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1.  High-resolution Global Genomic Survey of 178 Gliomas Reveals Novel Regions of Copy Number Alteration and Allelic Imbalances 
Cancer research  2006;66(19):9428-9436.
Primary brain tumors are the fourth leading cause of cancer mortality in adults under the age of 54 years and the leading cause of cancer mortality in children in the United States. Therapy for the most common type of primary brain tumors, gliomas, remains suboptimal. The development of new and more effective treatments will likely require a better understanding of the biology of these tumors. Here, we show that use of the high-density 100K single-nucleotide polymorphism arrays in a large number of primary tumor samples allows for a much higher resolution survey of the glioma genome than has been previously reported in any tumor type. We not only confirmed alterations in genomic areas previously reported to be affected in gliomas, but we also refined the location of those sites and uncovered multiple, previously unknown regions that are affected by copy number alterations (amplifications, homozygous and heterozygous deletions) as well as allelic imbalances (loss of heterozygosity/gene conversions). The wealth of genomic data produced may allow for the development of a more rational molecular classification of gliomas and serve as an important starting point in the search for new molecular therapeutic targets.
doi:10.1158/0008-5472.CAN-06-1691
PMCID: PMC4316202  PMID: 17018597
2.  Empty Virions In AAV8 Vector Preparations Reduce Transduction Efficiency And May Cause Total Viral Particle Dose-Limiting Side-Effects 
Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs). The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin, hA1AT) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ATL levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side-effects of rAAV8EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.
doi:10.1038/mtm.2013.9
PMCID: PMC4255953  PMID: 25485285
rAAV; empty virions; liver gene transfer; side-effect
3.  Large-Scale Functional Organization of Long-Range Chromatin Interaction Networks 
Cell reports  2012;2(5):1207-1219.
SUMMARY
Chromatin interactions play important roles in transcription regulation. To better understand the underlying evolutionary and functional constraints of these interactions, we implemented a systems approach to examine RNA polymerase-II-associated chromatin interactions in human cells. We found that 40% of the total genomic elements involved in chromatin interactions converged to a giant, scale-free-like, hierarchical network organized into chromatin communities. The communities were enriched in specific functions and were syntenic through evolution. Disease-associated SNPs from genome-wide association studies were enriched among the nodes with fewer interactions, implying their selection against deleterious interactions by limiting the total number of interactions, a model that we further reconciled using somatic and germline cancer mutation data. The hubs lacked disease-associated SNPs, constituted a nonrandomly interconnected core of key cellular functions, and exhibited lethality in mouse mutants, supporting an evolutionary selection that favored the nonrandom spatial clustering of the least-evolving key genomic domains against random genetic or transcriptional errors in the genome. Altogether, our analyses reveal a systems-level evolutionary framework that shapes functionally compartmentalized and error-tolerant transcriptional regulation of human genome in three dimensions.
doi:10.1016/j.celrep.2012.09.022
PMCID: PMC4181841  PMID: 23103170
4.  Macular hole secondary to Valsalva retinopathy after doing push-up exercise 
BMC Ophthalmology  2014;14:98.
Background
Valsalva retinopathy and traumatic macular hole are common conditions, but macular hole secondary to Valsalva retinopathy is rarely reported.
Case presentation
A 34-year-old healthy man suffered Valsalva retinopathy after doing push-up exercise. During his follow-up visits, the best-corrected visual acuity (BCVA) measurements, fundus examinations and spectral-domain optical coherence tomography (SD-OCT) tests were performed. Three months later, the premacular hemorrhage was noticeably absorbed with an improvement of visual acuity. SD-OCT showed a lamellar macular hole with intact but thickened internal limiting membrane (ILM) with vitreal tractions on surface of the macular. Nine months after the first visit, his vision acuity was 20/25. The fundus examination showed a complete absorption of the macular hemorrhage. SD-OCT showed that the lamellar macular hole has enlarged, with thickened ILM on the surface. Seventeen months after the onset, the BCVA, fundus examination results and OCT findings were stable.
