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1.  Molecular characterization and copy number of SMN1, SMN2 and NAIP in Chinese patients with spinal muscular atrophy and unrelated healthy controls 
Spinal muscular atrophy (SMA) is caused by SMN1 dysfunction, and the copy number of SMN2 and NAIP can modify the phenotype of SMA. The aim of this study was to analyze the copy numbers and gene structures of SMA-related genes in Chinese SMA patients and unrelated healthy controls.
Forty-two Chinese SMA patients and two hundred and twelve unrelated healthy Chinese individuals were enrolled in our study. The copy numbers and gene structures of SMA-related genes were measured by MLPA assay.
We identified a homozygous deletion of SMN1 in exons 7 and 8 in 37 of 42 patients (88.1%); the other 5 SMA patients (11.9%) had a single copy of SMN1 exon 8. The proportions of the 212 unrelated healthy controls with different copy numbers for the normal SMN1 gene were 1 copy in 4 individuals (1.9%), 2 copies in 203 (95.7%) and 3 copies in 5 (2.4%). Three hybrid SMN genes and five genes that lack partial sequences were found in SMA patients and healthy controls. Distributions of copy numbers for normal SMN2 and NAIP were significantly different (P < 0.001) in people with and without SMA.
The copy numbers and gene structures of SMA-related genes were different in Chinese SMA patients and healthy controls.
PMCID: PMC4328246
Chinese; MLPA; SMA; Gene copy number
2.  Establishment of a Model of Renal Impairment with Mild Renal Insufficiency Associated with Atrial Fibrillation in Canines 
PLoS ONE  2014;9(8):e105974.
Chronic kidney disease and occurrence of atrial fibrillation (AF) are closely related. No studies have examined whether renal impairment (RI) without severe renal dysfunction is associated with the occurrence of AF.
Unilateral RI with mild renal insufficiency was induced in beagles by embolization of small branches of the renal artery in the left kidney for 2 weeks using gelatin sponge granules in the model group (n = 5). The sham group (n = 5) underwent the same procedure, except for embolization. Parameters associated with RI and renal function were tested, cardiac electrophysiological parameters, blood pressure, left ventricular pressure, and AF vulnerability were investigated. The activity of the sympathetic nervous system, renin-angiotensin-aldosterone system, inflammation, and oxidative stress were measured. Histological studies associated with atrial interstitial fibrosis were performed.
Embolization of small branches of the renal artery in the left kidney led to ischemic RI with mild renal insufficiency. The following changes occurred after embolization. Heart rate and P wave duration were increased. Blood pressure and left ventricular systolic pressure were elevated. The atrial effective refractory period and antegrade Wenckebach point were shortened. Episodes and duration of AF, as well as atrial and ventricular rate during AF were increased in the model group. Plasma levels of norepinephrine, renin, and aldosterone were increased, angiotensin II and aldosterone levels in atrial tissue were elevated, and atrial interstitial fibrosis was enhanced after 2 weeks of embolization in the model group.
We successfully established a model of RI with mild renal insufficiency in a large animal. We found that RI with mild renal insufficiency was associated with AF in this model.
PMCID: PMC4144969  PMID: 25157494
3.  Discovery of common sequences absent in the human reference genome using pooled samples from next generation sequencing 
BMC Genomics  2014;15(1):685.
Sequences up to several megabases in length have been found to be present in individual genomes but absent in the human reference genome. These sequences may be common in populations, and their absence in the reference genome may indicate rare variants in the genomes of individuals who served as donors for the human genome project. As the reference genome is used in probe design for microarray technology and mapping short reads in next generation sequencing (NGS), this missing sequence could be a source of bias in functional genomic studies and variant analysis. One End Anchor (OEA) and/or orphan reads from paired-end sequencing have been used to identify novel sequences that are absent in reference genome. However, there is no study to investigate the distribution, evolution and functionality of those sequences in human populations.
