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1.  High Resolution Molecular Genomic Autopsy Reveals Complex SUDEP Risk Profile 
Epilepsia  2013;55(2):e6-12.
SUMMARY
Advanced variant detection in genes underlying risk of sudden unexpected death in epilepsy (SUDEP) can uncover extensive epistatic complexity and improve diagnostic accuracy of epilepsy related mortality. However, the sensitivity and clinical utility of diagnostic panels based solely on established cardiac arrhythmia genes in the molecular autopsy of SUDEP is unknown.
We applied the established clinical diagnostic panels, followed by sequencing and a high density copy number variant (CNV) detection array of an additional 253 related ion channel subunit genes to analyze the overall genomic variation in a SUDEP of the three year old proband with severe myoclonic epilepsy of infancy (SMEI).
We uncovered complex combinations of single nucleotide polymorphisms and CNVs in genes expressed in both neuro-cardiac and respiratory control pathways, including SCN1A, KCNA1, RYR3, and HTR2C.
Our findings demonstrate the importance of comprehensive high resolution variant analysis in the assessment of personally relevant SUDEP risk. In this case, the combination of de novo SNPs and CNVs in the SCN1A and KCNA1 genes respectively is suspected to be the principal risk factor for both epilepsy and premature death. However, consideration of the overall biologically relevant variant complexity with its extensive functional epistatic interactions reveals potential personal risk more accurately.
doi:10.1111/epi.12489
PMCID: PMC4195652  PMID: 24372310
SUDEP; SMEI; Epileptic Encephalopathy; Dravet Syndrome; Gene; Risk; Molecular Autopsy
2.  Genomic duplication resulting in increased copy number of genes encoding the sister chromatid cohesion complex conveys clinical consequences distinct from Cornelia de Lange 
Journal of medical genetics  2008;46(9):626-634.
Cornelia de Lange Syndrome (CdLS) is a multisystem congenital anomaly disorder. Heterozygous point mutations in three genes (NIPBL, SMC3 and SMC1A), encoding components of the sister chromatid cohesion apparatus, are responsible for ∼ 50-60% of CdLS cases. Recent studies have revealed a high degree of genomic rearrangements (e.g. deletions and duplications) in the human genome, which result in gene copy number variations (CNV). CNVs have been associated with a wide range of both Mendelian and complex traits including disease phenotypes such as Charcot-Marie-Tooth type 1A, Pelizaeus-Merzbacher, Parkinson, Alzheimer, autism and schizophrenia. Increased versus decreased copy number of the same gene can potentially cause either similar or different clinical features. We identified duplications on chromosomes 5 or X using genome wide array Comparative Genomic Hybridization (aCGH). The duplicated regions contain either the NIPBL or the SMC1A genes. Junction sequences analyses revealed the involvement of three genomic rearrangement mechanisms. The patients share some common features including mental retardation, developmental delay, sleep abnormalities, and crainofacial and limb defects. The systems affected are the same as in CdLS, but clinical manifestations are distinct from CdLS; particularly the absence of the CdLS facial gestalt. Our results confirm the notion that duplication CNV of genes can be a common mechanism for human genetic diseases. Defining the clinical consequences for a specific gene dosage alteration represents a new “reverse genomics” trend in medical genetics that is reciprocal to the traditional approach of delineation of the common clinical phenotype preceding the discovery of the genetic etiology.
doi:10.1136/jmg.2008.062471
PMCID: PMC4302738  PMID: 19052029
Copy number changes; CNV; CdLS; NIPBL; cohesion complex
3.  Employment and financial burden of families with preschool children diagnosed with autism spectrum disorders in urban China: results from a descriptive study 
BMC Psychiatry  2015;15:3.
Background
Autism spectrum disorder (ASD) affects many aspects of family life, such as social and economic burden. Little investigation of this phenomenon has been carried out in China. We designed this study to evaluate the employment and financial burdens of families with ASD-diagnosed preschoolers.
Methods
Four hundred and fifty-nine nuclear families of children with ASD, 418 with some other disability (OD) and 424 with typically developing (TD) children were recruited for this study. Employment and financial burdens of families were evaluated using a structured questionnaire; logistic regression was used to examine differences in job change measures by group, and ordinal logistic regression was used to investigate the association between household income and group.
Results
Fifty-eight percent of families with ASD children and 19% of families with OD children reported that childcare problems had greatly affected their employment decisions, compared with 9% of families with TD children (p < 0.001). Age of child, parental education and parental age notwithstanding, having a child with ASD and having a child with OD were both associated with increased odds of reporting that childcare greatly interfered with employment (ASD, OR: 15.936; OD, OR: 2.502; all p < 0.001) and decreased the odds of living in a higher-income household (ASD, estimate = -1.271; OD, estimate = -0.569; all p < 0.001). The average loss of annual income associated with having a child with ASD was Chinese RenMinBi (RMB) 44,077 ($7,226), compared with RMB 20,788 ($3,408) for families of OD children.