Conclusions
Macular hole secondary to Valsalva retinopathy had been rarely reported and its mechanism needs further understanding. SD-OCT can be used to observe the evolvement of Valsalva retinopathy.
doi:10.1186/1471-2415-14-98
PMCID: PMC4136644  PMID: 25117955
Optical coherence tomography; Valsalva retinopathy; Macular hole
5.  Hydrolysis of green tea residue protein using proteolytic enzyme derived from Aspergillus oryzae 
Free amino acids are important chemical components which impact the taste of green tea infusion. The hydrolysis of water-insoluble protein in the green tea residue helps to increase the contents of free amino acids components except theanine. Studies indicate that the hydrolysis of the tea protein could be restricted due to interaction of polyphenols with protein. The experiment indicates that the hydrolysis of tea protein by protease is the main trend when the polyphenols concentration is lower than 5 mg ml−1, however, the proteins (including tea protein and protease) would interact with polyphenoles instead of hydrolysis when the concentration of polyphenols is higher than 5 mg ml−1. The hydrolysis of tea protein is absolutely restrained when concentration comes to 10 mg ml−1.
doi:10.1007/s13197-011-0239-x
PMCID: PMC3550956  PMID: 24425904
Free amino acids; Protease; Tea protein; Green tea residue; Hydrolysis
6.  A novel Norrie disease pseudoglioma gene mutation, c.-1_2delAAT, responsible for Norrie disease in a Chinese family 
AIM
To investigate the genetic findings and phenotypic characteristics of a Chinese family with Norrie disease (ND).
METHODS
Molecular genetic analysis and clinical examinations were performed on a Chinese family with ND. Mutations in the Norrie disease pseudoglioma (NDP) gene were detected by direct sequencing. Haplotypes were constructed and compared with the phenotypes in the family. Evolutionary comparisons and mutant open reading frame (ORF) prediction were also undertaken.
RESULTS
Two family members with ocular manifestations were diagnosed with ND. No signs of sensorineural hearing loss were observed in either patient, while one of them showed signs of mild mental retardation. A novel heterozygous mutation in the NDP gene, c.-1_2delAAT, was detected in both patients. The mutation and the mutation bearing haplotype co-segregated with the ND phenotype in males and was transmitted from their mothers and/or grandmothers (II:2). The male without ND did not harbor the mutation. The mutation occurred at the highly conserved nucleotides. ORF finder predicted that the mutation would lead to the production of a truncated protein that lacks the first 11 N-terminal amino acids.
CONCLUSION
A novel mutation, c.-1_2delAAT in the NDP gene, was identified in a Chinese family with ND. This mutation caused ND without obvious sensorineural hearing loss. Mental disorder was found in one but not the other patients. The clinical heterogeneity in the family indicated that other genetic variants and epigenetic factors may also play a role in the disease presentation.
doi:10.3980/j.issn.2222-3959.2013.06.01
PMCID: PMC3874509  PMID: 24392318
Norrie disease; pseudoglioma; mutation; Chinese
7.  Targeted Delivery of an Antigenic Peptide to the Endoplasmic Reticulum: Application for Development of a Peptide Therapy for Ankylosing Spondylitis 
PLoS ONE  2013;8(10):e77451.
The development of suitable methods to deliver peptides specifically to the endoplasmic reticulum (ER) can provide some potential therapeutic applications of such peptides. Ankylosing spondylitis (AS) is strongly associated with the expression of human leukocytic antigen-B27 (HLA-B27). HLA-B27 heavy chain (HC) has a propensity to fold slowly resulting in the accumulation of misfolded HLA-B27 HC in the ER, triggering the unfolded protein response, and forming a homodimer, (B27-HC)2. Natural killer cells and T-helper 17 cells are then activated, contributing to the major pathogenic potentials of AS. The HLA-B27 HC is thus an important target, and delivery of an HLA-B27-binding peptide to the ER capable of promoting HLA-B27 HC folding is a potential mechanism for AS therapy. Here, we demonstrate that a His6-ubiquitin-tagged Tat-derived peptide (THU) can deliver an HLA-B27-binding peptide to the ER promoting HLA-B27 HC folding. The THU-HLA-B27-binding peptide fusion protein crossed the cell membrane to the cytosol through the Tat-derived peptide. The HLA-B27-binding peptide was specifically cleaved from THU by cytosolic ubiquitin C-terminal hydrolases and subsequently transported into the ER by the transporter associated with antigen processing. This approach has potential application in the development of peptide therapy for AS.
