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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  Unsupervised Analysis of Transcriptomic Profiles Reveals Six Glioma Subtypes 
Cancer research  2009;69(5):2091-2099.
Gliomas are the most common type of primary brain tumors in adults and a significant cause of cancer-related mortality. Defining glioma subtypes based on objective genetic and molecular signatures may allow for a more rational, patient-specific approach to therapy in the future. Classifications based on gene expression data have been attempted in the past with varying success and with only some concordance between studies, possibly due to inherent bias that can be introduced through the use of analytic methodologies that make a priori selection of genes before classification. To overcome this potential source of bias, we have applied two unsupervised machine learning methods to genome-wide gene expression profiles of 159 gliomas, thereby establishing a robust glioma classification model relying only on the molecular data. The model predicts for two major groups of gliomas (oligodendroglioma-rich and glioblastoma-rich groups) separable into six hierarchically nested subtypes. We then identified six sets of classifiers that can be used to assign any given glioma to the corresponding subtype and validated these classifiers using both internal (189 additional independent samples) and two external data sets (341 patients). Application of the classification system to the external glioma data sets allowed us to identify previously unrecognized prognostic groups within previously published data and within The Cancer Genome Atlas glioblastoma samples and the different biological pathways associated with the different glioma subtypes offering a potential clue to the pathogenesis and possibly therapeutic targets for tumors within each subtype.
doi:10.1158/0008-5472.CAN-08-2100
PMCID: PMC2845963  PMID: 19244127
22.  Epigenetic-Mediated Dysfunction of the Bone Morphogenetic Protein Developmental Pathway Inhibits Differentiation of Human Glioblastoma Tumor Initiating Cells 
Cancer cell  2008;13(1):69-80.
SUMMARY
Despite similarities between tumor initiating cells with stem-like properties (TICs) and normal neural stem cells, we hypothesized that there may be differences in their differentiation potentials. We now demonstrate that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Jak/STAT-dependent astroglial differentiation is impaired due to EZH2-dependent epigenetic silencing of BMP receptor 1B (BMPR1B) in a subset of glioblastoma TICs. Forced expression of BMPR1B either by transgene expression or demethylation of the promoter restores their differentiation capabilities and induces loss of their tumorigenicity. We propose that deregulation of the BMP developmental pathway in a subset of glioblastoma TICs contributes to their tumorigenicity both by desensitizing TICs to normal differentiation cues, and by converting otherwise cytostatic signals to pro-proliferative signals.
SIGNIFICANCE
Elucidation of the differentiation pathways operative and/or aberrant in both normal stem cells and TICs will be critical to fully understand the pathogenesis of primary human tumors and may help lead to better therapies. To this end, we utilized several TICs isolated from primary glioblastomas and compared them to normal human NSCs and mouse NSCs from various developmental stages. We demonstrate a major differentiation block in a subset of glioblastoma TICs is caused by the Polycomb repressor complex (PRC) mediated epigenetic silencing of the BMPR1B promoter, analogous to early embryonic NSCs. We provide here an example of a temporally deregulated and aberrantly fixed developmental block to differentiation contributing to the pathogenesis of a subset of human GBMs.
doi:10.1016/j.ccr.2007.12.005
PMCID: PMC2835498  PMID: 18167341
23.  Correlation Analysis between SNP and Expression Arrays in Gliomas Identify Potentially Relevant Targets Genes1 
Cancer research  2009;69(4):1596-1603.
Primary brain tumors are a major cause of cancer mortality in the United States. Therapy for gliomas, the most common type of primary brain tumors, remains suboptimal. The development of improved therapeutics will require greater knowledge of the biology of gliomas at both the genomic and transcriptional levels. We have previously reported whole genome profiling of chromosome copy number alterations (CNA) in gliomas, and now present our findings on how those changes may affect transcription of genes that may be involved in tumor induction and progression. By calculating correlation values of mRNA expression vs. DNA copy number average in a moving window around a given RNA probeset, biologically relevant information can be gained that is obscured by the analysis of a single data type. Correlation coefficients ranged from −0.6 to 0.7; highly significant when compared to previously studies. Most correlated genes are located on chromosomes 1, 7, 9, 10, 13, 14, 19, 20 and 22, chromosomes known to have genomic alterations in gliomas. Additionally, we were able to identify CNAs whose gene expression correlation suggests possible epigenetic regulation. This analysis revealed a number of interesting candidates such as CXCL12, PTER, LRRN6C, among others. The results have been verified using real-time PCR and methylation sequencing assays. These data will further help differentiate genes involved in the induction and/or maintenance of the tumorigenic process from those that are mere passenger mutations, thereby enriching for a population of potentially new therapeutic molecular targets.