Conclusions
ASD is associated with severe employment and financial burdens, much more than for OD, in families with preschool children.
doi:10.1186/s12888-015-0382-4
PMCID: PMC4307683  PMID: 25608486
Employment burden; Financial burden; Autism spectrum disorder; Chinese
4.  Lower irritation microemulsion-based rotigotine gel: formulation optimization and in vitro and in vivo studies 
Background
Rotigotine is a potent and selective D1, D2, and D3 dopaminergic receptor agonist. Due to an extensive first-pass effect, it has a very low oral bioavailability (approximately 0.5% in rats).
Purpose
The present investigation aimed to develop a microemulsion-based hydrogel for transdermal rotigotine delivery with lower application site reactions.
Methods
Pseudoternary phase diagrams were constructed to determine the region of oil in water (o/w)-type microemulsion. Central composite design was used to support the pseudoternary phase diagrams and to select homogeneous and stable microemulsions with an optimal amount of rotigotine permeation within 24 hours. In vitro skin permeation experiments were performed, using Franz diffusion cells, to compare rotigotine-loaded microemulsions with rotigotine solutions in oil. The optimized formulation was used to prepare a microemulsion-based hydrogel, which was subjected to bioavailability and skin irritancy studies.
Results
The selected formulations of rotigotine-loaded microemulsions had enhanced flux and permeation coefficients compared with rotigotine in oil. The optimum microemulsion contained 68% water, 6.8% Labrafil®, 13.44% Cremophor® RH40, 6.72% Labrasol®, and 5.04% Transcutol® HP; the drug-loading rate was 2%. To form a microemulsion gel, 1% Carbomer 1342 was added to the microemulsion. The bioavailability of the rotigotine-loaded microemulsion gel was 105.76%±20.52% with respect to the marketed rotigotine patch (Neupro®). The microemulsion gel irritated the skin less than Neupro.
Conclusion
A rotigotine microemulsion-based hydrogel was successfully developed, and an optimal formulation for drug delivery was identified. This product could improve patient compliance and have broad marketability.
doi:10.2147/IJN.S74079
PMCID: PMC4298334  PMID: 25609965
pseudoternary phase diagrams; central composite design; transdermal
5.  Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5’ sites 
Cell reports  2014;8(1):284-296.
N6-methyladenosine (m6A) is a common modification of mRNA, with potential roles in fine-tuning the RNA life-cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them, WTAP, METTL14 and KIAA1429, are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps, and their classification into WTAP-dependent and independent sites. WTAP-dependent sites are located at internal positions in transcripts, are topologically static across a variety of systems we surveyed, and are inversely correlated with mRNA stability, consistent with a role in establishing ‘basal’ degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure, and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data sheds new light on proteomic and transcriptional underpinnings of this epitranscriptomic modification.
doi:10.1016/j.celrep.2014.05.048
PMCID: PMC4142486  PMID: 24981863
6.  Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells 
Nature biotechnology  2014;32(7):670-676.
Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA- guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs tested targets dCas9 to tens to thousands of genomic sites, characterized by a 5-nucleotide seed region in the sgRNA, in addition to an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility prevents dCas9 binding to other sites with matching seed sequences, and consequently 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.
doi:10.1038/nbt.2889
PMCID: PMC4145672  PMID: 24752079
7.  The clinical significance of CDK1 expression in oral squamous cell carcinoma 
Objectives: To evaluate the clinical significance of cyclin-dependent kinase 1 (CDK1) in 77 oral squamous cell carcinomas (OSCC) using immunohistochemical methods. Study Design: Immunohistochemical expression of CDK1 was compared with various clinicopathological features in 77 OSCC and 60 controlled epithelia adjacent to the tumours. In addition, correlation of CDK1 expression and prognostic and the 5-year accumulative survival rate of OSCC were investigated. Results: The CDK1 protein was expressed in 52 cases of 77 tumor tissues (67.5%), compared with 21 cases of 60 controlled (35.0%). The expression of CDK1 was significantly correlated with the histological grade of OSCC (P<0.05). The CDK1 protein was over-expressed in recurrent tumors or in those with lymph node metastasis. Statistical analysis showed a significant reduction in the 5-year accumulative survival rate in CDK1 positive cases compared with CDK1 negative cases (P<0.05). Namely, the CDK1 positive patients had poor prognosis. Conclusions: The expression of CDK1 might serve as malignant degree and prognostic markers for the survival of OSCC.
