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1.  Aromatic–Aromatic Interactions Enhance Interfiber Contacts for Enzymatic Formation of a Spontaneously Aligned Supramolecular Hydrogel 
Anisotropy or alignment is a critical feature of functional soft materials in living organisms, but it remains a challenge for spontaneously generating anisotropic gel materials. Here we report a molecular design that increases intermolecular aromatic–aromatic interactions of hydrogelators during enzymatic hydrogelation for spontaneously forming an anisotropic hydrogel. This process, relying on both aromatic–aromatic interactions and enzyme catalysis, results in spontaneously aligned supramolecular nanofibers as the matrices of a monodomain hydrogel that exhibits significant birefringence. This work, as the first example of monodomain hydrogels formed via an enzymatic reaction, illustrates a new biomimetic approach for generating aligned anisotropic soft materials.
PMCID: PMC3986011  PMID: 24512553
2.  Evaluation of the Intestinal Transport of a Phenylethanoid Glycoside-Rich Extract from Cistanche deserticola across the Caco-2 Cell Monolayer Model 
PLoS ONE  2015;10(2):e0116490.
Phenylethanoid glycosides (PhGs), a class of polyphenolic compounds, are considered one of major bioactive constituents of Cistanche deserticola Y.C. Ma (CD), whose extract is orally used in traditional Chinese medicine. Although previous pharmacological studies have reported that PhGs exert many activities, their intestinal transport profiles have not been clarified. In this study, we investigated the intestinal permeability of a PhG-rich extract (PRE) from CD as an integrated system in the Caco-2 cell monolayer model using a bioassay system. The results showed that PRE is primarily transported via poorly absorbed passive diffusion down a concentration gradient without efflux, which provides the pharmacokinetic basis for the clinical application of PhGs in CD. We also determined the intestinal permeability of three major PhGs [acteoside (AC), isoacteoside (IS) and echinacoside (EC)] by HLPC. Furthermore, we developed a novel HPLC-fluorescence detection method to accurately determine the flux amount of AC and IS. As expected, the transport characteristics of the three PhGs are consistent with those of PRE, indicating that the present bioassay system is appropriate and reliable for the evaluation of the transport characteristics of active ingredient groups (AIG) in PRE. Moreover, this system may also be suitable for other plant extracts given appropriate bioactivity.
PMCID: PMC4315399  PMID: 25646971
3.  Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes 
Aging Cell  2014;14(1):35-48.
Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10−6). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.
PMCID: PMC4326912  PMID: 25510159
Alzheimer (disease); C. elegans; Huntington (disease); neurodegeneration; (protein) aggregation; proteasome
4.  Dynamic migration and cell-cell interactions of early reprogramming revealed by high resolution time-lapse imaging 
Stem cells (Dayton, Ohio)  2013;31(5):895-905.
Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multi-color time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as five minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct 2-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate the requirement of E-cadherin for proper cellular interactions from an early stage of reprogramming, including the 2-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution.
PMCID: PMC4309553  PMID: 23335078
5.  CIRI: an efficient and unbiased algorithm for de novo circular RNA identification 
Genome Biology  2015;16(1):4.
Recent studies reveal that circular RNAs (circRNAs) are a novel class of abundant, stable and ubiquitous noncoding RNA molecules in animals. Comprehensive detection of circRNAs from high-throughput transcriptome data is an initial and crucial step to study their biogenesis and function. Here, we present a novel chiastic clipping signal-based algorithm, CIRI, to unbiasedly and accurately detect circRNAs from transcriptome data by employing multiple filtration strategies. By applying CIRI to ENCODE RNA-seq data, we for the first time identify and experimentally validate the prevalence of intronic/intergenic circRNAs as well as fragments specific to them in the human transcriptome.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0571-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4316645  PMID: 25583365
6.  Genome-wide antagonism between 5-hydroxymethylcytosine and DNA methylation in the adult mouse brain 
Frontiers in biology  2014;9(1):66-74.