doi:10.1371/journal.pone.0077451
PMCID: PMC3796468  PMID: 24155957
8.  STB5 Is a Negative Regulator of Azole Resistance in Candida glabrata 
The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals (J. E. Bennett, K. Izumikawa, and K. A. Marr. Antimicrob. Agents Chemother. 48:1773–1777, 2004). Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown that Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata, the open reading frame (ORF) designated CAGL0I02552g has 38.8% amino acid identity with STB5 (YHR178w) and shares an N-terminal Zn2Cys6 binuclear cluster domain and a fungus-specific transcriptional factor domain, prompting us to test for homologous function and a possible role in azole resistance. Complementation of a Δyhr178w (Δstb5) mutant with CAGL0I02552g resolved the increased sensitivity to cold, hydrogen peroxide, and caffeine of the mutant, for which reason we designated CAGl0I02552g CgSTB5. Overexpression of CgSTB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 caused a modest increase in resistance. Expression analysis found that CgSTB5 shares many transcriptional targets with CgPDR1 but, unlike the latter, is a negative regulator of pleiotropic drug resistance, including the ABC transporter genes CDR1, PDH1, and YOR1.
doi:10.1128/AAC.01278-12
PMCID: PMC3553707  PMID: 23229483
9.  Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis 
Cell Research  2013;23(8):994-1006.
Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1β (IL-1β), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis.
doi:10.1038/cr.2013.91
PMCID: PMC3731568  PMID: 23835476
Mlkl; necroptosis; apoptosis; TNF; Rip3; mice
10.  Analysis of cream formation in green tea concentrates with different solid concentrations 
The formation of tea cream in the green tea concentrates of different solid concentrations (5, 10, 20, 30, 40, 50 and 60°Brix) was investigated. The results showed a good positive correlation (γ = 0.98, p ≤ 0.05) between the amount of tea cream and the solid concentrations from 5 to 40°Brix, while the amount of tea cream in the tea concentrates of 50 and 60°Brix decreased acutely. Total sugar, caffeine and catechins were found to be the main chemical components of tea cream in the green tea concentrate. The large decrease of the amount of tea cream in the tea concentrates of 50 and 60°Brix may be induced by a sharp increase of the viscosity of the tea concentrates, which helped to improve the stability of tea concentrate. It may be indicated that the stability of green tea concentrate enhanced when the concentration higher than 50°Brix, which helped to restrain the formation of tea cream.
doi:10.1007/s13197-011-0281-8
PMCID: PMC3614051  PMID: 23729857
Cream formation; Green tea concentrate; Solid concentration; Chemical components; Viscosity
11.  Skewed X-chromosome inactivation in patients with esophageal carcinoma 
Diagnostic Pathology  2013;8:55.
