of human chorionic gonadotropin(free-β-hCG),in 50 cases of DS pregnancy identified among 60 931 pregnant women received prenatal screening from November 1997 to April 2008 in Nanjing Maternal and Child Health Hospital.Results Among the 50 DS cases,the detection rate of DS was 50% (25/50) when taking free-β-hCG≥2.5 MoM as the cut-off,with the positive rate of screening was 6.6%.And the detection rate of DS would be 18.0%(9/25) when taking AFP≤0.5 MoM as the cut-off,with the positive rate of screening was 4.6%.When the risk cut-off value of DS was set at 1/270,the detection rate changed to 52.0%,and the positive rate of screening was 4.7%;and the two figures changed to 62.0% and 5.5%,respectively,when the risk cut-off was set to 1/300.Thirteen DS cases showed the risk value between 1/1000 and 1/300,among which two were monomarker abnormality.Thirteen (26.0%) of the 50 DS fetus were found to have one or two abnormality markers by ultrasound scan,among which one was DS low risk,and the other 12 were DS high risk in serum screening.Conclusions The second-trimester prenatal screening using AFP or free β-hCG for Down's syndrome is effective in identifying DS pregnancy with limited specificity and sensitivity.But the detection rate can be elevated by the combination of these two markers.The second trimester systemic ultrasound scan is not ideal for DS identification,but it can increase the specificity and sensitivity of serum prenatal screening.

Objective To perform prenatal diagnosis for a fetus with hydrocephalus and congenital heart disease by whole exome se-quencing ( WES) , and then provide genetic counseling for the next pregnancy. Methods DNAs from amniotic fluid cells of the fetus and peripheral blood of his/her parents were extracted, respectively, and then performed WES. After the process of library construc-tion, hybrid capture and sequencing, the obtained data were compared with the database from human genome and literatures and ana-lyzed by software. The pathogenic mutations were searched based on the American College of Medical Genetics and Genomics ( ACMG, 2015) guideline and verified by the Sanger sequencing. Results The WES results found that the compound heterozygous mutations ex-isted in POMT1 gene of the fetus, which were inherited from the splice site mutation c.605+1G>A( IVS7) of his/her mother and the frameshift mutation c.1367 c.1368 ( exon 15) insGA, p. L456Lfs?80 of his/her father, respectively. The Sanger sequencing results were consistent with that of WES. The fetus was affected by Walker-Warburg syndrome, and his/her parents decided to terminate the pregnancy finally. Conclusion The WES may diagnose Walker-Warburg syndrome rapidly and accurately, which may play an impor-tant role in clinical management and genetic counseling.

Objective: To evaluate the efficacy of cell free DNA (cf-DNA) screening in prenatal care by analyzing the follow-up information and pregnancy outcomes. Methods: All cf-DNA cases conducted in Women′s Hospital of Nanjing Medical University from August 2011 to December 2017 were enrolled. The general information of the pregnancies, cf-DNA results, confirmatory testing results, and the follow-up results were collected. The pregnancy outcomes were analyzed in cases with low risk cf-DNA results as well as with high risk results for common trisomies, which were trisomy 21 (T21), trisomy 18 (T18), and trisomy 13 (T13). The sensitivity, specificity, positive predictive value and negative predictive value of cf-DNA screening were calculated. Results: (1) A total of 43 615 cf-DNA cases were involved, with 44 cases (0.10%, 44/43 615) test failure results, 314 cases (0.72%, 314/43 571) high risk results for common trisomies and 43 257 cases (99.27%, 43 257/43 571) low risk results. (2) Among 277 cases (88.21%, 277/314) high risk cases were successfully followed up, and 228 cases (82.31%, 228/277) underwent invasive confirmatory prenatal diagnosis. In the low risk results, 36 826 cases (85.13%, 36 826/43 257) were successfully followed up, and 572 (1.55%, 572/36 826) cases were found to have adverse pregnancy outcomes, among which 4 false negative cf-DNA results were confirmed. (3) In the 37 103 successfully followed up cf-DNA cases, the sensitivity for T21, T18, T13 were calculated as 97.96%, 96.67% and 100.00%, respectively; the specificity for T21, T18, T13 were calculated as 99.96%, 99.95% and 99.95%, respectively. The positive predictive value for T21, T18, T13 were calculated as 90.57%, 63.04% and 17.39%, respectively. The negative predictive value for T21, T18, T13 were calculated as 99.99%, 99.98% and 100.00%. Conclusions Cf-DNA is effective in detecting common trisomies, with a high sensitivity and specificity. However, the follow-up information revealed several potential limitations in current clinical practice, such as a number of cases with high risk results rejected invasive confirmatory testing, as well as the genetic diagnostic results for most low risk cases with an adverse pregnancy outcome aren′t obtained. Genetic counseling and the follow-up for all the cf-DNA cases should be emphasized in the future.

