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1.  Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior 
Nature neuroscience  2016;19(11):1497-1505.
Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). The RTT missense MECP2R306C mutation prevents MeCP2 interaction with NCoR/Histone deacetylase 3 (HDAC3); however, the neuronal function of HDAC3 is incompletely understood. We report that neuronal deletion of Hdac3 in mice elicits abnormal locomotor coordination, sociability, and cognition. Transcriptional and chromatin profiling revealed HDAC3 positively regulates a subset of genes and is recruited to active gene promoters via MeCP2. HDAC3-associated promoters are enriched for the FOXO transcription factors, and FOXO acetylation is elevated in Hdac3 KO and Mecp2 KO neurons. Human RTT patient-derived MECP2R306C neural progenitor cells have deficits in HDAC3 and FOXO recruitment and gene expression. Gene editing of MECP2R306C cells to generate isogenic controls rescued HDAC3-FOXO-mediated impairments in gene expression. Our data suggests that HDAC3 interaction with MeCP2 positively regulates a subset of neuronal genes through FOXO deacetylation, and disruption of HDAC3 contributes to cognitive and social impairment.
doi:10.1038/nn.4347
PMCID: PMC5083138  PMID: 27428650
2.  A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation 
Nature Communications  2016;7:13916.
Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats.
Development of a safe and efficacious vaccine for respiratory syncytial virus (RSV) has been challenging. Here the authors generate a live-attenuated RSV vaccine that shows increased thermostability and immunogenicity, and protects cotton rats from RSV challenge.
doi:10.1038/ncomms13916
PMCID: PMC5187593  PMID: 28000669
3.  Consolidated Bioprocessing for Butyric Acid Production from Rice Straw with Undefined Mixed Culture 
Lignocellulosic biomass is a renewable source with great potential for biofuels and bioproducts. However, the cost of cellulolytic enzymes limits the utilization of the low-cost bioresource. This study aimed to develop a consolidated bioprocessing without the need of supplementary cellulase for butyric acid production from lignocellulosic biomass. A stirred-tank reactor with a working volume of 21 L was constructed and operated in batch and semi-continuous fermentation modes with a cellulolytic butyrate-producing microbial community. The semi-continuous fermentation with intermittent discharging of the culture broth and replenishment with fresh medium achieved the highest butyric acid productivity of 2.69 g/(L· d). In semi-continuous operation mode, the butyric acid and total carboxylic acid concentrations of 16.2 and 28.9 g/L, respectively, were achieved. Over the 21-day fermentation period, their cumulative yields reached 1189 and 2048 g, respectively, corresponding to 41 and 74% of the maximum theoretical yields based on the amount of NaOH pretreated rice straw fed in. This study demonstrated that an undefined mixed culture-based consolidated bioprocessing for butyric acid production can completely eliminate the cost of supplementary cellulolytic enzymes.
doi:10.3389/fmicb.2016.01648
PMCID: PMC5076434  PMID: 27822203
butyric acid production; rice straw; consolidated bioprocessing; undefined mixed culture; carboxylate platform
4.  Molecular hydrogen decelerates rheumatoid arthritis progression through inhibition of oxidative stress 
Rheumatoid arthritis (RA) is a chronic inflammatory disease which results in progressive destruction of the joint. In this study, we examined if the hydrogen could inhibit inflammation in a mouse model of collagen-induced arthritis (CIA) via oxidative stress on RA-FLSs. Moreover, to identify the mechanisms of action, we evaluated the effect of hydrogen on RA-FLSs development and the expression of pro-inflammatory cytokines and signaling pathways. Based on our result, H2 enriched medium can increase super oxide dismutase (SOD) level following H2O2 treatment and decrease 8-hydroxy-2’-deoxyguanosine (8-OHdG) level. Since H2O2 treatment activates MAPK, NF-κB and TGF-β1 in cells, our study suggested that H2 could inhibit H2O2 activated MAPK and NF-κB activation as well as TGF-β1 expression in treated cells. Taken together, our data suggested that H2 can directly neutralize OH and ONOO- to reduce oxidative stress. Moreover, MAPK and NF-κB pathway also play roles in oxidative damage caused by H2O2 in RA-FLSs. H2 can provide protection to cells against inflammation, which may be related to inhibition of the activation of MAPK and NF-κB.