Key words:Cyclin-dependent kinase 1 (CDK1), oral squamous cell carcinoma (OSCC), immunohistochemistry, cell proliferation.
doi:10.4317/medoral.19841
PMCID: PMC4320424  PMID: 25129248
8.  An HIV-1 Envelope Immunogen with W427S Mutation in CD4 Binding Site Induced More T Follicular Helper Memory Cells and Reduced Non-Specific Antibody Responses 
PLoS ONE  2014;9(12):e115047.
The CD4 binding site (CD4BS) of the HIV-1 envelope glycoprotein (Env) contains epitopes for broadly neutralizing antibody (nAb) and is the target for the vaccine development. However, the CD4BS core including residues 425-430 overlaps the B cell superantigen site and may be related to B cell exhaustion in HIV-1 infection. Furthermore, production of nAb and high-affinity plasma cells needs germinal center reaction and the help of T follicular helper (Tfh) cells. We believe that strengthening the ability of Env CD4BS in inducing Tfh response and decreasing the effects of the superantigen are the strategies for eliciting nAb and development of HIV-1 vaccine. We constructed a gp120 mutant W427S of an HIV-1 primary R5 strain and examined its ability in the elicitation of Ab and the production of Tfh by immunization of BALB/c mice. We found that the trimeric wild-type gp120 can induce more non-specific antibody-secreting plasma cells, higher serum IgG secretion, and more Tfh cells by splenocyte. The modified W427S gp120 elicits higher levels of specific binding antibodies as well as nAbs though it produces less Tfh cells. Furthermore, higher Tfh cell frequency does not correlate to the specific binding Abs or nAbs indicating that the wild-type gp120 induced some non-specific Tfh that did not contribute to the production of specific Abs. This gp120 mutant led to more memory Tfh production, especially, the effector memory Tfh cells. Taken together, W427S gp120 could induce higher level of specific binding and neutralizing Ab production that may be associated with the reduction of non-specific Tfh but strengthening of the memory Tfh.
doi:10.1371/journal.pone.0115047
PMCID: PMC4278894  PMID: 25546013
9.  Diffusion-Weighted MRI Monitoring of Pancreatic Cancer Response to Radiofrequency Heat-Enhanced Intratumor Chemotherapy 
NMR in biomedicine  2013;26(12):10.1002/nbm.3014.
PURPOSE
To evaluate the feasibility of using diffusion-weighted MRI to monitor the early response of pancreatic cancers to radiofrequency heat (RFH)-enhanced chemotherapy.
MATERIALS AND METHODS
Human pancreatic carcinoma cells (PANC-1) in different groups and twenty four mice with pancreatic cancer xenografts in four groups were treated by phosphate buffered saline (PBS) as a control, RFH at 42 °C, gemcitabine and gemcitabine plus RFH at 42°C. One day before and 1, 7, and 14 days after the treatment, diffusion-weighted MR imaging and T2 weighted imaging were applied to monitor the apparent diffusion coefficients (ADCs) of tumors and tumors growth. MRI findings were correlated with results of tumors apoptosis analysis.
RESULTS
Of the in vitro experiments, quantitative viability assay showed lower relative cell viabilities treated by gemcitabine plus RFH at 42°C, compared to those by RFH only and gemcitabine only (37% ± 5% vs 65% ± 4% and 58% ± 8%, p < 0.05). Of the in vivo experiments, the combination therapy resulted in smaller relative tumor volume than RFH-only and chemotherapy-only (0.82 ± 0.17 vs 2.23 ± 0.90 and 1.64 ± 0.44, p = 0.003). In vivo 14T MRI demonstrated a remarkable decrease of ADCs at day 1 and increased ADCs at days 7 and 14 in the combination therapy group. The apoptosis index in the combination therapy group was significantly higher than those in the groups of chemotherapy-only, RFH-only and PBS treatments (37% ± 6% vs 20% ± 5%, 8% ± 2%, and 3% ± 1%, p < 0.05).
CONCLUSION
This study confirms that it is feasible to use MRI to monitor RFH-enhanced chemotherapy on pancreatic cancers, which may present new options for efficient treatment of pancreatic malignancies using MR/RF-integrated local chemotherapy.
doi:10.1002/nbm.3014
PMCID: PMC3838434  PMID: 24038282
10.  Genome Architecture and Its Roles in Human Copy Number Variation 
Genomics & Informatics  2014;12(4):136-144.