To systematically identify and study the missing common sequences (micSeqs), we extended the previous method by pooling OEA reads from large number of individuals and applying strict filtering methods to remove false sequences. The pipeline was applied to data from phase 1 of the 1000 Genomes Project. We identified 309 micSeqs that are present in at least 1% of the human population, but absent in the reference genome. We confirmed 76% of these 309 micSeqs by comparison to other primate genomes, individual human genomes, and gene expression data. Furthermore, we randomly selected fifteen micSeqs and confirmed their presence using PCR validation in 38 additional individuals. Functional analysis using published RNA-seq and ChIP-seq data showed that eleven micSeqs are highly expressed in human brain and three micSeqs contain transcription factor (TF) binding regions, suggesting they are functional elements. In addition, the identified micSeqs are absent in non-primates and show dynamic acquisition during primate evolution culminating with most micSeqs being present in Africans, suggesting some micSeqs may be important sources of human diversity.
76% of micSeqs were confirmed by a comparative genomics approach. Fourteen micSeqs are expressed in human brain or contain TF binding regions. Some micSeqs are primate-specific, conserved and may play a role in the evolution of primates.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-685) contains supplementary material, which is available to authorized users.
PMCID: PMC4148959  PMID: 25129063
Missing common sequence; De novo assembling; Next generation sequencing; Expression in brain; Transcription factor binding; Genome evolution
4.  Full-length soluble CD147 promotes MMP-2 expression and is a potential serological marker in detection of hepatocellular carcinoma 
As a surface glycoprotein, CD147 is capable of stimulating the production of matrix metalloproteinases (MMPs) from neighboring fibroblasts. The aim of the present study is to explore the role of soluble CD147 on MMPs secretion from hepatocellular carcinoma (HCC) cells, and to investigate the diagnostic value of serum soluble CD147 in the HCC detection.
We identified the form of soluble CD147 in cell culture supernate of HCC cells and serum of patients with HCC, and explored the role of soluble CD147 on MMPs secretion. Serum CD147 levels were detected by the enzyme-linked immunosorbent assay, and the value of soluble CD147 as a marker in HCC detection was analyzed.
Full length soluble CD147 was presented in the culture medium of HCC cells and serum of patients with HCC. The extracellular domain of soluble CD147 promoted the expression of CD147 and MMP-2 from HCC cells. Knockdown of CD147 markedly diminished the up-regulation of CD147 and MMP-2 which induced by soluble CD147. Soluble CD147 activated ERK, FAK, and PI3K/Akt pathways, leading to the up-regulation of MMP-2. The level of soluble CD147 in serum of patients with HCC was significantly elevated compared with healthy individuals (P < 0.001). Soluble CD147 levels were found to be associated with HCC tumor size (P = 0.007) and Child-Pugh grade (P = 0.007). Moreover, soluble CD147 showed a better performance in distinguishing HCC compared with alpha-fetoprotein.
The extracellular domain of soluble CD147 enhances the secretion of MMP-2 from HCC cells, requiring the cooperation of membrane CD147 and activation of ERK, FAK, and PI3K/Akt signaling. The measurement of soluble CD147 may offer a useful approach in diagnosis of HCC.
PMCID: PMC4227008  PMID: 24996644
Hepatocellular carcinoma; Soluble CD147; Matrix metalloproteinases; Alpha-fetoprotein; Serological marker
5.  Gastric nNOS reduction accompanied by natriuretic peptides signaling pathway upregulation in diabetic mice 
AIM: To investigate the relationship between neuronal nitric oxide synthase (nNOS) expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin (STZ)-induced diabetic mice.
METHODS: Diabetic mice were induced by injection of STZ solution. Immunofluorescence labeling of HuC/D, nNOS and natriuretic peptide receptor-A, B, C (NPRs) in the gastric fundus (GF) was used to observe nNOS expression and whether NPRs exist on enteric neurons. The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting. An isometric force transducer recorded the electric field stimulation (EFS)-induced relaxation and contraction in the diabetic GF. An intracellular recording method assessed EFS-induced inhibitory junction potentials (IJP) on the GF. GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide (CNP) for 24 h, after which their nNOS expressions were detected by western blotting.
RESULTS: Eight weeks after injection, 43 diabetic mice were obtained from mouse models injected with STZ. Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF. The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced, while the contraction was enhanced in the diabetic GF. EFS-induced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice. However, both NPR-A and NPR-B were detected on enteric neurons, and their expression levels were upregulated in the diabetic GF. The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP.
CONCLUSION: These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.