Abstract
Skewed X-chromosome inactivation (SXCI) was found in some apparently healthy females mainly from Western countries. It has been linked to development of ovarian, breast and pulmonary carcinomas. The present study aimed to observe the SXCI frequencies in apparently healthy Chinese females and patients with esophageal carcinoma. DNA was extracted from the peripheral blood cells from 401 Chinese females without a detectable tumor and 143 female patients with esophageal carcinoma. Exon 1 of androgen receptor (AR) gene was amplified, and the products of different CAG alleles were resolved on denaturing polyacrylamide gels and visualized after silver staining. The corrected ratios (CR) of the products before and after HpaII digestion were calculated. As to the healthy females, when CR ≥ 3 was used as a criterion, SXCI was found in two (4.3%) of the 46 neonates, 13 (7.8%) of the 166 younger adults (16–50 years) and 37 (25.7%) of the 144 elderly females (51–96 years), with the frequency higher in the elderly subjects than in the two former groups (P < 0.05). When a more stringent criterion (CR ≥ 10) was used, SXCI was found in one (2.2%), two (1.2%) and 16 (11.1%) of the subjects in the three age groups, respectively, itsfrequency being higher in the elderly than in the younger age groups (P < 0.05). Occurrence of SXCI was detected in both the patients and controls at similar frequencies. However, the phenomenon, as defined as CR ≥ 3, was more frequent in the patients aging <40 years (35.7%) compared to the corresponding reference group (7.6%, P = 0.006). When CR ≥ 10 was adopted, the frequencies were 7.1% and 1.2%, respectively. Their difference did not attain statistical significance (P = 0. 217). SXCI also occurs in apparently healthy Chinese females, and is associated with age. It may be considered as a predisposing factor for the early development of esophageal carcinoma.
Virtual slides
The virtual slide(s) for this article can be found here http://www.diagnosticpathology.diagnomx.eu/vs/1542364337927656
doi:10.1186/1746-1596-8-55
PMCID: PMC3640911  PMID: 23556484
Skewed X-chromosome inactivation; Androgen receptor gene; Carcinoma; Esophagus; Cancer predisposition
12.  Extensive Promoter-centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation 
Cell  2012;148(1-2):84-98.
Summary
Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intra-genic, extra-genic and inter-genic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated non-coding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into the transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.
doi:10.1016/j.cell.2011.12.014
PMCID: PMC3339270  PMID: 22265404
13.  The Influence of the Severity of Chronic Virus-Related Liver Disease on Propofol Requirements during Propofol-Remifentanil Anesthesia 
Yonsei Medical Journal  2012;54(1):231-237.
Purpose
The purpose of this study was to investigate the influence of chronic virus-related liver disease severity on propofol requirements.
Materials and Methods
In this study, 48 male patients with chronic hepatitis B infection were divided into three groups according to Child-Turcotte-Pugh classification of liver function (groups A, B, and C with mild, moderate and severe liver disease, respectively). After intubation, propofol concentration was adjusted by ±0.3 µg/mL increments to maintain bispectral index in the range of 40-60. Target propofol concentrations at anesthesia initiation, pre-intubation and pre-incision were recorded.
Results
The initial concentration used in group C was significantly lower than that used in group A or B (p<0.05), whereas no difference was observed between groups A and B. At pre-intubation, the actual required concentration of propofol increased significantly (3.2 µg/mL) in group A (p<0.05), which lead to significant differences between the groups (p<0.05). At pre-incision, the requirements for propofol decreased significantly in both groups A and B (3.0 µg/mL and 2.7 µg/mL, respectively) compared with those at pre-intubation (p<0.05), and were significantly different for all three groups (p<0.05), with group C demonstrating the lowest requirement (2.2 µg/mL). The required concentrations of propofol at pre-incision were similar to those at induction.
Conclusion
In this study, propofol requirements administered by target-controlled infusion to maintain similar depths of hypnosis were shown to depend on the severity of chronic virus-related liver dysfunction. In other words, patients with the most severe liver dysfunction required the least amount of propofol.
doi:10.3349/ymj.2013.54.1.231
PMCID: PMC3521282  PMID: 23225825
Propofol; liver disease; electroencephalography
14.  The prognostic value of pulmonary embolism severity index in acute pulmonary embolism: a meta-analysis 
Respiratory Research  2012;13(1):111.
Background
Prognostic assessment is important for the management of patients with acute pulmonary embolism (APE). Pulmonary Embolism Severity Index (PESI) and simple PESI (sPESI) are new emerged prognostic assessment tools for APE. The aim of this meta-analysis is to assess the accuracy of the PESI and the sPESI to predict prognostic outcomes (all-cause and PE-related mortality, serious adverse events) in APE patients, and compare between these two PESIs.