Besides single-nucleotide variants in the human genome, large-scale genomic variants, such as copy number variations (CNVs), are being increasingly discovered as a genetic source of human diversity and the pathogenic factors of diseases. Recent experimental findings have shed light on the links between different genome architectures and CNV mutagenesis. In this review, we summarize various genomic features and discuss their contributions to CNV formation. Genomic repeats, including both low-copy and high-copy repeats, play important roles in CNV instability, which was initially known as DNA recombination events. Furthermore, it has been found that human genomic repeats can also induce DNA replication errors and consequently result in CNV mutations. Some recent studies showed that DNA replication timing, which reflects the high-order information of genomic organization, is involved in human CNV mutations. Our review highlights that genome architecture, from DNA sequence to high-order genomic organization, is an important molecular factor in CNV mutagenesis and human genomic instability.
doi:10.5808/GI.2014.12.4.136
PMCID: PMC4330246
DNA copy number variations; DNA replication; genetic recombination; genomic instability
11.  Toxic Effects of Levofloxacin on Rat Annulus Fibrosus Cells: An In-vitro Study 
Background
Fluoroquinolones are in wide clinical use as safe and effective antibiotics. Articular cartilage, tendons, and epiphyseal growth plates have been recognized as targets of fluoroquinolone-induced connective tissue toxicity. However, the effects of fluoroquinolones on annulus fibrosus (AF) cells are still unknown.
Material/Methods
The main objective of this study was to investigate the effects of levofloxacin, a typical fluoroquinolone antibiotic drug, on rat AF cells in vitro. Rat annulus fibrosus (RAF) cells were treated with levofloxacin at different concentrations (0, 10, 20, 30, 40, 60, 80, and 90 μg/ml) and were assessed to determine the possible cytotoxic effects of levofloxacin. Inverted phase-contrast microscopy was used to accomplish the morphological observation of apoptosis of treated cells. Western blot and real-time quantitative RT-PCR (qPCR) was used to explore the expression of active caspase-3 and MMP-3. Flow cytometry was used to measure the apoptotic incidences.
Results
Our study showed that levofloxacin, with concentrations at 30, 60, and 90 μg/ml, induced dose-dependent RAF cell apoptosis and higher expression of caspase-3 and MMP-3. More apoptotic cells were observed by inverted phase-contrast microscopy. Moreover, levofloxacin increased the activity of caspase-3, and it also reduced cell viability with different concentrations ranging from 10 to 80 μg/ml.
Conclusions
Our study results suggest that levofloxacin has cytotoxic effects on RAF cells, characterized by enhancing apoptosis and reducing cell viability, and indicate a potential toxic effect of fluoroquinolones on RAF cells.
doi:10.12659/MSM.892610
PMCID: PMC4237079  PMID: 25380657
Apoptosis; Caspase 3; Cell Survival; Levofloxacin
12.  N-acetylcysteine attenuates reactive-oxygen-species-mediated endoplasmic reticulum stress during liver ischemia-reperfusion injury 
World Journal of Gastroenterology : WJG  2014;20(41):15289-15298.
AIM: To investigate the effects of N-acetylcysteine (NAC) on endoplasmic reticulum (ER) stress and tissue injury during liver ischemia reperfusion injury (IRI).
METHODS: Mice were injected with NAC (300 mg/kg) intraperitoneally 2 h before ischemia. Real-time polymerase chain reaction and western blotting determined ER stress molecules (GRP78, ATF4 and CHOP). To analyze the role of NAC in reactive oxygen species (ROS)-mediated ER stress and apoptosis, lactate dehydrogenase (LDH) was examined in cultured hepatocytes treated by H2O2 or thapsigargin (TG).
RESULTS: NAC treatment significantly reduced the level of ROS and attenuated ROS-induced liver injury after IRI, based on glutathione, malondialdehyde, serum alanine aminotransferase levels, and histopathology. ROS-mediated ER stress was significantly inhibited in NAC-treated mice. In addition, NAC treatment significantly reduced caspase-3 activity and apoptosis after reperfusion, which correlated with the protein expression of Bcl-2 and Bcl-xl. Similarly, NAC treatment significantly inhibited LDH release from hepatocytes treated by H2O2 or TG.
CONCLUSION: This study provides new evidence for the protective effects of NAC treatment on hepatocytes during IRI. Through inhibition of ROS-mediated ER stress, NAC may be critical to inhibit the ER-stress-related apoptosis pathway.
doi:10.3748/wjg.v20.i41.15289
PMCID: PMC4223262  PMID: 25386077
N-acetylcysteine; Reactive oxygen species; Endoplasmic reticulum stress; Apoptosis; Liver ischemia-reperfusion
13.  GJB1/Connexin 32 whole gene deletions in patients with X-linked Charcot–Marie–Tooth disease 
Neurogenetics  2010;11(4):465-470.