doi:10.1158/0008-5472.CAN-08-2496
PMCID: PMC2644341  PMID: 19190341
24.  Clonality and allelotype analyses of focal nodular hyperplasia compared with hepatocellular adenoma and carcinoma 
AIM: To identify clonality and genetic alterations in focal nodular hyperplasia (FNH) and the nodules derived from it.
METHODS: Twelve FNH lesions were examined. Twelve hepatocellular adenomas (HCAs) and 22 hepatocellular carcinomas (HCCs) were used as references. Nodules of different types were identified and isolated from FNH by microdissection. An X-chromosome inactivation assay was employed to describe their clonality status. Loss of heterozygosity (LOH) was detected, using 57 markers, for genetic alterations.
RESULTS: Nodules of altered hepatocytes (NAH), the putative precursors of HCA and HCC, were found in all the FNH lesions. Polyclonality was revealed in 10 FNH lesions from female patients, and LOH was not detected in any of the six FNH lesions examined, the results apparently showing their polyclonal nature. In contrast, monoclonality was demonstrated in all the eight HCAs and in four of the HCCs from females, and allelic imbalances were found in the HCAs (9/9) and HCCs (15/18), with chromosomal arms 11p, 13q and 17p affected in the former, and 6q, 8p, 11p, 16q and 17p affected in the latter lesions in high frequencies (≥ 30%). Monoclonality was revealed in 21 (40%) of the 52 microdissected NAH, but was not found in any of the five ordinary nodules. LOH was found in all of the 13 NAH tested, being highly frequent at six loci on 8p, 11p, 13q and 17p.
CONCLUSION: FNH, as a whole, is polyclonal, but some of the NAH lesions derived from it are already neoplastic and harbor similar allelic imbalances as HCAs.
doi:10.3748/wjg.15.4695
PMCID: PMC2754518  PMID: 19787833
Clonality analysis; Focal nodular hyperplasia; Hepatocellular adenoma; Liver tumorigenesis; Loss of heterozygosity; Nodules of altered hepatocytes
25.  Notch-1 regulates transcription of the epidermal growth factor receptor through p53 
Carcinogenesis  2008;29(5):918-925.
The Notch pathway plays a key role in the development and is increasingly recognized for its importance in cancer. We demonstrated previously the overexpression of Notch-1 and its ligands in gliomas and showed that their knockdown inhibits glioma cell proliferation and survival. To elucidate the mechanisms downstream of Notch-1 in glioma cells, we performed microarray profiling of glioma cells transfected with Notch-1 small interfering RNA. Notable among downregulated transcripts was the epidermal growth factor receptor (EGFR), known to be overexpressed or amplified in gliomas and prominent in other cancers as well. Further studies confirmed that Notch-1 inhibition decreased EGFR messenger RNA (mRNA) and EGFR protein in glioma and other cell lines. Transfection with Notch-1 increased EGFR expression. Additionally, we found a significant correlation in levels of EGFR and Notch-1 mRNA in primary high-grade human gliomas. Subsequent experiments showed that p53, an activator of the EGFR promoter, is regulated by Notch-1. Experiments with p53-positive and -null cell lines confirmed that p53 partially mediates the effects of Notch-1 on EGFR expression. These results show for the first time that Notch-1 upregulates EGFR expression and also demonstrate Notch-1 regulation of p53 in gliomas. These observations have significant implications for understanding the mechanisms of Notch in cancer and development.
doi:10.1093/carcin/bgn079
PMCID: PMC2902388  PMID: 18359760

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