Methods
MEDLINE and EMBASE database were searched up to June 2012 using the terms “Pulmonary Embolism Severity Index” and “pulmonary embolism”. Summary odds ratio (OR) with 95% confidence intervals (CIs) for prognostic outcomes in low risk PESI versus high risk PESI were calculated. Summary receiver operating characteristic curve (SROC) used to estimate overall predicting accuracies of prognostic outcomes.
Results
Twenty-one studies were included in this meta-analysis. The results showed low-risk PESI was significantly associated with lower all-cause mortality (OR 0.13; 95% CI 0.12 to 0.15), PE-related mortality (OR 0.09; 95% CI 0.05 to 0.17) and serious adverse events (OR 0.34; 95% CI 0.29 to 0.41), with no homogeneity across studies. In sPESI subgroup, the OR of all-cause mortality, PE-related mortality, and serious adverse events was 0.10 (95% CI 0.08 to 0.14), 0.09 (95% CI 0.03 to 0.26) and 0.40 (95% CI 0.31 to 0.51), respectively; while in PESI subgroup, the OR was 0.14 (95% CI 0.13 to 0.16), 0.09 (95% CI 0.04 to 0.21), and 0.30 (95% CI 0.23 to 0.38), respectively. For accuracy analysis, the pooled sensitivity, the pooled specificity, and the overall weighted AUC for PESI predicting all-cause mortality was 0.909 (95% CI: 0.900 to 0.916), 0.411 (95% CI: 0.407 to 0.415), and 0.7853±0.0058, respectively; for PE-related mortality, it was 0.953 (95% CI: 0.913 to 0.978), 0.374 (95% CI: 0.360 to 0.388), and 0.8218±0.0349, respectively; for serious adverse events, it was 0.821 (95% CI: 0.795 to 0.845), 0.389 (95% CI: 0.384 to 0.394), and 0.6809±0.0208, respectively. In sPESI subgroup, the AUC for predicting all-cause mortality, PE-related mortality, and serious adverse events was 0.7920±0.0117, 0.8317±0.0547, and 0.6454±0.0197, respectively. In PESI subgroup, the AUC was 0.7856±0.0075, 0.8158±0.0451, and 0.6609±0.0252, respectively.
Conclusions
PESI has discriminative power to predict the short-term death and adverse outcome events in patients with acute pulmonary embolism, the PESI and the sPESI have similar accuracy, while sPESI is easier to use. However, the calibration for predicting prognosis can’t be calculated from this meta-analysis, some prospective studies for accessing PESI predicting calibration can be recommended.
doi:10.1186/1465-9921-13-111
PMCID: PMC3571977  PMID: 23210843
Acute pulmonary embolism; Pulmonary embolism severity index; Prognosis
15.  Long-term, efficient inhibition of microRNA function in mice using rAAV vectors 
Nature methods  2012;9(4):403-409.
Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA `Tough Decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuD depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122—but not anti-let-7—TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuD RNAs induce miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.
doi:10.1038/nmeth.1903
PMCID: PMC3420816  PMID: 22388288
16.  Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation 
Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14.
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8.
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.
doi:10.3791/3770
PMCID: PMC3466657  PMID: 22564980
Genetics;  Issue 62;  ChIP;  ChIA-PET;  Chromatin Interactions;  Genomics;  Next-Generation Sequencing
17.  Myosin Light Chain Kinase Mediates Intestinal Barrier Disruption following Burn Injury 
PLoS ONE  2012;7(4):e34946.
Background
Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.
Methodology/Principal Findings
Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.