PMCID: PMC5095341  PMID: 27830032
Rheumatoid arthritis; hydrogen; SOD; NF-κB; TGF-β1
5.  The Rural-Urban Difference in BMI and Anemia among Children and Adolescents 
There is growing concern over the double burden of over- and under-nutrition in individuals, especially in children and adolescents, which could dwarf their growth and development. This study aims to explore the rural-urban difference in BMI and anemia among children and adolescents. A stratified cluster sampling technique was employed. Dietary data were collected through interviews, and anthropometric values were measured. There were 1534 children and adolescents who participated in this study, including 775 male and 759 female participants. The prevalence of obesity among children living in a city, township and rural area was 10.3%, 8.5% and 5.5%, and that among adolescents was 1.4%, 2.9% and 2.8%. The prevalence of anemia among children and living in a city, township and rural area was 4.3%, 2.5% and 4.5%, while that among adolescents was 6.1%, 3.7% and 11.3%, respectively, with significant difference (χ2 = 10.824, p = 0.004). The prevalence of being overweight, obesity and anemia was significant when comparing children with adolescents (χ2 = 37.861, p = 0.000; χ2 = 19.832, p = 0.000; χ2 = 8.611, p = 0.003). Findings of this study indicate the double burden of malnutrition in Zhejiang province, characterized by a high prevalence of being overweight, obesity and anemia among children and a high prevalence of anemia among adolescents living in townships.
doi:10.3390/ijerph13101020
PMCID: PMC5086759  PMID: 27763565
obesity; wasting; anemia; children; adolescent
6.  Body Mass Index and Mortality: A 10-Year Prospective Study in China 
Scientific Reports  2016;6:31609.
Although several studies have evaluated the role of body weight as a risk factor for mortality, most studies have been conducted in Western populations and the findings remain controversial. We performed a prospective study to examine the association between body mass index (BMI) and all-cause mortality in Yinzhou District, Ningbo, China. At baseline, 384,533 subjects were recruited through the Yinzhou Health Information System between 2004 and 2009. The final analysis was restricted to 372,793 participants (178,333 men and 194,460 women) aged 18 years and older. Cox proportional hazards models were used to estimate hazard ratios(HRs) and 95% confidence intervals(CIs). We found an increased risk of all-cause mortality among individuals with BMI levels <22.5–24.9, although several groups were not statistically significant—adjusted HRs for persons with BMIs of <15.0, 15.0–17.4, 17.5–19.9, and 20.0–22.4 were 1.61(95% CI: 1.17–2.23), 1.07(0.94–1.20), 1.04(0.98–1.10), 1.06(1.02–1.11), respectively. In the upper BMI range, subjects with BMIs of 25.0–34.9 had a reduced risk of all-cause mortality. Sensitivity analyses excluding smokers, those with prevalent chronic disease or those with less than four years of follow-up did not materially alter these results. Our findings provide evidence for an inverse association of BMI and mortality in this population.
doi:10.1038/srep31609
PMCID: PMC4992855  PMID: 27546611
7.  A hierarchical model for clustering m6A methylation peaks in MeRIP-seq data 
BMC Genomics  2016;17(Suppl 7):520.
Background
The recent advent of the state-of-art high throughput sequencing technology, known as Methylated RNA Immunoprecipitation combined with RNA sequencing (MeRIP-seq) revolutionizes the area of mRNA epigenetics and enables the biologists and biomedical researchers to have a global view of N6-Methyladenosine (m6A) on transcriptome. Yet there is a significant need for new computation tools for processing and analysing MeRIP-Seq data to gain a further insight into the function and m6A mRNA methylation.