Mounting evidence points to critical roles for DNA modifications, including 5-methylcytosine (5mC) and its oxidized forms, in the development, plasticity and disorders of the mammalian nervous system. The novel DNA base 5-hydroxymethylcytosine (5hmC) is known to be capable of initiating passive or active DNA demethylation, but whether and how extensively 5hmC functions in shaping the post-mitotic neuronal DNA methylome is unclear. Here we report the genome-wide distribution of 5hmC in dentate granule neurons from adult mouse hippocampus in vivo. 5hmC in the neuronal genome is highly enriched in gene bodies, especially in exons, and correlates with gene expression. Direct genome-wide comparison of 5hmC distribution between embryonic stem cells and neurons reveals extensive differences, reflecting the functional disparity between these two cell types. Importantly, integrative analysis of 5hmC, overall DNA methylation and gene expression profiles of dentate granule neurons in vivo reveals the genome-wide antagonism between these two states of cytosine modifications, supporting a role for 5hmC in shaping the neuronal DNA methylome by promoting active DNA demethylation.
PMCID: PMC4284063  PMID: 25568643
dentate granule neuron; active DNA demethylation; TET; methylome
7.  Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Interacts with Host KAP1 To Facilitate Establishment of Viral Latency 
Journal of Virology  2014;88(13):7331-7344.
Kaposi's sarcoma-associated herpesvirus (KSHV) typically displays two different phases in its life cycle, the default latent phase and the lytic phase. There is a short period of lytic gene expression in the early stage of KSHV primary infection. The factors involved in the shutdown process of lytic gene expression are poorly identified. It has been shown that the latency-associated nuclear antigen (LANA) encoded by KSHV plays an important role in the establishment of viral latency. In screening, we identified a host protein, Krüppel-associated box domain-associated protein 1 (KAP1), that bound to LANA. We validated the interaction between LANA and KAP1 in vivo and in vitro, as well as their colocalization in the nucleus. We mapped out that LANA interacted with both the N- and C-terminal domains of KAP1. Based on the interface of LANA-KAP1 interaction determined, we proved that LANA recruited KAP1 to the RTA promoter region of the KSHV genome. We revealed that KAP1 was involved in transcriptional repression by LANA. We found multiple cooccupation sites of LANA and KAP1 on the whole KSHV genome by chromatin immunoprecipitation for sequencing (ChIP-seq) and demonstrated that LANA-recruited KAP1 played a critical role in the shutdown of lytic gene expression during the early stage of KSHV primary infection. Taken together, our data suggest that LANA interacts with KAP1 and represses lytic gene expression to facilitate the establishment of KSHV latency.
IMPORTANCE Our study revealed the mechanism of transcriptional repression by LANA during KSHV primary infection, providing new insights into the process of KSHV latency establishment.
PMCID: PMC4054432  PMID: 24741090
8.  A Novel Medical Image Protection Scheme Using a 3-Dimensional Chaotic System 
PLoS ONE  2014;9(12):e115773.
Recently, great concerns have been raised regarding the issue of medical image protection due to the increasing demand for telemedicine services, especially the teleradiology service. To meet this challenge, a novel chaos-based approach is suggested in this paper. To address the security and efficiency problems encountered by many existing permutation-diffusion type image ciphers, the new scheme utilizes a single 3D chaotic system, Chen's chaotic system, for both permutation and diffusion. In the permutation stage, we introduce a novel shuffling mechanism, which shuffles each pixel in the plain image by swapping it with another pixel chosen by two of the three state variables of Chen's chaotic system. The remaining variable is used for quantification of pseudorandom keystream for diffusion. Moreover, the selection of state variables is controlled by plain pixel, which enhances the security against known/chosen-plaintext attack. Thorough experimental tests are carried out and the results indicate that the proposed scheme provides an effective and efficient way for real-time secure medical image transmission over public networks.
PMCID: PMC4277340  PMID: 25541941
9.  Imaging Self-assembly Dependent Spatial Distribution of Small Molecules in Cellular Environment 
Self-assembly of small molecules, as a more common phenomenon than one previously thought, can be either beneficial or detrimental to cells. Despite its profound biological implications, how the self-assembly of small molecules behave in cellular environment is largely unknown and barely explored. This work studies four fluorescent molecules that consist of the same peptidic backbone (e.g., Phe-Phe-Lys) and enzyme trigger (e.g., a phosphotyrosine residue), but bear different fluorophores on the side chain of the lysine residue of the peptidic motif. These molecules, however, exhibit different ability of self-assembly before and after enzymatic transformation (e.g., dephosphorylation). Fluorescent imaging reveals that self-assembly directly affects the distribution of these small molecules in cellular environment. Moreover, cell viability tests suggest that the states and the location of the molecular assemblies in the cellular environment control the phenotypes of the cells. For example, the molecular nanofibers of one of the small molecules apparently stabilize actin filaments and alleviate the insult of an F-actin toxin (e.g., latrunculin A). Combining fluorescent imaging and enzyme-instructed self-assembly of small peptidic molecules, this work not only demonstrates that self-assembly as a key factor for dictating the spatial distribution of small molecules in cellular environment. In addition, it illustrates a useful approach, based on enzyme-instructed self-assembly of small molecules, to modulate spatiotemporal profiles of small molecules in cellular environment, which allows the use of the emergent properties of small molecules to control the fate of cells.