The X-linked form of Charcot–Marie–Tooth disease (CMTX) is the second most common form of this genetically heterogeneous inherited peripheral neuropathy. CMT1X is caused by mutations in the GJB1 gene. Most of the mutations causative for CMT1X are missense mutations. In addition, a few disease causative nonsense mutations and frameshift deletions that lead to truncated forms of the protein have also been reported to be associated with CMT1X. Previously, there have been reports of patients with deletions of the coding sequence of GJB1; however, the size and breakpoints of these deletions were not assessed. Here, we report five patients with deletions that range in size from 12.2 to 48.3 kb and that completely eliminate the entire coding sequence of the GJB1 gene, resulting in a null allele for this locus. Analyses of the breakpoints of these deletions showed that they are nonrecurrent and that they can be generated by different mechanisms. In addition to PMP22, GJB1 is the second CMT gene for which both point mutations and genomic rearrangements can cause a neuropathy phenotype, stressing the importance of CMT as a genomic disorder.
doi:10.1007/s10048-010-0247-4
PMCID: PMC4222676  PMID: 20532933
Charcot–Marie–Tooth disease; GJB1; Connexin32; Gene deletion; Genomic rearrangement
14.  Transgenic Overproduction of Omega-3 Polyunsaturated Fatty Acids Provides Neuroprotection and Enhances Endogenous Neurogenesis after Stroke 
Current molecular medicine  2013;13(9):1465-1473.
Strokes are devastating as there are no current therapies to prevent the long term neurological deficits that they cause. Soon after ischemic stroke, there is proliferation and differentiation of neural stem/progenitor cells as an important mechanism for neuronal restoration. However, endogenous neurogenesis by itself is insufficient for effective brain repair after stroke as most newborn neurons do not survive. One fascinating strategy for stroke treatment would thus be maintaining the survival and/or promoting the differentiation of endogenous neural stem/progenitor cells. Using transgenic (Tg) mice over-expressing the C. elegans fat-1 gene encoding an enzyme that converts endogenous omega-6 to omega-3 polyunsaturated fatty acids (n-3 PUFAs), we showed that fat-1 Tg mice with chronically elevated brain levels of n-3 PUFAs exhibited less brain damage and significantly improved long-term neurological performance compared to wild type littermates. Importantly, post-stroke neurogenesis occurred more robustly in fat-1 Tg mice after focal ischemia. This was manifested by enhanced neural stem cell proliferation/differentiation and increased migration of neuroblasts to the ischemic sites where neuroblasts matured into resident neurons. Moreover, these neurogenic effects were accompanied by significantly increased oligodendrogenesis. Our results suggest that n-3 PUFA supplementation is a potential neurogenic and oligodendrogenic treatment to naturally improve post-stroke brain repair and long-term functional recovery.
PMCID: PMC4083810  PMID: 23971733
omega-3 polyunsaturated fatty acids; stroke; neuroprotection; neurogenesis; oligodendrogenesis
15.  RNA-Seq-Based Transcriptome Analysis of Aflatoxigenic Aspergillus flavus in Response to Water Activity 
Toxins  2014;6(11):3187-3207.
Aspergillus flavus is one of the most important producers of carcinogenic aflatoxins in crops, and the effect of water activity (aw) on growth and aflatoxin production of A. flavus has been previously studied. Here we found the strains under 0.93 aw exhibited decreased conidiation and aflatoxin biosynthesis compared to that under 0.99 aw. When RNA-Seq was used to delineate gene expression profile under different water activities, 23,320 non-redundant unigenes, with an average length of 1297 bp, were yielded. By database comparisons, 19,838 unigenes were matched well (e-value < 10−5) with known gene sequences, and another 6767 novel unigenes were obtained by comparison to the current genome annotation of A. flavus. Based on the RPKM equation, 5362 differentially expressed unigenes (with |log2Ratio| ≥ 1) were identified between 0.99 aw and 0.93 aw treatments, including 3156 up-regulated and 2206 down-regulated unigenes, suggesting that A. flavus underwent an extensive transcriptome response during water activity variation. Furthermore, we found that the expression of 16 aflatoxin producing-related genes decreased obviously when water activity decreased, and the expression of 11 development-related genes increased after 0.99 aw treatment. Our data corroborate a model where water activity affects aflatoxin biosynthesis through increasing the expression of aflatoxin producing-related genes and regulating development-related genes.
doi:10.3390/toxins6113187
PMCID: PMC4247253  PMID: 25421810
RNA-Seq; transcriptome; Aspergillus flavus; water activity; aflatoxin
16.  Transurethral holmium laser vaporization to the urethral tumour through a ureteroscope 
Canadian Urological Association Journal  2014;8(11-12):E913-E915.