Results
We developed a novel algorithm and an open source R package (http://compgenomics.utsa.edu/metcluster) for uncovering the potential types of m6A methylation by clustering the degree of m6A methylation peaks in MeRIP-Seq data. This algorithm utilizes a hierarchical graphical model to model the reads account variance and the underlying clusters of the methylation peaks. Rigorous statistical inference is performed to estimate the model parameter and detect the number of clusters. MeTCluster is evaluated on both simulated and real MeRIP-seq datasets and the results demonstrate its high accuracy in characterizing the clusters of methylation peaks. Our algorithm was applied to two different sets of real MeRIP-seq datasets and reveals a novel pattern that methylation peaks with less peak enrichment tend to clustered in the 5′ end of both in both mRNAs and lncRNAs, whereas those with higher peak enrichment are more likely to be distributed in CDS and towards the 3′end of mRNAs and lncRNAs. This result might suggest that m6A’s functions could be location specific.
Conclusions
In this paper, a novel hierarchical graphical model based algorithm was developed for clustering the enrichment of methylation peaks in MeRIP-seq data. MeTCluster is written in R and is publicly available.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2913-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-016-2913-x
PMCID: PMC5001242  PMID: 27556597
8.  G-1 exerts neuroprotective effects through G protein-coupled estrogen receptor 1 following spinal cord injury in mice 
Bioscience Reports  2016;36(4):e00373.
Spinal cord injury (SCI) always occurs accidently and leads to motor dysfunction because of biochemical and pathological events. Estrogen has been shown to be neuroprotective against SCI through estrogen receptors (ERs), but the underlying mechanisms have not been fully elucidated. In the present study, we investigated the role of a newly found membrane ER, G protein-coupled estrogen receptor 1 (GPR30 or GPER1), and discussed the feasibility of a GPR30 agonist as an estrogen replacement. Forty adult female C57BL/6J mice (10–12 weeks old) were divided randomly into vehicle, G-1, E2, G-1 + G-15 and E2 + G-15 groups. All mice were subjected to SCI using a crushing injury approach. The specific GPR30 agonist, G-1, mimicked the effects of E2 treatment by preventing SCI-induced apoptotic cell death and enhancing motor functional recovery after injury. GPR30 activation regulated phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK/extracellular signal-regulated kinase (ERK) signalling pathways, increased GPR30 and anti-apoptosis proteins Bcl-2 and brain derived neurotrophic factor (BDNF), but decreased the pro-apoptosis factor Bax and cleaved caspase-3. However, the neuroprotective effects of G-1 and E2 were blocked by the specific GPR30 antagonist, G-15. Thus, GPR30 rather than classic ERs is required to induce estrogenic neuroprotective effects. Given that estrogen replacement therapy may cause unexpected side effects, especially on the reproductive system, GPR30 agonists may represent a potential therapeutic approach for treating SCI.
doi:10.1042/BSR20160134
PMCID: PMC5006313  PMID: 27407175
apoptosis; GPR30; motor functional recovery; neuroprotection
9.  Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein 
Journal of Virology  2015;90(1):245-253.
ABSTRACT
Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection.
IMPORTANCE RSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought.
doi:10.1128/JVI.02140-15
PMCID: PMC4702574  PMID: 26468535
10.  A novel algorithm for calling mRNA m6A peaks by modeling biological variances in MeRIP-seq data 
Bioinformatics  2016;32(12):i378-i385.
Motivation: N6-methyl-adenosine (m6A) is the most prevalent mRNA methylation but precise prediction of its mRNA location is important for understanding its function. A recent sequencing technology, known as Methylated RNA Immunoprecipitation Sequencing technology (MeRIP-seq), has been developed for transcriptome-wide profiling of m6A. We previously developed a peak calling algorithm called exomePeak. However, exomePeak over-simplifies data characteristics and ignores the reads’ variances among replicates or reads dependency across a site region. To further improve the performance, new model is needed to address these important issues of MeRIP-seq data.