PMCID: PMC3895920  PMID: 24266765
10.  Self-delivery Multifunctional Anti-HIV Hydrogels for Sustained Release 
Advanced healthcare materials  2013;2(12):1586-1590.
None of the clinical trials of anti-HIV gels based on conventional polymers or lipid emulsions is successful, suggesting the need of new molecular design of the anti-HIV gels. This paper reports the conversion of anti-HIV prodrugs into self-delivery supramolecular hydrogels. By covalently conjugating reverse transcriptase inhibitors to a versatile self-assembly motif, we obtained the hydrogelators that self-assemble to form supramolecular nanofibers as the matrices of hydrogels in a weak acidic condition. Upon the treatment of prostate acid phosphatase (PAP), the hydrogels exhibit drastically enhanced elasticity. The hydrogelators are biocompatible and able to release the inhibitors under physiological condition. The use of the self-assembly motif as a self-delivery agent containing non-steroid anti-inflammatory drug (NSAID) renders this hydrogel to be both anti-inflammatory and anti-HIV. This work illustrates an unprecedented approach for designing multifunctional supramolecular hydrogels that may serve as potential anti-HIV hydrogels for sustained drug release.
PMCID: PMC3934007  PMID: 23616384
Anti-HIV hydrogels; self-delivery; self-assembly; supramolecular interaction; nanotechnology
11.  Multi-platform and cross-methodological reproducibility of transcriptome profiling by RNA-seq in the ABRF Next-Generation Sequencing Study 
Nature biotechnology  2014;32(9):915-925.
High-throughput RNA sequencing (RNA-seq) dramatically expands the potential for novel genomics discoveries, but the wide variety of platforms, protocols and performance has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We tested replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (polyA-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies’ PGM and Proton, Pacific Biosciences RS and Roche’s 454). The results show high intra-platform and inter-platform concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. These data also demonstrate that ribosomal RNA depletion can both enable effective analysis of degraded RNA samples and be readily compared to polyA-enriched fractions. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.
PMCID: PMC4167418  PMID: 25150835
12.  The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells 
Nucleic Acids Research  2014;42(22):13799-13811.
The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.
PMCID: PMC4267628  PMID: 25399420
13.  Inhibition of angiogenesis by a novel neutralizing antibody targeting human VEGFR-3 
mAbs  2013;5(6):956-961.
Vascular endothelial growth factor receptor 3 (VEGFR-3) is a receptor for the vascular endothelial growth factor C and D (VEGF-C and D) and plays a critical role in the development of embryonic vascular system and regulation of tumor lymphangiogenesis. In this report, we generated a novel panel of 17 monoclonal antibodies (mAbs) against human VEGFR-3 and determined their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. Furthermore, in chick embryo CAM angiogenesis experiments, the angiogenesis induced by recombinant glutathione-S-transferase-VEGF-D was decreased in the presence of antibody BDD073. These data suggest that this novel neutralizing antibody against human VEGFR-3 could be a tool for the investigations into the biology of VEGFR-3, and potentially a reagent for blocking VEGF-D-induced angiogenesis and lymphogenesis.
PMCID: PMC3896609  PMID: 23995616
VEGFR-3; VEGF-D; antibody; angiogenesis
14.  Isolation and Multiple Differentiation Potential Assessment of Human Gingival Mesenchymal Stem Cells 
The aim of this study was to isolate human mesenchymal stem cells (MSCs) from the gingiva (GMSCs) and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs) and dermal stem cells (DSCs) cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F) and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP) activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN) and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation). Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM) was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.