PMCID: PMC4000499  PMID: 24782615
Diabetic gastroparesis; Natriuretic peptides; Nitric oxide synthase; Enteric neuron
6.  Voltage Dependent Potassium Channel Remodeling in Murine Intestinal Smooth Muscle Hypertrophy Induced by Partial Obstruction 
PLoS ONE  2014;9(2):e86109.
Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.
PMCID: PMC3916336  PMID: 24516526
7.  Immune-Tolerizing Procedure for Preparation of Monoclonal Antibodies Against Glycoprotein E of Pseudorabies Virus 
The glycoprotein E (gE) of pseudorabies virus (PRV) is known to be an important marker protein in the control and eradication of Aujeszky's disease. In this study, BALB/c mice were immunized with gE-deleted PRV as tolerogen and with wild-type PRV as immunogen. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. Two hybridoma cell lines that could stably secrete the monoclonal antibody (MAb) against gE were achieved by using indirect ELISA screening and subcloning three times; they were named 1D2 and 2B2. Indirect immunofluorescence assay (IFA) revealed that the MAbs were specifically against gE of PRV. MAbs 1D2 and 2B2 were subgroup IgG1. The MAbs obtained in this study provide useful tools for the development of differential diagnostic methods for PRV.
PMCID: PMC4014296  PMID: 23600501
8.  The Diagnostic Performance of Coronary Artery Angiography with 64-MSCT and Post 64-MSCT: Systematic Review and Meta-Analysis 
PLoS ONE  2014;9(1):e84937.
To comprehensively investigate the diagnostic performance of coronary artery angiography with 64-MDCT and post 64-MDCT.
Materials and Methods
PubMed was searched for all published studies that evaluated coronary arteries with 64-MDCT and post 64-MDCT. The clinical diagnostic role was evaluated by applying the likelihood ratios (LRs) to calculate the post-test probability based on Bayes' theorem.
91 studies that met our inclusion criteria were ultimately included in the analysis. The pooled positive and negative LRs at patient level were 8.91 (95% CI, 7.53, 10.54) and 0.02 (CI, 0.01, 0.03), respectively. For studies that did not claim that non-evaluable segments were included, the pooled positive and negative LRs were 11.16 (CI, 8.90, 14.00) and 0.01 (CI, 0.01, 0.03), respectively. For studies including uninterruptable results, the diagnostic performance decreased, with the pooled positive LR 7.40 (CI, 6.00, 9.13) and negative LR 0.02 (CI, 0.01, 0.03). The areas under the summary ROC curve were 0.98 (CI, 0.97 to 0.99) for 64-MDCT and 0.96 (CI, 0.94 to 0.98) for post 64-MDCT, respectively. For references explicitly stating that the non-assessable segments were included during analysis, a post-test probability of negative results >95% and a positive post-test probability <95% could be obtained for patients with a pre-test probability of <73% for coronary artery disease (CAD). On the other hand, when the pre-test probability of CAD was >73%, the diagnostic role was reversed, with a positive post-test probability of CAD >95% and a negative post-test probability of CAD <95%.
The diagnostic performance of post 64-MDCT does not increase as compared with 64-MDCT. CTA, overall, is a test of exclusion for patients with a pre-test probability of CAD<73%, while for patients with a pre-test probability of CAD>73%, CTA is a test used to confirm the presence of CAD.
PMCID: PMC3897406  PMID: 24465453
9.  Continuous-flow Mass Production of Silicon Nanowires via Substrate-Enhanced Metal-Catalyzed Electroless Etching of Silicon with Dissolved Oxygen as an Oxidant 
Scientific Reports  2014;4:3667.
Silicon nanowires (SiNWs) are attracting growing interest due to their unique properties and promising applications in photovoltaic devices, thermoelectric devices, lithium-ion batteries, and biotechnology. Low-cost mass production of SiNWs is essential for SiNWs-based nanotechnology commercialization. However, economic, controlled large-scale production of SiNWs remains challenging and rarely attainable. Here, we demonstrate a facile strategy capable of low-cost, continuous-flow mass production of SiNWs on an industrial scale. The strategy relies on substrate-enhanced metal-catalyzed electroless etching (MCEE) of silicon using dissolved oxygen in aqueous hydrofluoric acid (HF) solution as an oxidant. The distinct advantages of this novel MCEE approach, such as simplicity, scalability and flexibility, make it an attractive alternative to conventional MCEE methods.