Results: We propose a novel, graphical model-based peak calling method, MeTPeak, for transcriptome-wide detection of m6A sites from MeRIP-seq data. MeTPeak explicitly models read count of an m6A site and introduces a hierarchical layer of Beta variables to capture the variances and a Hidden Markov model to characterize the reads dependency across a site. In addition, we developed a constrained Newton’s method and designed a log-barrier function to compute analytically intractable, positively constrained Beta parameters. We applied our algorithm to simulated and real biological datasets and demonstrated significant improvement in detection performance and robustness over exomePeak. Prediction results on publicly available MeRIP-seq datasets are also validated and shown to be able to recapitulate the known patterns of m6A, further validating the improved performance of MeTPeak.
Availability and implementation: The package ‘MeTPeak’ is implemented in R and C ++, and additional details are available at https://github.com/compgenomics/MeTPeak
Contact: yufei.huang@utsa.edu or xdchoi@gmail.com
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btw281
PMCID: PMC4908365  PMID: 27307641
11.  Biochemical Effect of Resistance Mutations against Synergistic Inhibitors of RSV RNA Polymerase 
PLoS ONE  2016;11(5):e0154097.
ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.
doi:10.1371/journal.pone.0154097
PMCID: PMC4862670  PMID: 27163448
12.  EGFR Interacts with the Fusion Protein of Respiratory Syncytial Virus Strain 2-20 and Mediates Infection and Mucin Expression 
PLoS Pathogens  2016;12(5):e1005622.
Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2–20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2–20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2–20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2–20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from “mucogenic” strains. RSV 2–20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.
Author Summary
Respiratory syncytial virus (RSV) is responsible for severe lower respiratory disease in infants and young children. Overabundant airway mucus contributes to airway obstruction in RSV bronchiolitis, and a better understanding of RSV pathogenesis may contribute to needed therapies and vaccines. We reported previously that RSV clinical isolate strain 2–20 induces more airway mucin expression in mice than prototypic RSV strains and that the 2–20 fusion (F) protein mediates mucin induction. Epidermal growth factor receptor (EGFR) has been shown to play a role in lung mucin expression. We identified a functional interaction between 2–20 F and EGFR, in that 2–20 F expression activated EGFR and, reciprocally, EGFR expression increased 2–20 F fusion activity. RSV F and EGFR co-localized in infected cells. EGFR co-immunoprecipitated with RSV F protein from various RSV strains, and the strength of this in vitro interaction correlated with strain-specific airway pathogenicity in mice. EGFR inhibition abrogated 2–20 F-mediated infection in vitro and mucin expression induction in vivo. These data identify EGFR as a novel strain-specific co-factor of RSV infection and suggest EGFR may be a target for ameliorating RSV disease.
doi:10.1371/journal.ppat.1005622
PMCID: PMC4859522  PMID: 27152417
13.  Molecular characterization and antimicrobial susceptibility of Acinetobacter baumannii isolates obtained from two hospital outbreaks in Los Angeles County, California, USA 
BMC Infectious Diseases  2016;16:194.
Background
Antibiotic resistant strains of Acinetobacter baumannii have been responsible for an increasing number of nosocomial infections including bacteremia and ventilator-associated pneumonia. In this study, we analyzed 38 isolates of A. baumannii obtained from two hospital outbreaks in Los Angeles County for the molecular epidemiology, antimicrobial susceptibility and resistance determinants.
Methods
Pulsed field gel electrophoresis, tri-locus multiplex PCR and multi-locus sequence typing (Pasteur scheme) were used to examine clonal relationships of the outbreak isolates. Broth microdilution method was used to determine antimicrobial susceptibility of these isolates. PCR and subsequent DNA sequencing were employed to characterize antibiotic resistance genetic determinants.
Results
Trilocus multiplex PCR showed these isolates belong to Global Clones I and II, which were confirmed to ST1 and ST2, respectively, by multi-locus sequence typing. Pulsed field gel electrophoresis analysis identified two clonal clusters, one with 20 isolates (Global Clone I) and the other with nine (Global Clone II), which dominated the two outbreaks. Antimicrobial susceptibility testing using 14 antibiotics indicated that all isolates were resistant to antibiotics belonging to four or more categories of antimicrobial agents. In particular, over three fourth of 38 isolates were found to be resistant to both imipenem and meropenem. Additionally, all isolates were found to be resistant to piperacillin, four cephalosporin antibiotics, ciprofloxacin and levofloxacin. Resistance phenotypes of these strains to fluoroquinolones were correlated with point mutations in gyrA and parC genes that render reduced affinity to target proteins. ISAba1 was detected immediately upstream of the blaOXA–23 gene present in those isolates that were found to be resistant to both carbapenems. Class 1 integron-associated resistance gene cassettes appear to contribute to resistance to aminoglycoside antibiotics.