PMCID: PMC4264207  PMID: 25405732
mesenchymal stem cells; gingiva; osteogenesis; adipogenesis; odontogenesis
15.  Probing Nanoscale Self-Assembly of Non-Fluorescent Small Molecules inside Live Mammalian Cells 
ACS nano  2013;7(10):10.1021/nn403664n.
Like cellular proteins that form fibrillar nanostructures, small hydrogelator molecules self-assemble in water to generate molecular nanofibers. In contrast to the well-defined (dys)functions of endogenous protein filaments, the fate of intracellular assembly of small molecules remains largely unknown. Here we demonstrate the imaging of enzyme-triggered self-assembly of non-fluorescent small molecules by doping the molecular assemblies with a fluorescent hydrogelator. The cell fractionation experiments, fluorescent imaging, and electron microscopy indicate that the hydrogelators self-assemble and localize to the endoplasmic reticulum (ER) and are likely processed via the cellular secretory pathway (i.e., ER-Golgi-lysosomes/secretion). This work, as the first example of the use of correlative light and electron microscopy (CLEM) for probing the selfassembly of non-fluorescent small molecules inside live mammalian cells, not only establishes a general strategy to provide the spatiotemporal profile of the assemblies of small molecules inside cells, but may lead to a new paradigm for regulating cellular functions based on the interactions between the assemblies of small molecules (e.g., molecular nanofibers) and subcellular organelles.
PMCID: PMC3845088  PMID: 24067160
self-assembly; small molecule; nanofibers; intracellular; localization; enzyme
16.  Split-Cre Complementation Restores Combination Activity on Transgene Excision in Hair Roots of Transgenic Tobacco 
PLoS ONE  2014;9(10):e110290.
The Cre/loxP system is increasingly exploited for genetic manipulation of DNA in vitro and in vivo. It was previously reported that inactive ‘‘split-Cre’’ fragments could restore Cre activity in transgenic mice when overlapping co-expression was controlled by two different promoters. In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the in vitro recombination reaction in which only the N-terminal domain (NCre) of split-Cre protein was expressed, whereas recombination activity was obtained when the C-terminal (CCre) or both NCre and CCre fragments were supplied. We have also determined the recombination efficiency of split-Cre proteins which were co-expressed in hair roots of transgenic tobacco. No Cre recombination event was observed in hair roots of transgenic tobacco when the NCre or CCre genes were expressed alone. In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes. Moreover, the restored recombination efficiency of split-Cre proteins fused with the nuclear localization sequence (NLS) was higher than that of intact Cre in transgenic lines. Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants.
PMCID: PMC4201524  PMID: 25329460
17.  Chronotype and a PERIOD3 variable number tandem repeat polymorphism in Han Chinese pilots 
An association has been determined between variable number tandem-repeat (VNTR) polymorphisms in the PERIOD3 gene (PER3, rs57875989) and chronotype. An association has been found in which the longer PER3(5) allele is correlated with diurnal preference and shorter PER3(4) allele is linked with preference for evening, respectively. In this study, we explored the genotype frequency and relationship to the chronotype of a PER3 VNTR polymorphism in Han Chinese pilots compared to other populations to further develop aviation safety research. DNA samples were genotyped with respect to the 4-repeat and 5-repeat alleles of the PER3 VNTR polymorphism. We compared and analyzed PER3 VNTR genotype frequencies of a general Han Chinese population and Han Chinese pilots. The chronotypes of our subjects were evaluated by the morningness-eveningness questionnaire (MEQ). The distribution of PER3 VNTR genotype frequencies from 240 Han Chinese was determined (PER3(4/4), 78.3%; PER3(4/5), 20.0%; PER3(5/5), 1.7%) and compared to the genotype frequencies of 126 Han Chinese pilots (PER3(4/4), 71.4%; PER3(4/5), 26.1%; PER3(5/5), 2.4%). Statistical analysis revealed no significant difference between the general Han Chinese population and Han Chinese pilots regarding the PER3 VNTR genotype and allele frequencies (x2 = 2.170, p > 0.05). Furthermore, MEQ results showed no association between the PER3 VTNR polymorphism and chronotype. However, PER3 VNTR genotype frequencies differed significantly between Han Chinese and other ethnic groups previously reported, such as Caucasians, African Americans and Italians. These data indicate that the proposed role of the PER3 VNTR needs further clarification and the role of PER3(5) allele in sleep regulation needs to be investigated in more detail. In particular, a study of PER3 polymorphisms with a larger sample size of Han Chinese individuals and Han Chinese pilots may be required.