PMCID: PMC3888973  PMID: 24413157
10.  Dual-sided composite mesh repair of hiatal hernia: Our experience and a review of the Chinese literature 
AIM: To summarize our experience in the application of Crurasoft® for antireflux surgery and hiatal hernia (HH) repair and to introduce the work of Chinese doctors on this topic.
METHODS: Twenty-one patients underwent HH repair with Crurasoft® reinforcement. Gastroesophageal reflux disease (GERD) and HH-related symptoms including heartburn, regurgitation, chest pain, dysphagia, and abdominal pain were evaluated preoperatively and 6 mo postoperatively. A patient survey was conducted by phone by one of the authors. Patients were asked about “recurrent reflux or heartburn” and “dysphagia”. An internet-based Chinese literature search in this field was also performed. Data extracted from each study included: number of patients treated, hernia size, hiatorrhaphy, antireflux surgery, follow-up period, recurrence rate, and complications (especially dysphagia).
RESULTS: There were 8 type I, 10 type II and 3 type III HHs in this group. Mean operative time was 119.29 min (range 80-175 min). Intraoperatively, length and width of the hiatal orifice were measured, (4.33 ± 0.84 and 2.85 ± 0.85 cm, respectively). Thirteen and eight Nissen and Toupet fundoplications were performed, respectively. The intraoperative complication rate was 9.52%. Despite dysphagia, GERD-related symptoms improved significantly compared with those before surgery. The recurrence rate was 0% during the 6-mo follow-up period, and long-term follow-up disclosed a recurrence rate of 4.76% with a mean period of 16.28 mo. Eight patients developed new-onset dysphagia. The Chinese literature review identified 12 papers with 213 patients. The overall recurrence rate was 1.88%. There was no esophageal erosion and the rate of dysphagia ranged from 0% to 24%.
CONCLUSION: The use of Crurasoft® mesh for HH repair results in satisfactory symptom control with a low recurrence rate. Postoperative dysphagia continues to be an issue, and requires more research to reduce its incidence.
PMCID: PMC3761107  PMID: 24023497
Hiatal hernia; Gastroesophageal reflux disease; Anti-reflux surgery; Mesh; Prosthetic
11.  Anticancer Activity of Amauroderma rude 
PLoS ONE  2013;8(6):e66504.
More and more medicinal mushrooms have been widely used as a miraculous herb for health promotion, especially by cancer patients. Here we report screening thirteen mushrooms for anti-cancer cell activities in eleven different cell lines. Of the herbal products tested, we found that the extract of Amauroderma rude exerted the highest activity in killing most of these cancer cell lines. Amauroderma rude is a fungus belonging to the Ganodermataceae family. The Amauroderma genus contains approximately 30 species widespread throughout the tropical areas. Since the biological function of Amauroderma rude is unknown, we examined its anti-cancer effect on breast carcinoma cell lines. We compared the anti-cancer activity of Amauroderma rude and Ganoderma lucidum, the most well-known medicinal mushrooms with anti-cancer activity and found that Amauroderma rude had significantly higher activity in killing cancer cells than Ganoderma lucidum. We then examined the effect of Amauroderma rude on breast cancer cells and found that at low concentrations, Amauroderma rude could inhibit cancer cell survival and induce apoptosis. Treated cancer cells also formed fewer and smaller colonies than the untreated cells. When nude mice bearing tumors were injected with Amauroderma rude extract, the tumors grew at a slower rate than the control. Examination of these tumors revealed extensive cell death, decreased proliferation rate as stained by Ki67, and increased apoptosis as stained by TUNEL. Suppression of c-myc expression appeared to be associated with these effects. Taken together, Amauroderma rude represented a powerful medicinal mushroom with anti-cancer activities.
PMCID: PMC3688780  PMID: 23840494
12.  Evidence of recent natural selection on the Southeast Asian deletion (--SEA) causing α-thalassemia in South China 
The Southeast Asian deletion (--SEA) is the most commonly observed mutation among diverse α-thalassemia alleles in Southeast Asia and South China. It is generally argued that mutation --SEA, like other variants causing hemoglobin disorders, is associated with protection against malaria that is endemic in these regions. However, little evidence has been provided to support this claim.