In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA), these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potential epitope regions were located at Codons 469-882 in the S protein, and one epitope site was located at Codons 599-620. Identification of antigenic regions in the SARS-CoV S protein may be important for the functional studies of this virus or the development of clinical diagnosis.
Antigens, Viral ; immunology ; Chromatography, High Pressure Liquid ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Humans ; Mass Spectrometry ; Membrane Glycoproteins ; genetics ; immunology ; metabolism ; Molecular Weight ; Peptide Fragments ; chemistry ; Recombinant Proteins ; genetics ; immunology ; SARS Virus ; genetics ; immunology ; metabolism ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo investigate potential mutation of the ASS1 gene in a male infant with acute citrullinemia type I.

METHODSGenomic DNA was prepared from peripheral blood samples of the family members. Mutation analysis of the 14 ASS1 exons was carried out by PCR and direct DNA sequencing.

RESULTSA homozygous missense mutation of c.970G>A located in exon 13, which results in p.G324S, was identified in the child. Sequencing of the parents showed a heterozygous status for the same mutation.

CONCLUSIONA missense mutation of c.970G>A in the ASS1 gene is responsible for the pathogenesis of the disease in the infant.

Amino Acid Sequence ; Amino Acid Substitution ; Argininosuccinate Synthase ; chemistry ; genetics ; Base Sequence ; Citrullinemia ; genetics ; Gene Order ; Humans ; Infant ; Male ; Models, Molecular ; Molecular Sequence Data ; Mutation, Missense ; Protein Conformation ; Sequence Alignment ; Sequence Analysis, DNA

Objective To obtain myoclonic epilepsy with ragged-red fibers (MERRF)-specific cardiomyocytes by the differentiation in vitro of inducing pluripotent stem cells (iPSC) derived from a MERRF patient and evaluate the application of the prepared cardiomyocytes in construction of MERRF syndrome model.Methods The patient-derived iPSCs and H9 embryonic stem (ES) cells,the control cell line,were unidirectionally differentiated into cardiomyocytes n in vitro.The obtained cardiomyocytes were identified and validated by detecting the presence of cardiomyocyte-specific markers using immunofluorescence staining and RT-PCR.The beating frequencies were recorded to compare the functional evaluation for the two groups of cardiomyocyte.Results Both the patient-derived iPSC and H9 ES cells were differentiated into cardiomyocytes successfully.The average beating frequencies of MERRF-induced cardiomyocytes (iCMs) were 13,24,15 and 18 times/min on the day 10,13,15 and 16 during the cell differentiation process.The average beating frequencies of H9-iCMs were 80,96,120 and 120 times/min,respectively.The beating ability of iPSC-differentiated cardiomyocytes was significantly lower than that of corresponding control (all P ＜ 0.05).Conclusion The patient-derived iPSCs may differentiated into cardiomyocytes.Based on the functional evaluation for these cardiomyocytes,the model for MERRF syndrome with mitochondrial mutations was generated and characterized in vitro.