We present 2 cases of urethral cancers: one is recurrent bladder transitional cell carcinoma accompanied by urethral metastatic carcinoma located on the right side of verumontanum, and the other is primary bladder and metastatic urethral adenocarcinoma. The urethral tumour was treated by transurethral holmium laser vaporization to the urethral tumour through a ureteroscope and the bladder tumour was treated with transurethral resection and degeneration of the bladder tumour (TURD-Bt). After the second or third therapy, patients were free of urethral or bladder tumour recurrence; they also did not experience urethral stricture or urinary incontinence during the 24- to 36-month follow-up. Transurethral holmium laser vaporization and TURD-Bt could be performed to treat non-invasive urethral cancer accompanied with bladder cancer and preserve the urethra and bladder.
doi:10.5489/cuaj.2026
PMCID: PMC4277533  PMID: 25553166
17.  Hepatocyte Growth Factor Modification Enhances the Anti-Arrhythmic Properties of Human Bone Marrow-Derived Mesenchymal Stem Cells 
PLoS ONE  2014;9(10):e111246.
Background/Aims
Chronic myocardial infarction (MI) results in the formation of arrhythmogenic substrates, causing lethal ventricular arrhythmia (VA). We aimed to determine whether mesenchymal stem cells (MSCs) carrying a hepatocyte growth factor (HGF) gene modification (HGF-MSCs) decrease the levels of arrhythmogenic substrates and reduce the susceptibility to developing VA compared with unmodified MSCs and PBS in a swine infarction model.
Methods
The left descending anterior artery was balloon-occluded to establish an MI model. Four weeks later, the randomly grouped pigs were administered MSCs, PBS or HGF-MSCs via thoracotomy. After an additional four weeks, dynamic electrocardiography was performed to assess heart rate variability, and programmed electrical stimulation was conducted to evaluate the risk for VA. Then, the pigs were euthanized for morphometric, immunofluorescence and western blot analyses. Results: The HGF-MSC group displayed the highest vessel density and Cx43 expression levels, and the lowest levels of apoptosis, and tyrosine hydroxylase (TH) and growth associated protein 43 (GAP43) expression. Moreover, the HGF-MSC group exhibited a decrease in the number of sympathetic nerve fibers, substantial decreases in the low frequency and the low-/high- frequency ratio and increases in the root mean square of successive differences (rMSSD) and the percentage of successive normal sinus R-R intervals longer than 50 ms (pNN50), compared with the other two groups. Finally, the HGF-MSC group displayed the lowest susceptibility to developing VA.
Conclusion
HGF-MSCs displayed potent antiarrhythmic effects, reducing the risk for VA.
doi:10.1371/journal.pone.0111246
PMCID: PMC4216066  PMID: 25360679
18.  Differentiation of peripheral nerve functions and properties with spectral analysis and Karnovsky-Roots staining: a preliminary study 
Objective: The purpose of this study was to explore the possibility for analyzing and differentiating between motor and sensory functions of peripheral nerve axons using spectral technology. Methods: 10 μm slide section of S1 anterior and posterior rabbit spinal nerve roots were made and then stained with Karnovsky-Roots method for molecular hyperspectral imaging microscopy analysis. In addition, Raman spectra data of nerve axons on each slide was collected after Karnovsky-Roots staining for 30 minutes. Results: Motor axons were differentiated from sensory axons in a nerve axon section hyperspectral image via Spectral angle mapper algorithm. Raman scatterings could be detected near 2110 cm-1, and 2155 cm-1 in motor axons after Karnvosky-Roots staining. The value of I2100/I1440 in motor axons are significantly different (P0.001) than in sensory axons after staining for 30 minutes. Conclusions: Motor and sensory nerve axons can be differentiated from their counterparts in 30 minutes by using Raman micro-spectroscopy analysis assisted with Karnovsky-Roots staining.
PMCID: PMC4238542  PMID: 25419356
Peripheral nerve; molecular hyperspectral imaging; Raman spectroscopy
19.  East China Sea Storm Surge Modeling and Visualization System: The Typhoon Soulik Case 
The Scientific World Journal  2014;2014:626421.