We first examined the genetic imprint of recent positive selection on the --SEA allele and flanking sequences in the human α-globin cluster, covering a genomic region spanning ~410 kb, by genotyping 28 SNPs in a Chinese population consisting of 76 --SEA heterozygotes and 138 normal individuals. The pattern of linkage disequilibrium (LD) and the long-range haplotype test revealed a signature of positive selection. The network of inferred haplotypes suggested a single origin of the --SEA allele.
Thus, our data support the hypothesis that the --SEA allele has been subjected to recent balancing selection, triggered by malaria.
PMCID: PMC3626844  PMID: 23497175
13.  Ergosterol Peroxide Isolated from Ganoderma lucidum Abolishes MicroRNA miR-378-Mediated Tumor Cells on Chemoresistance 
PLoS ONE  2012;7(8):e44579.
Due to an altered expression of oncogenic factors and tumor suppressors, aggressive cancer cells have an intrinsic or acquired resistance to chemotherapeutic agents. This typically contributes to cancer recurrence after chemotherapy. microRNAs are short non-coding RNAs that are involved in both cell self-renewal and cancer development. Here we report that tumor cells transfected with miR-378 acquired properties of aggressive cancer cells. Overexpression of miR-378 enhanced both cell survival and colony formation, and contributed to multiple drug resistance. Higher concentrations of chemotherapeutic drugs were needed to induce death of miR-378-transfected cells than to induce death of control cells. We found that the biologically active component isolated from Ganoderma lucidum could overcome the drug-resistance conferred by miR-378. We purified and identified the biologically active component of Ganoderma lucidum as ergosterol peroxide. We demonstrated that ergosterol peroxide produced greater activity in inducing death of miR-378 cells than the GFP cells. Lower concentrations of ergosterol peroxide were needed to induce death of the miR-378-transfected cells than in the control cells. With further clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells.
PMCID: PMC3431381  PMID: 22952996
14.  Characterization of Basigin Isoforms and the Inhibitory Function of Basigin-3 in Human Hepatocellular Carcinoma Proliferation and Invasion ▿  
Molecular and Cellular Biology  2011;31(13):2591-2604.
Basigin, which has four isoforms, plays an important role in invasion of hepatocellular carcinoma (HCC). Detailed transcriptional regulation and functions of the basigin isoforms have not been reported except in the case of the predominant isoform basigin-2, which act as inducer of matrix metalloproteinases (MMPs). Here we determined that basigin-2, basigin-3, and basigin-4 were the most abundant transcript variants in human cell lines. GeneRacer PCR and luciferase reporter assays showed that basigin-3 and basigin-4 were initiated from an alternative promoter. Basigin-3 and basigin-4 were widely expressed in various normal human tissues at the mRNA level and were upregulated in HCC tissues compared to in normal tissues. Western blotting and confocal imaging showed that glycosylated basigin-3 and basigin-4 were expressed and localized to the plasma membrane. However, in cultured cell lines, only native basigin-3, and not basigin-4, was detected at protein level. Overexpression of basigin-3 inhibited HCC cell proliferation, MMP induction, and cell invasion in vitro and in vivo. Bimolecular fluorescence complementation assays and nuclear magnetic resonance (NMR) analysis indicated that basigin-3 interacted with basigin-2 to form hetero-oligomers. In conclusion, we systematically investigated the alternative splicing of basigin and found that basigin-3 could inhibit HCC proliferation and invasion, probably through interaction with basigin-2 as an endogenous inhibitor via hetero-oligomerization.
PMCID: PMC3133368  PMID: 21536654
15.  Reliable Detection of Paternal SNPs within Deletion Breakpoints for Non-Invasive Prenatal Exclusion of Homozygous α0-Thalassemia in Maternal Plasma 
PLoS ONE  2011;6(9):e24779.
Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude α-thalassemia major that uses SNPs linked to the normal paternal α-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (−−SEA) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (−−SEA) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (−−SEA) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4–78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in α-thalassemia major, and could further be employed to test for other diseases due to gene deletion.