Conclusion
The two outbreaks were found to be dominated by two clonal clusters of A. baumannii belonging to MLST ST1 and ST2. All isolates were resistant to antibiotics of at least four categories of antimicrobial agents, and their antimicrobial susceptibility profiles correlate well with genetic determinants. The results of this study will facilitate our understanding of the molecular epidemiology, antimicrobial susceptibility and mechanisms of resistance of A. baumannii obtained from Los Angeles hospitals.
doi:10.1186/s12879-016-1526-y
PMCID: PMC4857389  PMID: 27146090
Acinetobacter baumannii; Nosocomial outbreak; Epidemiology; Antimicrobial susceptibility; Mechanism of resistance
14.  CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells 
The Journal of General Virology  2015;96(Pt 9):2543-2556.
Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.
doi:10.1099/vir.0.000218
PMCID: PMC4635495  PMID: 26297201
15.  Guitar: An R/Bioconductor Package for Gene Annotation Guided Transcriptomic Analysis of RNA-Related Genomic Features 
BioMed Research International  2016;2016:8367534.
Biological features, such as genes and transcription factor binding sites, are often denoted with genome-based coordinates as the genomic features. While genome-based representation is usually very effective in correlating various biological features, it can be tedious to examine the relationship between RNA-related genomic features and the landmarks of RNA transcripts with existing tools due to the difficulty in the conversion between genome-based coordinates and RNA-based coordinates. We developed here an open source Guitar R/Bioconductor package for sketching the transcriptomic view of RNA-related biological features represented by genome based coordinates. Internally, Guitar package extracts the standardized RNA coordinates with respect to the landmarks of RNA transcripts, with which hundreds of millions of RNA-related genomic features can then be efficiently analyzed within minutes. We demonstrated the usage of Guitar package in analyzing posttranscriptional RNA modifications (5-methylcytosine and N6-methyladenosine) derived from high-throughput sequencing approaches (MeRIP-Seq and RNA BS-Seq) and show that RNA 5-methylcytosine (m5C) is enriched in 5′UTR. The newly developed Guitar R/Bioconductor package achieves stable performance on the data tested and revealed novel biological insights. It will effectively facilitate the analysis of RNA methylation data and other RNA-related biological features in the future.
doi:10.1155/2016/8367534
PMCID: PMC4864564  PMID: 27239475
16.  Decomposition of RNA methylome reveals co-methylation patterns induced by latent enzymatic regulators of epitranscriptome 
Molecular bioSystems  2014;11(1):262-274.
Biochemical modifications to mRNA, especially N6-methyladenosine (m6A) and 5-methylcytosine (m5C), are recently shown to be associated with crucial biological functions. Despite the intriguing advancements, little is known so far about the dynamic landscape of RNA methylome across different cell types and how the epitranscriptome is regulated at system level by enzymes, i.e., RNA methyltransferases and demethylases. To investigate this issue, a meta-analysis of m6A MeRIP-Seq datasets from 10 different experimental conditions (cell type/tissue or treatment) are collected, and the combinatorial epitranscriptome, which consists of 42758 m6A sites, is extracted and divided into 3 clusters, in which the methylation sites are likely to be hyper- or hypo-methylated simultaneously (or co-methylated), indicating the sharing of a common methylation regulator. Four different clustering approaches are used, including, K-means, hierarchical clustering (HC), Bayesian factor regression model (BFRM) and nonnegative matrix factorization (NMF) to unveil the co-methylation patterns.