PMCID: PMC4238505  PMID: 25419431
PER3 VNTR; chronotype; circadian rhythm; sleep deprivation; diurnal preference; gene frequency; Han Chinese
18.  External Validation of the A2DS2 Score to Predict Stroke-Associated Pneumonia in a Chinese Population: A Prospective Cohort Study 
PLoS ONE  2014;9(10):e109665.
Background and Purpose
The A2DS2 score was recently developed from the Berlin Stroke Registry for predicting in-hospital pneumonia after acute ischemic stroke and performed well in an external validation in the North-west Germany Stroke Registry. It could be a useful tool for risk stratification in clinical practice or stroke trials. We aimed to prospectively validate the predictive value of A2DS2 score in a Chinese stroke population.
The prognostic model was used to predict stroke-associated pneumonia (SAP) from Henan Province Stroke Registry (HNSR) in which data were prospectively collected. The receiver-operating characteristic curves were plotted, and the C statistics were calculated to assess the discrimination ability. The Hosmer–Lemeshow goodness-of-fit test and the plot of observed versus predicted SAP risk were used to assess model calibration.
Among 1142 eligible patients, the overall in-hospital SAP was 18.8%, which ranged from 9.0% in patients with lower A2DS2 scores (0–4) to 65.0% in those with higher scores of 5 to 10 (P for trend <0.001). The C statistic was 0.836 (95% confidence interval, 0.803–0.868) through the A2DS2 score, suggesting excellent discrimination in the HNSR. The A2DS2 score also showed excellent calibration (Cox and Snell R2 = 0.243) in the external validation sample from the HNSR.
The A2DS2 score could reliably predict in-hospital SAP in Chinese stroke patients. It might be helpful for the assessment of increased risk monitoring and prophylactic treatment in identified high-risk patients for SAP in clinical routine.
PMCID: PMC4192306  PMID: 25299226
19.  The Majority of HIV Type 1 DNA in Circulating CD4+ T Lymphocytes Is Present in Non-Gut-Homing Resting Memory CD4+ T Cells 
AIDS Research and Human Retroviruses  2013;29(10):1330-1339.
Memory CD4+ T lymphocytes in peripheral blood that express integrins α4ß7 preferentially recirculate through gut-associated lymphoid tissue (GALT), a proposed site of significant HIV-1 replication. Tregs and activated CD4+ T cells in GALT could also be particularly susceptible to infection. We therefore hypothesized that infection of these subsets of memory CD4+ T cells may contribute disproportionately to the HIV-1 reservoir. A cross-sectional study of CD4+ T cell subsets of memory CD45RO+ cells in peripheral blood mononuclear cells (PBMCs) was conducted using leukapheresis from eight subjects with untreated chronic HIV-1 infection. Real-time polymerase chain reaction (PCR) was used to quantify total and integrated HIV-1 DNA levels from memory CD4+ T cells sorted into integrin β7+ vs. β7−, CD25+CD127low Treg vs. CD127high, and activated CD38+ vs. CD38−. More than 80% of total HIV-1 DNA was found to reside in the integrin β7-negative non-gut-homing subset of CD45RO+ memory CD4+ T cells. Less than 10% was found in highly purified Tregs or CD38+ activated memory cells. Similarly, integrated HIV-1 DNA copies were found to be more abundant in resting non-gut-homing memory CD4+ T cells (76%) than in their activated counterparts (23%). Our investigations showed that the majority of both total and integrated HIV-1 DNA was found within non-gut-homing resting CD4+ T cells.
PMCID: PMC3785810  PMID: 23971972
20.  Differential effects of common variants in SCN2A on general cognitive ability, brain physiology and mRNA expression in schizophrenia cases and controls 
JAMA psychiatry  2014;71(6):647-656.
One approach to understanding the genetic complexity of schizophrenia is to study associated behavioral and biological phenotypes that may be more directly linked to genetic variation.
To identify single nucleotide polymorphisms associated with general cognitive ability (“g”) in people with schizophrenia and controls.