Conclusions/Significance
The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.
doi:10.1371/journal.pone.0034946
PMCID: PMC3329538  PMID: 22529961
18.  Identification and Characterization of the Actin-Binding Motif of Phostensin 
Phostensin, a protein phosphatase 1 F-actin cytoskeleton-targeting subunit encoded by KIAA1949, consists of 165 amino acids and caps the pointed ends of actin filaments. Sequence alignment analyses suggest that the C-terminal region of phostensin, spanning residues 129 to 155, contains a consensus actin-binding motif. Here, we have verified the existence of an actin-binding motif in the C-terminal domain of phostensin using colocalization, F-actin co-sedimentation and single filament binding assays. Our data indicate that the N-terminal region of phostensin (1–129) cannot bind to actin filaments and cannot retard the pointed end elongation of gelsolin-actin seeds. Furthermore, the C-terminal region of phostensin (125–165) multiply bind to the sides of actin filaments and lacks the ability to block the pointed end elongation, suggesting that the actin-binding motif is located in the C-terminal region of the phostensin. Further analyses indicate that phostensin binding to the pointed end of actin filament requires N-terminal residues 35 to 51. These results suggest that phostensin might fold into a rigid structure, allowing the N-terminus to sterically hinder the binding of C-terminus to the sides of actin filament, thus rendering phostensin binding to the pointed ends of actin filaments.
doi:10.3390/ijms131215967
PMCID: PMC3546673  PMID: 23443105
phostensin; actin filament; KIAA1949; protein phosphatase 1
19.  Icariin and its Derivative Icariside II Extend Healthspan via Insulin/IGF-1 Pathway in C. elegans 
PLoS ONE  2011;6(12):e28835.
Compounds that delay aging might also postpone age-related diseases and extend healthspan in humans. Icariin is a flavonol extracted from several plant species of the Epimedium family. The icariin and its metabolic derivatives have been shown to exert wide protective effects in age-related diseases. However, whether icariin and its derivatives have the potency of delaying aging remains unclear. Here, we report that icariin and its derivative icariside II extend C. elegans lifespan. Using HPLC, we found high level of icariside II in the animals treated with icariin, suggesting icariside II is the bioactive form in vivo of icariin. Icariside II also increased the thermo and oxidative stress tolerance, slowed locomotion decline in late adulthood and delayed the onset of paralysis mediated by polyQ and Aβ1–42 proteotoxicity. The lifespan extension effect of icariside II is dependent on the insulin/IGF-1 signaling (IIS) since the daf-16(mu86) and daf-2(e1370) failed to show any lifespan extension upon icariside II treatment. Consistently, icariside II treatment upregulates the expression of DAF-16 targets in the wild-type. Moreover, our data suggests that the heat shock transcription factor HSF-1 has a role in icariside II-dependent lifespan extension further implicating the IIS pathway. In conclusion, we demonstrate a novel natural compound, icariside II as the bioactive form of icariin, extends the healthspan via IIS pathway in C. elegans.
doi:10.1371/journal.pone.0028835
PMCID: PMC3244416  PMID: 22216122
20.  Identification of PRRT2 as the causative gene of paroxysmal kinesigenic dyskinesias 
Brain  2011;134(12):3490-3498.
Paroxysmal kinesigenic dyskinesias is a paroxysmal movement disorder characterized by recurrent, brief attacks of abnormal involuntary movements induced by sudden voluntary movements. Although several loci, including the pericentromeric region of chromosome 16, have been linked to paroxysmal kinesigenic dyskinesias, the causative gene has not yet been identified. Here, we identified proline-rich transmembrane protein 2 (PRRT2) as a causative gene of paroxysmal kinesigenic dyskinesias by using a combination of exome sequencing and linkage analysis. Genetic linkage mapping with 11 markers that encompassed the pericentromeric of chromosome 16 was performed in 27 members of two families with autosomal dominant paroxysmal kinesigenic dyskinesias. Then, the whole-exome sequencing was performed in three patients from these two families. By combining the defined linkage region (16p12.1–q12.1) and the results of exome sequencing, we identified an insertion mutation c.649_650InsC (p.P217fsX7) in one family and a nonsense mutation c.487C>T (p.Q163X) in another family. To confirm our findings, we sequenced the exons and flanking introns of PRRT2 in another three families with paroxysmal kinesigenic dyskinesias. The c.649_650InsC (p.P217fsX7) mutation was identified in two of these families, whereas a missense mutation, c.796C>T (R266W), was identified in another family with paroxysmal kinesigenic dyskinesias. All of these mutations completely co-segregated with the phenotype in each family. None of these mutations was identified in 500 normal unaffected individuals of matched geographical ancestry. Thus, we have identified PRRT2 as the first causative gene of paroxysmal kinesigenic dyskinesias, warranting further investigations to understand the pathogenesis of this disorder.