To validate whether the patterns are corresponding to enzymatic regulators, i.e., RNA methyltransferases or demethylases, the target sites of a known m6A regulator, fat mass and obesity-associated protein (FTO), are identified from an independent mouse MeRIP-Seq dataset and lifted to human. Our study shows that, 3 out of the 4 clustering approaches used can successfully identify a group of methylation sites overlapping with FTO target sites at significance level 0.05 (after multiple hypothesis adjustment), among which, the result of NMF is the most significant (p-value 2.81e-06). We defined a new approach evaluating the consistency between two clustering results and shows that clustering results of different methods are highly correlated indicating strongly the existence of co-methylation patterns. Consistent with recent studies, a number of cancer and neuronal disease-related bimolecular functions are enriched in the identified clusters, which are biological functions can be regulated at epitranscriptional level, indicating the pharmaceutical prospect of RNA N6-methyladenosine-related studies.
This result successfully reveals the linkage between the global RNA co-methylation patterns embedded in the epitranscriptomic data under multiple experimental conditions and the latent enzymatic regulators, suggesting a promising direction towards more comprehensive understanding of the epitranscriptome.
doi:10.1039/c4mb00604f
PMCID: PMC4253022  PMID: 25370990
17.  Recent Progress in Light-Triggered Nanotheranostics for Cancer Treatment 
Theranostics  2016;6(7):948-968.
Treatments of high specificity are desirable for cancer therapy. Light-triggered nanotheranostics (LTN) mediated cancer therapy could be one such treatment, as they make it possible to visualize and treat the tumor specifically in a light-controlled manner with a single injection. Because of their great potential in cancer therapy, many novel and powerful LTNs have been developed, and are mainly prepared from photosensitizers (PSs) ranging from small organic dyes such as porphyrin- and cyanine-based dyes, semiconducting polymers, to inorganic nanomaterials such as gold nanoparticles, transition metal chalcogenides, carbon nanotubes and graphene. Using LTNs and localized irradiation in combination, complete tumor ablation could be achieved in tumor-bearing animal models without causing significant toxicity. Given their great advances and promising future, we herein review LTNs that have been tested in vivo with a highlight on progress that has been made in the past a couple of years. The current challenges faced by these LTNs are also briefly discussed.
doi:10.7150/thno.15217
PMCID: PMC4876621  PMID: 27217830
Nanoparticle; theranostics; imaging; photodynamic; photothermal.
18.  lncRScan-SVM: A Tool for Predicting Long Non-Coding RNAs Using Support Vector Machine 
PLoS ONE  2015;10(10):e0139654.
Functional long non-coding RNAs (lncRNAs) have been bringing novel insight into biological study, however it is still not trivial to accurately distinguish the lncRNA transcripts (LNCTs) from the protein coding ones (PCTs). As various information and data about lncRNAs are preserved by previous studies, it is appealing to develop novel methods to identify the lncRNAs more accurately. Our method lncRScan-SVM aims at classifying PCTs and LNCTs using support vector machine (SVM). The gold-standard datasets for lncRScan-SVM model training, lncRNA prediction and method comparison were constructed according to the GENCODE gene annotations of human and mouse respectively. By integrating features derived from gene structure, transcript sequence, potential codon sequence and conservation, lncRScan-SVM outperforms other approaches, which is evaluated by several criteria such as sensitivity, specificity, accuracy, Matthews correlation coefficient (MCC) and area under curve (AUC). In addition, several known human lncRNA datasets were assessed using lncRScan-SVM. LncRScan-SVM is an efficient tool for predicting the lncRNAs, and it is quite useful for current lncRNA study.
doi:10.1371/journal.pone.0139654
PMCID: PMC4593643  PMID: 26437338
19.  A protocol for RNA methylation differential analysis with MeRIP-Seq data and exomePeak R/Bioconductor package 
Methods (San Diego, Calif.)  2014;69(3):274-281.
Despite the prevalent studies of DNA/Chromatin related epigenetics, such as, histone modifications and DNA methylation, RNA epigenetics has not drawn deserved attention until a new affinity-based sequencing approach MeRIP-Seq was developed and applied to survey the global mRNA N6-methyladenosine (m6A) in mammalian cells. As a marriage of ChIP-Seq and RNA-Seq, MeRIP-Seq has the potential to study the transcriptome-wide distribution of various post-transcriptional RNA modifications.