PMCID: PMC3182989  PMID: 21980356
16.  Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population 
Screening for carriers of spinal muscular atrophy (SMA) is necessary for effective clinical/prenatal diagnosis and genetic counseling. However, a population-based study of SMA prevalence in mainland China has not yet been conducted. In this study, the copy number of survival motor neuron (SMN) genes was determined in 1712 newborn cord blood samples collected from southern China and from 25 core families, which included 26 SMA patients and 44 parents, to identify SMA carriers. The results presented 13 groups with different SMN1/SMN2 ratios among 1712 newborn individuals, which corresponded to 1535 subjects with two copies of SMN1, 119 with three copies of SMN1, 17 with four copies of SMN1, and 41 with a heterozygous deletion of SMN1 exon 7. Simultaneously, two ‘2+0' genotypes and two point mutations were found among the 44 obligate carriers in the core families, including a novel SMN1 splice-site mutation that was identified in the junction between intron 6 and exon 7 (c. 835–1G>A). These results indicated that the carrier frequency is 1/42 in the general Chinese population and that duplicated SMN1 alleles and de novo deletion mutations are present in a small number of SMA carriers. In addition, we developed and validated a new alternative screening method using a reverse dot blot assay for rapid genotyping of deletional SMA. Our research elucidated the genetic load and SMN gene variants that are present in the Chinese population, and could serve as the basis for a nationwide program of genetic counseling and clinical/prenatal diagnosis to prevent SMA in China.
PMCID: PMC2987421  PMID: 20442745
SMA; SMN copy number; carrier screening; RDB; DHPLC
17.  Preclinical transplantation and safety of HS/PCs expanded from human umbilical cord blood 
World Journal of Stem Cells  2011;3(5):43-52.
AIM: To expand hematopoietic/progenitor stem cells (HS/PCs) from umbilical cord blood (UCB) and prepare the HS/PC product, and analyze preclinical transplantation and safety of HS/PC product.
METHODS: Human bone marrow-derived mesenchymal stem cells (MSCs) were used as feeder cells to expand HS/PCs from UCB in a serum-free culture system. The proliferation potential of HS/PCs was analyzed. The expanded HS/PCs were suspended in the L-15 medium to prepare the HS/PC product. The contamination of bacteria, fungi and mycoplasmas, the infection of exogenous virus, the concentration of bacterial endotoxin, and the SCF residual in HS/PC product were determined. Finally, cells from the HS/PC product with or without bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the irradiated NOD/SCID mice to determine the in vivo engraftment potential.
RESULTS: After co-culture for 10 d, the total nuclear cells (TNCs) increased 125-fold, and CD34+ cells increased 43-fold. The granulocyte-macrophage colony- forming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) increased 3.3- and 4.7-fold respectively. The expanded cells were collected and prepared as the expanded product of HS/PCs by re-suspending cells in L-15 medium. For preclinical safety, the HS/PC product was analysed for contamination by bacteria, fungi and mycoplasmas, the bacterial endotoxin concentration and the SCF content. The results showed that the HS/PC product contained no bacteria, fungi or mycoplasmas. The bacterial endotoxin concentration was less than the detection limit of 6 EU/mL, and residual SCF was 75 pg/mL. Based on clinical safety, the HS/PC product was qualified for clinical transplantation. Finally, the HS/PC product was transplanted the irradiated mice where it resulted in rapid engraftment of hematopoietic cells.
CONCLUSION: HSPC product prepared from UCB in the serum-free culture system with hMSCs as feeder cells should be clinically safe and effective for clinical transplantation.
PMCID: PMC3110917  PMID: 21666821
Hematopoietic stem cells; Ex vivo expansion; Preclinical safeties; Transplantation
18.  Modification of the Epworth Sleepiness Scale in Central China 
Quality of Life Research  2011;20(10):1721-1726.
The well-known excessive daytime sleepiness (EDS) assessment, Epworth Sleepiness Scale (ESS), is not consistently qualified for patients with diverse living habits. This study is aimed to build a modified ESS (mESS) and then to verify its feasibility in the assessment of EDS for patients with suspected sleep-disordered breathing (SDB) in central China.