East China Sea (ECS) Storm Surge Modeling System (ESSMS) is developed based on Regional Ocean Modeling System (ROMS). Case simulation is performed on the Typhoon Soulik, which landed on the coastal region of Fujian Province, China, at 6 pm of July 13, 2013. Modeling results show that the maximum tide level happened at 6 pm, which was also the landing time of Soulik. This accordance may lead to significant storm surge and water level rise in the coastal region. The water level variation induced by high winds of Soulik ranges from −0.1 to 0.15 m. Water level generally increases near the landing place, in particular on the left hand side of the typhoon track. It is calculated that 0.15 m water level rise in this region can cause a submerge increase of ~0.2 km2, which could be catastrophic to the coastal environment and the living. Additionally, a Globe Visualization System (GVS) is realized on the basis of World Wind to better provide users with the typhoon/storm surge information. The main functions of GVS include data indexing, browsing, analyzing, and visualization. GVS is capable of facilitating the precaution and mitigation of typhoon/storm surge in ESC in combination with ESSMS.
doi:10.1155/2014/626421
PMCID: PMC4216692  PMID: 25386608
20.  Tracking stem cell differentiation in the setting of automated optogenetic stimulation 
Stem cells (Dayton, Ohio)  2011;29(1):78-88.
Membrane depolarization has been shown to play an important role in the neural differentiation of stem cells as well as in the survival and function of mature neurons. Here we introduce a microbial opsin into embryonic stem cells and develop optogenetic technology for stem cell engineering applications, with an automated system for noninvasive modulation of embryonic stem cell differentiation employing fast optogenetic control of ion flux. Mouse embryonic stem cells (ESCs) were stably transduced with ChR2-YFP and purified by FACS. Illumination of resulting ChR2-ESCs with pulses of blue light triggered inward currents. These labeled ESCs retained the capability to differentiate into functional mature neurons, assessed by the presence of voltage-gated sodium currents, action potentials, fast excitatory synaptic transmission, and expression of mature neuronal proteins and neuronal morphology. We designed and tested an apparatus for optically stimulating ChR2-ESCs during chronic neuronal differentiation, with high-speed optical switching on a custom robotic stage with environmental chamber for automated stimulation and imaging over days, with tracking for increased expression of neural and neuronal markers. These data point to potential uses of ChR2 technology for chronic and temporally precise noninvasive optical control of embryonic stem cells both in vitro and in vivo, ranging from noninvasive control of stem cell differentiation to causal assessment of the specific contribution of transplanted cells to tissue and network function.
doi:10.1002/stem.558
PMCID: PMC4182948  PMID: 21280159
embryonic stem cells; optogenetics; Channelrhodopsin-2; neuronal differentiation
21.  Perceived Value of a Urinary Catheter Self-Management Program in the Home 
Home healthcare nurse  2013;31(9):465-473.
A long-term indwelling urinary catheter intervention was tested in a randomized trial that is described in this article. The perceived value of the intervention to the catheter users, one of the study’s specific aims, was assessed at the end of their 12-month participation and is reported here. Study participants’ responses, our findings, and implications for home healthcare are discussed.
doi:10.1097/NHH.0b013e3182a89791
PMCID: PMC3915410  PMID: 24081128
22.  Randomized Controlled Trial to Compare Growth Parameters and Nutrient Adequacy in Children with Picky Eating Behaviors Who Received Nutritional Counseling With or Without an Oral Nutritional Supplement 
In this study, changes in growth parameters and nutrient intake were compared in Chinese children (ages 30–60 months) with picky eating (PE) behaviors and weight-for-height ≤25th percentile, who were randomized to receive nutrition counseling alone (NC; n = 76) or with a nutritional milk supplement (NC + NS; n = 77) for 120 days. Increases in weight-for-height z-scores were significantly greater in the NC + NS group at days 30 and 90 and over the entire study period (all P < 0.05), but not at day 120. Increases in weight-for-age z-scores were significantly greater in the NC + NS group at day 90 (P = 0.025) and over the entire study period (P = 0.046). Mean intakes of energy, protein, carbohydrate, docosahexaenoic acid, arachidonic acid, calcium, phosphorous, iron, zinc, and vitamins A, C, D, E, and B6 were significantly higher in the NC + NS group at days 60 and 120 (all P < 0.01). Thus, in young children with PE behaviors, nutritional supplementation given as an adjunct to NC resulted in greater improvements in nutrient intake compared with NC alone. Growth parameters differed between groups at several timepoints during the study, but not at day 120.
doi:10.4137/NMI.S15097
PMCID: PMC4196879  PMID: 25342910
picky eating; eating behavior; growth; nutrient intake; preschool children
23.  Comparison of the Clinical Effects of Open and Closed Chain Exercises after Medial Patellofemoral Ligament Reconstruction 
Journal of Physical Therapy Science  2014;26(10):1557-1560.