doi:10.1093/brain/awr289
PMCID: PMC3235563  PMID: 22120146
proline-rich transmembrane protein 2; paroxysmal kinesigenic dyskinesias; whole-exome sequencing; linkage analysis
21.  Inactivation of Rheb by PRAK-mediated phosphorylation is essential for energy depletion-induced suppression of mTORC1 
Nature cell biology  2011;13(3):263-272.
Cell growth can be suppressed by stressful environments, but the role of stress pathways in this process is largely unknown. Here we show that a cascade of p38β mitogen activated protein kinase and p38 regulated/activated kinase (PRAK) plays a role in energy starvation-induced suppression of mammalian target of rapamycin (mTOR), that energy starvation activates the p38β-PRAK cascade, and that p38β- or PRAK-deletion diminishes energy depletion-induced suppression of mTORC1 and reduction of cell size. We show that p38β-PRAK operates independent from the known mTORC1 inactivation pathways – phosphorylation of tuberous sclerosis protein 2 (TSC2) and raptor by AMP activated protein kinase (AMPK), and surprisingly, PRAK directly regulates Ras homolog enriched in brain (Rheb), a key component of the mTORC1 pathway by phosphorylation. Phosphorylation of Rheb at serine 130 by PRAK impairs Rheb’s nucleotide-binding ability and inhibits Rheb-mediated mTORC1 activation. The direct regulation of Rheb by PRAK integrates a stress pathway with the mTORC1 pathway in response to energy depletion.
doi:10.1038/ncb2168
PMCID: PMC3070924  PMID: 21336308
22.  MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression 
Recombinant adeno-associated viruses (rAAVs) that can cross the blood-brain-barrier and achieve efficient and stable transvascular gene transfer to the central nervous system (CNS) hold significant promise for treating CNS disorders. However, following intravascular delivery, these vectors also target liver, heart, skeletal muscle, and other tissues, which may cause untoward effects. To circumvent this, we used tissue-specific, endogenous microRNAs (miRNAs) to repress rAAV expression outside the CNS, by engineering perfectly complementary miRNA-binding sites into the rAAV9 genome. This approach allowed simultaneous multi-tissue regulation and CNS-directed stable transgene expression without detectably perturbing the endogenous miRNA pathway. Regulation of rAAV expression by miRNA was primarily via site-specific cleavage of the transgene mRNA, generating specific 5′ and 3′ mRNA fragments. Our findings promise to facilitate the development of miRNA-regulated rAAV for CNS-targeted gene delivery and other applications.
doi:10.1038/mt.2010.279
PMCID: PMC3048189  PMID: 21179009
rAAV; intravascular delivery; CNS transduction; miRNA-binding site; miRNA regulation
23.  MicroRNA-regulated, Systemically Delivered rAAV9: A Step Closer to CNS-restricted Transgene Expression 
Molecular Therapy  2010;19(3):526-535.