A Ten-item Sleepiness Questionnaire (10-ISQ) was built by adding two backup items to the original ESS. Then the 10-ISQ was administered to 122 patients in central China with suspected SDB [among them, 119 cases met the minimal diagnostic criteria for obstructive sleep apnea by sleep study, e.g., apnea and hypopnea index (AHI) ≥ 5 h−1] and 117 healthy central Chinese volunteers without SDB. Multivariate exploratory techniques were used for item validation. The unreliable item in the original ESS was replaced by the eligible backup item, thus a modified ESS (mESS) was built, and then verified.
Item 8 proved to be the only unreliable item in central Chinese patients, with the least factor loading on the main factor and the lowest item-total correlation both in the 10-ISQ and in the original ESS, deletion of it would increase the Cronbach’s alpha (from 0.86 to 0.87 in the 10-ISQ; from 0.83 to 0.85 in the original ESS). The mESS was subsequently built by replacing item 8 in the original ESS with item 10 in the 10-ISQ. Verification with patients’ responses revealed that the mESS was a single-factor questionnaire with good internal consistency (Cronbach’s alpha = 0.86). The sum score of the mESS not only correlated with AHI (P < 0.01) but was also able to discriminate the severity of obstructive apnea (P < 0.01). Nasal CPAP treatment for severe OSA reduced the score significantly (P < 0.001). The performance of the mESS was poor in evaluating normal subjects.
The mESS improves the validity of ESS for our patients. Therefore, it is justified to use it instead of the original one in assessment of EDS for patients with SDB in central China.
PMCID: PMC3220816  PMID: 21484529
Epworth Sleepiness Scale; Excessive daytime sleepiness; Sleep-disordered breathing
19.  Diagnosis and molecular characterization of rabies virus from a buffalo in China: a case report 
Virology Journal  2011;8:101.
Rabies virus (RABV) can infect many different species of warm-blooded animals. Glycoprotein G plays a key role in viral pathogenicity and neurotropism, and includes antigenic domains that are responsible for membrane fusion and host cell receptor recognition.
Case presentation
A case of buffalo rabies in China was diagnosed by direct fluorescent antibody test, G gene reverse-transcriptase polymerase chain reaction, and RABV mouse inoculation test. Molecular characterization of the RABV was performed using DNA sequencing, phylogenetic analysis and amino acid sequence comparison based on the G gene from different species of animals.
The results confirmed that the buffalo with suspected rabies was infected by RABV, which was genetically closely related to HNC (FJ602451) that was isolated from cattle in China in 2007. Comparison of the G gene among different species of animal showed that there were almost no amino acid changes among RABVs isolated from the same species of animals that distributed in a near region. However, there were many changes among RABVs that were isolated from different species of animal, or the same species from different geographic regions. This is believed to be the first case report of buffalo rabies in China, and the results may provide further information to understand the mechanism by which RABV breaks through the species barrier.
PMCID: PMC3061937  PMID: 21375773
20.  The Minimally Invasive Effect of Breast Approach Endoscopic Thyroidectomy: An Expert's Experience 
We evaluated the invasiveness of breast approach endoscopic thyroidectomy (BAET) carried out by surgeon very experienced in this procedure. Twenty-four patients who underwent BAET and 19 patients who underwent conventional thyroidectomy were the study population. Postoperative pain was assessed by a visual analog scale (VAS). The values 2, 12, and 24 h after surgery were significantly lower in the BAET group than those in the conventional group. Serum IL-6 and CRP levels were measured by an ELISA preoperatively and at 2, 12, 24 and 48 h after operation. Their values increased significantly after both procedures when compared to preoperative levels with significant differences between the two groups detected at the 24-hour and 48-hour time points. Subjective and objective evidence supported the notion that BAET could become a minimally invasive procedure if the surgeon gained sufficient experience.
PMCID: PMC2933855  PMID: 20827304
21.  Evidence-based appraisal in laparoscopic Nissen and Toupet fundoplications for gastroesophageal reflux disease 
AIM: To demonstrate the optimal surgical procedure for gastroesophageal reflux disease.
METHODS: The electronic databases of Medline, Elsevier, Springerlink and Embase over the last 16 years were searched. All clinical trials involved in the outcomes of laparoscopic Nissen fundoplication (LNF) and laparoscopic Toupet fundoplication (LTF) were identified. The data of assessment in benefits and adverse results of LNF and LTF were extracted and compared using meta-analysis.