Genome-wide association study (GWAS), followed by analyses in unaffected siblings and independent schizophrenia samples, functional magnetic resonance imaging studies of brain physiology in vivo, and RNA sequencing in post-mortem brain samples.
The discovery cohort and unaffected siblings were participants in the NIMH Clinical Brain Disorders Branch schizophrenia genetics studies. Additional schizophrenia cohorts were from psychiatric treatment settings in the United States, Japan, and Germany.
The discovery cohort comprised 339 with schizophrenia and 363 community controls. Follow-up analyses studied 147 unaffected siblings of the schizophrenia cases, and independent schizophrenia samples of 279, 95 and 294 participants. Imaging analyses included 87 schizophrenia cases and 397 controls. Brain tissue samples were available for 64 cases and 61 controls.
Main Outcome Measures
We studied genome-wide association with g, by group, in the discovery cohort. We used selected genotypes to test specific associations in unaffected siblings and independent schizophrenia samples. Imaging analyses focused on activation in prefrontal cortex during working memory. Brain tissue studies yielded mRNA expression levels for RefSeq transcripts.
The schizophrenia discovery cohort showed GWAS-significant association of g with polymorphisms in sodium channel gene SCN2A, accounting for 10.4% of g variance (rs10174400, P=9.27×10−10). Controls showed a trend for g/genotype association with reversed allelic directionality. The genotype-by-group interaction was also GWAS-significant (P=1.75×10−9). Siblings showed a genotype association with g parallel to the schizophrenia group, and the same interaction pattern. Parallel, but weaker, associations with cognition were found in independent schizophrenia samples. Imaging analyses showed a similar pattern of genotype associations by group and genotype-by-group interaction. RNA sequencing revealed reduced expression in 2 of 3 SCN2A alternative transcripts in the patient group, with genotype-by-group interaction, that again paralleled the cognition effects.
The findings implicate SCN2A and sodium channel biology in cognitive impairment in schizophrenia cases and unaffected relatives, and may facilitate development of cognition-enhancing treatments.
PMCID: PMC4160812  PMID: 24718902
21.  Naming Game on Networks: Let Everyone be Both Speaker and Hearer 
Scientific Reports  2014;4:6149.
To investigate how consensus is reached on a large self-organized peer-to-peer network, we extended the naming game model commonly used in language and communication to Naming Game in Groups (NGG). Differing from other existing naming game models, in NGG everyone in the population (network) can be both speaker and hearer simultaneously, which resembles in a closer manner to real-life scenarios. Moreover, NGG allows the transmission (communication) of multiple words (opinions) for multiple intra-group consensuses. The communications among indirectly-connected nodes are also enabled in NGG. We simulated and analyzed the consensus process in some typical network topologies, including random-graph networks, small-world networks and scale-free networks, to better understand how global convergence (consensus) could be reached on one common word. The results are interpreted on group negotiation of a peer-to-peer network, which shows that global consensus in the population can be reached more rapidly when more opinions are permitted within each group or when the negotiating groups in the population are larger in size. The novel features and properties introduced by our model have demonstrated its applicability in better investigating general consensus problems on peer-to-peer networks.
PMCID: PMC4139946  PMID: 25143140
22.  Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain 
Nature neuroscience  2013;17(2):215-222.
DNA methylation plays critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single-base resolution DNA methylome from adult mouse dentate neurons consists of both CpG (~75%) and CpH (~25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in low CpG-density regions, depleted at protein-DNA interaction sites, and anti-correlated with gene expression. Functionally, both mCpGs and mCpHs can repress transcription in vitro and are recognized by MeCP2 in neurons in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNMT3A for active maintenance in post-mitotic neurons. These characteristics of CpH methylation suggest a significantly expanded proportion of the neuronal genome under cytosine methylation regulation and provide a new foundation for understanding the role of this key epigenetic modification in the nervous system.
PMCID: PMC3970219  PMID: 24362762
23.  Pharmacological inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression 
Cell  2013;154(3):10.1016/j.cell.2013.06.048.
Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell cycle progression, senescence, metabolism, cancer progression and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes. It also phenocopies the effects observed upon genetic Skp2 deficiency, such as suppressing survival, Akt-mediated glycolysis as well as triggering p53-independent cellular senescence. Strikingly, we discovered a critical function of Skp2 in positively regulating cancer stem cell populations and self-renewal ability through genetic and pharmacological approaches. Notably, Skp2 inhibitor exhibits potent anti-tumor activities in multiple animal models and cooperates with chemotherapeutic agents to reduce cancer cell survival. Our study thus provides pharmacological evidence that Skp2 is a promising target for restricting cancer stem cell and cancer progression.