We have previously developed an R/Bioconductor package ‘exomePeak’ for detecting RNA methylation sites under a specific experimental condition or the identifying the differential RNA methylation sites in a case control study from MeRIP-Seq data. Compared with other relatively well studied data types such as ChIP-Seq and RNA-Seq, the study of MeRIP-Seq data is still at very early stage, and existing protocols are not optimized for dealing with the intrinsic characteristic of MeRIP-Seq data. We therein provide here a detailed and easy-to-use protocol of using exomePeak R/Bioconductor package along with other software programs for analysis of MeRIP-Seq data, which covers raw reads alignment, RNA methylation site detection, motif discovery, differential RNA methylation analysis, and functional analysis. Particularly, the rationales behind each processing step as well as the specific method used, the best practice, and possible alternative strategies are briefly discussed.
doi:10.1016/j.ymeth.2014.06.008
PMCID: PMC4194139  PMID: 24979058
20.  Tet1 is critical for neuronal activity-regulated gene expression and memory extinction 
Neuron  2013;79(6):1109-1122.
SUMMARY
The ten-eleven translocation (Tet) family of methylcytosine dioxygenases catalyze oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylacytosine (5hmC) and promote DNA demethylation. Despite the abundance of 5hmC and Tet proteins in the brain, little is known about the functions of the neuronal Tet enzymes. Here, we analyzed Tet1 knockout mice (Tet1KO) and found downregulation of multiple neuronal activity-regulated genes, including Npas4, c-Fos, and Arc. Furthermore, Tet1KO animals exhibited abnormal hippocampal long-term depression and impaired memory extinction. Analysis of the key regulatory gene, Npas4, indicated that its promoter region, containing multiple CpG dinucleotides, is hypermethylated in both naive Tet1KO mice and following extinction training. Such hypermethylation may account for the diminished expression of Npas4 itself and its downstream targets, impairing transcriptional programs underlying cognitive processes. In summary, we show that neuronal Tet1 regulates normal DNA methylation levels, expression of activity-regulated genes, synaptic plasticity, and memory extinction.
doi:10.1016/j.neuron.2013.08.003
PMCID: PMC4543319  PMID: 24050401
21.  Spatially Enhanced Differential RNA Methylation Analysis from Affinity-Based Sequencing Data with Hidden Markov Model 
BioMed Research International  2015;2015:852070.
With the development of new sequencing technology, the entire N6-methyl-adenosine (m6A) RNA methylome can now be unbiased profiled with methylated RNA immune-precipitation sequencing technique (MeRIP-Seq), making it possible to detect differential methylation states of RNA between two conditions, for example, between normal and cancerous tissue. However, as an affinity-based method, MeRIP-Seq has yet provided base-pair resolution; that is, a single methylation site determined from MeRIP-Seq data can in practice contain multiple RNA methylation residuals, some of which can be regulated by different enzymes and thus differentially methylated between two conditions. Since existing peak-based methods could not effectively differentiate multiple methylation residuals located within a single methylation site, we propose a hidden Markov model (HMM) based approach to address this issue. Specifically, the detected RNA methylation site is further divided into multiple adjacent small bins and then scanned with higher resolution using a hidden Markov model to model the dependency between spatially adjacent bins for improved accuracy. We tested the proposed algorithm on both simulated data and real data. Result suggests that the proposed algorithm clearly outperforms existing peak-based approach on simulated systems and detects differential methylation regions with higher statistical significance on real dataset.
doi:10.1155/2015/852070
PMCID: PMC4537718  PMID: 26301253
22.  Identification of miR-194-5p as a potential biomarker for postmenopausal osteoporosis 
PeerJ  2015;3:e971.