RESULTS: We ultimately identified a total of 32 references reporting nine randomized controlled trials, eight prospective cohort trials and 15 retrospective trials. These studies reported a total of 6236 patients, of whom 4252 (68.18%) underwent LNF and 1984 (31.82%) underwent LTF. There were no differences between LNF and LTF in patients’ satisfaction, perioperative complications, postoperative heartburn, reflux recurrence and re-operation. Both LNF and LTF enhanced the function of lower esophageal sphincter and improved esophagitis. The postoperative dysphagia, gas-bloating syndrome, inability to belch and the need for dilatation after LNF were more common than after LTF. Subgroup analyses showed that dysphagia after LNF and LTF was similar in patients with normal esophageal peristalsis (EP), but occurred more frequently in patients with weak EP after LNF than after LTF. Furthermore, patients with normal EP after LNF still had a higher risk of developing dysphagia than did patients with abnormal EP after LTF.
CONCLUSION: Compared with LNF, LTF offers equivalent symptom relief and reduces adverse results.
PMCID: PMC2890948  PMID: 20572311
Laparoscopic fundoplication; Nissen, Toupet; Gastroesophageal reflux disease; Anti-reflux surgery; Esophageal peristalsis; Meta-analysis
22.  Progress in laparoscopic anatomy research: A review of the Chinese literature 
The development of laparoscopic surgery has generated the new field of study, laparoscopic anatomy. This article reviews the reported literature on laparoscopic anatomy and explores how it has evolved along with advances in abdominal surgery. In addition, the principal concerns in current laparoscopic anatomy research are discussed, including: (1) types of special adjacent anatomical structures; and (2) special surgical planes and anatomical landmarks. Understanding of systematic laparoscopic anatomy can provide the junior surgeons a clear procedural approach, and would benefit laparoscopic surgeons in training.
PMCID: PMC2874137  PMID: 20480518
Anatomy; Landmark; Laparoscopy; Minimally invasive; Surgical plane
23.  1-Phenyl-1-[(1-phenyl­ethyl)sulfonyl­methyl­sulfon­yl]ethane 
There are two mol­ecules in the asymmetric unit of the title compound, C17H20O4S2. There are slight differences in the twist of the two rings relative to the S–C–S chain [dihedral angles of 48.41 (18) and 87.58 (16)° in the first mol­ecule and 45.98 (18) and 87.02 (18)° in the second] and the difference in the C—S—C—S torsion angles [176.68 (17) and −77.6 (2)° for the two independent mol­ecules].
PMCID: PMC2971820  PMID: 21578908
24.  Effect of lifestyle intervention on non-alcoholic fatty liver disease in Chinese obese children 
AIM: To investigate the effect of lifestyle intervention on non-alcoholic fatty liver disease (NAFLD) in Chinese obese children.
METHODS: Seventy-six obese children aged from 10 to 17 years with NAFLD were enrolled for a one-month intervention and divided randomly into three groups. Group1, consisting of 38 obese children, was an untreated control group without any intervention. Group 2, consisting of 19 obese children in summer camp, was strictly controlled only by life style intervention. Group 3, consisting of 19 obese children, received oral vitamin E therapy at a dose of 100 mg/d. The height, weight, fasting blood glucose (FBG), fasting serum insulin (FINS), plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TCHO) and homeostasis model assent-insulin resistance (HOMA-IR) were measured at baseline and after one month. All patients were underwent to an ultrasonographic study of the liver performed by one operator who was blinded to the groups.
RESULTS: The monitor indices of BMI, ALT, AST, TG, TCHO and HOMA-IR were successfully improved except in group 1. BMI and ALT in group 2 were reduced more significantly than in group 3 (2.44 ± 0.82 vs 1.45 ± 0.80, P = 0.001; 88.58 ± 39.99 vs 63.69 ± 27.05, P = 0.040, respectively).
CONCLUSION: Both a short-term lifestyle intervention and vitamin E therapy have an effect on NAFLD in obese children. Compared with vitamin E, lifestyle intervention is more effective. Therefore, lifestyle intervention should represent the first step in the management of children with NAFLD.
PMCID: PMC2693759  PMID: 18330955
Non-alcoholic fatty liver disease; Lifestyle intervention; Vitamin E; Obese; Children

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