PMCID: PMC3845452  PMID: 23911321
24.  Oncogenic Herpesvirus KSHV Hijacks BMP-Smad1-Id Signaling to Promote Tumorigenesis 
PLoS Pathogens  2014;10(7):e1004253.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), a malignancy commonly found in AIDS patients. Whether KS is a true neoplasm or hyperplasia has been a subject of intensive debate until recently when KSHV is unequivocally shown to efficiently infect, immortalize and transform rat primary mesenchymal precursor cells (MM). Moreover, KSHV-transformed MM cells (KMM) efficiently induce tumors with hallmark features of KS when inoculated into nude mice. Here, we showed Smad1 as a novel binding protein of KSHV latency-associated nuclear antigen (LANA). LANA interacted with and sustained BMP-activated p-Smad1 in the nucleus and enhanced its loading on the Id promoters. As a result, Ids were significantly up-regulated in KMM cells and abundantly expressed in human KS lesions. Strikingly, genetic and chemical inhibition of the BMP-Smad1-Id pathway blocked the oncogenic phenotype of KSHV-transformed cells in vitro and in vivo. These findings illustrate a novel mechanism by which a tumor virus hijacks and converts a developmental pathway into an indispensable oncogenic pathway for tumorigenesis. Importantly, our results demonstrate the efficacy of targeting the BMP-Smad1-Id pathway for inhibiting the growth of KSHV-induced tumors, and therefore identify the BMP pathway as a promising therapeutic target for KS.
Author Summary
Although KSHV exerts multiple mechanisms to promote cell survival by repressing TGF-β signaling, little is known whether KSHV manipulates BMP signaling and contributes to the pathogenesis of KSHV-induced malignancies. In the present study, we have identified Smad1 as a novel binding protein of LANA by tandem affinity purification. We demonstrated that LANA up-regulated Id transcription through BMP-Smad1-Id signaling pathway. Id proteins were significantly up-regulated in KSHV-transformed MM (KMM) cells, and were abundantly expressed in human KS lesions; therefore, they were probably relevant to the development of KS. Importantly, we have shown that Ids are required to maintain the oncogenic phenotype of KMM cells in vitro and in vivo. These findings illustrate a novel mechanism by which a tumor virus hijacks and converts a developmental pathway into an indispensable oncogenic pathway for tumorigenesis. Furthermore, we showed that BMP signaling inhibitors dramatically hampered the tumorigenicity of KMM cells in vitro and in vivo. Our results demonstrate that small inhibitors targeting BMP-Smad1-Id signaling pathway are promising candidates for the treatment of KS.
PMCID: PMC4092152  PMID: 25010525
25.  Dephosphorylation of D-Peptide Derivatives to Form Biofunctional, Supramolecular Nanofibers/Hydrogels and Their Potential Applications for Intracellular Imaging and Intratumoral Chemotherapy 
D-peptides, as the enantiomers of the naturally occurring L-peptides, usually resist endogenous proteases and presumably insensitive to most enzymes. But it is unclear whether or how a phosphatase catalyzes the dephosphorylation from D-peptides. In this work, we examine the formation of the nanofibers of D-peptides via enzymatic dephosphorylation. By comparing the enzymatic hydrogelation of L-peptide and D-peptide based hydrogelators, we find that the chirality of the precursors of the hydrogelators affects little on the enzymatic hydrogelation resulted from the removal of phosphate group from a tyrosine phosphate residue. The attachment of a therapeutic agent (e.g., taxol) or a fluorophore (e.g., 4-nitro-2,1,3-benzoxadiazole (NBD)) to the D-peptide based hydrogelators afford a new type of biostable or biocompatible hydrogelators, which may find applications in intratumoral chemotherapy or intracellular imaging, respectively. This work, as the first comprehensive and systematic study of the unexpected enzymatic dephosphorylation of D-peptides, illustrates a useful approach to generate supramolecular hydrogels that have both biostability and other desired functions.
PMCID: PMC3730259  PMID: 23742714

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