[Purpose] To compare the effects of open-chain exercise (OCE) and closed-chain exercise (CCE) for patients after medial patellofemoral ligament (MPFL) reconstruction. [Subjects and Methods] Forty patients after MPFL reconstruction were randomly divided into an OCE group and a CCE group. All the patients were evaluated at four different time points. [Results] The mean change of thigh circumference decrease in the CCE group was lower than that in the OCE group at both the 3rd and 6th month after surgery. The Lysholm score of the CCE group was higher than that of the OCE group at both the 3rd and 6th month. At the 3rd month after surgery, the visual analog scale score of the CCE group was lower than that of the OCE group. [Conclusion] CCE is better than OCE for both short and long term outcomes of patients after MPFL reconstruction.
doi:10.1589/jpts.26.1557
PMCID: PMC4210397  PMID: 25364112
Medial patellofemoral ligament reconstruction; Open-chain exercise; Closed-chain exercise
24.  Sequencing of SCN5A identifies rare and common variants associated with cardiac conduction 
Background
The cardiac sodium channel SCN5A regulates atrioventricular and ventricular conduction. Genetic variants in this gene are associated with PR and QRS intervals. We sought to further characterize the contribution of rare and common coding variation in SCN5A to cardiac conduction.
Methods and Results
In the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study (CHARGE), we performed targeted exonic sequencing of SCN5A (n=3699, European-ancestry individuals) and identified 4 common (minor allele frequency >1%) and 157 rare variants. Common and rare SCN5A coding variants were examined for association with PR and QRS intervals through meta-analysis of European ancestry participants from CHARGE, NHLBI’s Exome Sequencing Project (ESP, n=607) and the UK10K (n=1275) and by examining ESP African-ancestry participants (N=972). Rare coding SCN5A variants in aggregate were associated with PR interval in European and African-ancestry participants (P=1.3×10−3). Three common variants were associated with PR and/or QRS interval duration among European-ancestry participants and one among African-ancestry participants. These included two well-known missense variants; rs1805124 (H558R) was associated with PR and QRS shortening in European-ancestry participants (P=6.25×10−4 and P=5.2×10−3 respectively) and rs7626962 (S1102Y) was associated with PR shortening in those of African ancestry (P=2.82×10−3). Among European-ancestry participants, two novel synonymous variants, rs1805126 and rs6599230, were associated with cardiac conduction. Our top signal, rs1805126 was associated with PR and QRS lengthening (P=3.35×10−7 and P=2.69×10−4 respectively), and rs6599230 was associated with PR shortening (P=2.67×10−5).
Conclusions
By sequencing SCN5A, we identified novel common and rare coding variants associated with cardiac conduction.
doi:10.1161/CIRCGENETICS.113.000098
PMCID: PMC4177904  PMID: 24951663
PR interval; QRS interval; genetics; sequencing; cohort
25.  Screening of Duchenne Muscular Dystrophy (DMD) Mutations and Investigating Its Mutational Mechanism in Chinese Patients 
PLoS ONE  2014;9(9):e108038.
Duchenne muscular dystrophy (DMD) is a common X-linked recessive disease of muscle degeneration and death. In order to provide accurate and reliable genetic counseling and prenatal diagnosis, we screened DMD mutations in a cohort of 119 Chinese patients using multiplex ligation-dependent probe amplification (MLPA) and denaturing high performance liquid chromatography (DHPLC) followed by Sanger sequencing. In these unrelated DMD patients, we identified 11 patients with DMD small mutations (9.2%) and 81 patients with DMD deletions/duplications (del/dup) (68.1%), of which 64 (79.0%) were deletions, 16 (19.8%) were duplications, and one (1.2%) was both deletion and duplication. Furthermore, we analyzed the frequency of DMD breakpoint in the 64 deletion cases by calculating exon-deletion events of certain exon interval that revealed a novel mutation hotspot boundary. To explore why DMD rearrangement breakpoints were predisposed to specific regions (hotspot), we precisely characterized junction sequences of breakpoints at the nucleotide level in 21 patients with exon deleted/duplicated in DMD with a high-resolution SNP microarray assay. There were no exactly recurrent breakpoints and there was also no significant difference between single-exon del/dup and multiple-exon del/dup cases. The data from the current study provided a comprehensive strategy to detect DMD mutations for clinical practice, and identified two deletion hotspots at exon 43–55 and exon 10–23 by calculating exon-deletion events of certain exon interval. Furthermore, this is the first study to characterize DMD breakpoint at the nucleotide level in a Chinese population. Our observations provide better understanding of the mechanism for DMD gene rearrangements.
doi:10.1371/journal.pone.0108038
PMCID: PMC4171529  PMID: 25244321

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