Recombinant adeno-associated viruses (rAAVs) that can cross the blood–brain-barrier and achieve efficient and stable transvascular gene transfer to the central nervous system (CNS) hold significant promise for treating CNS disorders. However, following intravascular delivery, these vectors also target liver, heart, skeletal muscle, and other tissues, which may cause untoward effects. To circumvent this, we used tissue-specific, endogenous microRNAs (miRNAs) to repress rAAV expression outside the CNS, by engineering perfectly complementary miRNA-binding sites into the rAAV9 genome. This approach allowed simultaneous multi-tissue regulation and CNS-directed stable transgene expression without detectably perturbing the endogenous miRNA pathway. Regulation of rAAV expression by miRNA was primarily via site-specific cleavage of the transgene mRNA, generating specific 5′ and 3′ mRNA fragments. Our findings promise to facilitate the development of miRNA-regulated rAAV for CNS-targeted gene delivery and other applications.
doi:10.1038/mt.2010.279
PMCID: PMC3048189  PMID: 21179009
24.  Microarray and Molecular Analyses of the Azole Resistance Mechanism in Candida glabrata Oropharyngeal Isolates▿  
DNA microarrays were used to analyze Candida glabrata oropharyngeal isolates from seven hematopoietic stem cell transplant recipients whose isolates developed azole resistance while the recipients received fluconazole prophylaxis. Transcriptional profiling of the paired isolates revealed 19 genes upregulated in the majority of resistant isolates compared to their paired susceptible isolates. All seven resistant isolates had greater than 2-fold upregulation of C. glabrata PDR1 (CgPDR1), a master transcriptional regulator of the pleiotropic drug resistance (PDR) network, and all seven resistant isolates showed upregulation of known CgPDR1 target genes. The altered transcriptome can be explained in part by the observation that all seven resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 open reading frame. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, and S391L) and one in the activation domain (G943S), while two mutations (N764I and R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive isolate and five resistant isolates into a laboratory azole-hypersusceptible strain (Cgpdr1 strain) via integrative transformation. The Cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic backgrounds, upregulation of CgPDR1 and CgPDR1 target genes varied between the five transformants, independent of the domain locations in which the mutations occurred. In summary, gain-of-function mutations in CgPDR1 contributed to the clinical azole resistance, but different mutations had various degrees of impact on the CgPDR1 target genes.
doi:10.1128/AAC.00535-10
PMCID: PMC2916311  PMID: 20547810
25.  Clinicopathological features and prognosis assessment of extranodal follicular dendritic cell sarcoma 
AIM: To establish a model for prognosis assessment of extranodal follicular dendritic cell (FDC) sarcoma.
METHODS: Nine lesions were examined by routine and molecular approaches. Clinicopathological factors from the new cases and 97 reported cases were analyzed for their prognostic values.
RESULTS: The current lesions were found in five male and four female patients, located mainly in the head and neck area and averaging 7.2 cm in size. Six patients had recurrence or metastasis and three remained free of disease. The 106 patients (male/female ratio, 1.1:1) were aged from 9 to 82 years (median, 44 years). The tumor sizes ranged from 1.5 to 21 cm (mean, 7.4 cm). Abdominal/pelvic region was affected most frequently (43%). Surgical resection was performed in 100 patients, followed by radiation and/or chemotherapy in 35 of them. Follow-up data were available in 91 cases, covering a period of 3-324 mo (mean, 27 mo; median, 19 mo). Of the informative cases, 38 (42%) had recurrence or metastasis, and 12 (13%) died of the disease. These tumors were classified histologically into low- and high-grade lesions. A size ≥ 5 cm (P = 0.003), high-grade histology (P = 0.046) and a mitotic count ≥ 5/10 HPF (P = 0.013) were associated with tumor recurrence. The lesions were defined as low-, intermediate- and high-risk tumors, and their recurrence rates were 16%, 46% and 73%, and their mortality rates 0%, 4% and 45%, respectively.
CONCLUSION: Extranodal FDC tumors behave like soft tissue sarcomas. Their clinical outcomes are variable and can be evaluated according to their sizes and grades.
doi:10.3748/wjg.v16.i20.2504
PMCID: PMC2877180  PMID: 20503450
Extranodal follicular dendritic cell sarcoma; Prognosis assessment; Histologic grade; Immunohistochemistry; In situ hybridization; Mutation detection

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