The incidence of osteoporosis is high in postmenopausal women due to altered estrogen levels and continuous calcium loss that occurs with aging. Recent studies have shown that microRNAs (miRNAs) are involved in the development of osteoporosis. These miRNAs may be used as potential biomarkers to identify women at a high risk for developing the disease. In this study, whole blood samples were collected from 48 postmenopausal Chinese women with osteopenia or osteoporosis and pooled into six groups according to individual T-scores. A miRNA microarray analysis was performed on pooled blood samples to identify potential miRNA biomarkers for postmenopausal osteoporosis. Five miRNAs (miR-130b-3p, -151a-3p, -151b, -194-5p, and -590-5p) were identified in the microarray analysis. These dysregulated miRNAs were subjected to a pathway analysis investigating whether they were involved in regulating osteoporosis-related pathways. Among them, only miR-194-5p was enriched in multiple osteoporosis-related pathways. Enhanced miR-194-5p expression in women with osteoporosis was confirmed by quantitative reverse transcription–polymerase chain reaction analysis. For external validation, a significant correlation between the expression of miR-194-5p and T-scores was found in an independent patient collection comprised of 24 postmenopausal women with normal bone mineral density, 30 postmenopausal women with osteopenia, and 32 postmenopausal women with osteoporosis (p < 0.05). Taken together, the present findings suggest that miR-194-5p may be a viable miRNA biomarker for postmenopausal osteoporosis.
doi:10.7717/peerj.971
PMCID: PMC4451039  PMID: 26038726
Biomarker; Postmenopausal osteoporosis; MicroRNA; miR-194
23.  Linoleic acid induces red blood cells and hemoglobin damage via oxidative mechanism 
Hidden blood loss typically occurs following total hip arthroplasty (THA) and total knee arthroplasty (TKA) and is thought to be related to free fatty acid (FFA). To study the effect of linoleic acid on red blood cells and to examine the pathogenesis of hidden blood loss in vivo, we generated an animal model by injecting linoleic acid into the tail veins of rats. We collected blood samples and determined red blood cell count (RBC) and levels of hemoglobin (Hb), as well as the oxidation and reducing agents in the blood, including glutathione peroxidase (GSH-PX), total superoxide dismutase (T-SOD), hydrogen peroxide (H2O2), and ferryl hemoglobin (Fe4+ = O2-), which is generated by the oxidation of Hb. Hidden blood loss occurred when linoleic acid was administered at a concentration of 60 mmol/L; RBC and Hb levels were significantly reduced by 24 h post-injection. This was followed by erythrocyte deformation, reduced activity of GSH-PX and T-SOD, and decreased levels of H2O2. This was accompanied by an increase in ferryl species, which likely contributes to oxidative stress in vivo. Our findings suggest that linoleic acid enhances acute red blood cell injury. Hb and RBC began to increase by 72 h, potentially resulting from linoleic acid metabolism. Thus, elevated levels of linoleic acid in the blood cause acute oxidative damage to red blood cells, eventually leading to partial acute anemia. These findings highlight the pathophysiology underlying hidden blood loss.
PMCID: PMC4503070  PMID: 26191198
Linoleic acid; free fatty acid; erythrocyte damage; oxidative stress; hidden blood loss
24.  HEPeak: an HMM-based exome peak-finding package for RNA epigenome sequencing data 
BMC Genomics  2015;16(Suppl 4):S2.
Background
Methylated RNA Immunoprecipatation combined with RNA sequencing (MeRIP-seq) is revolutionizing the de novo study of RNA epigenomics at a higher resolution. However, this new technology poses unique bioinformatics problems that call for novel and sophisticated statistical computational solutions, aiming at identifying and characterizing transcriptome-wide methyltranscriptome.
Results
We developed HEP, a Hidden Markov Model (HMM)-based Exome Peak-finding algorithm for predicting transcriptome methylation sites using MeRIP-seq data. In contrast to exomePeak, our previously developed MeRIP-seq peak calling algorithm, HEPeak models the correlation between continuous bins in an m6A peak region and it is a model-based approach, which admits rigorous statistical inference. HEPeak was evaluated on a simulated MeRIP-seq dataset and achieved higher sensitivity and specificity than exomePeak. HEPeak was also applied to real MeRIP-seq datasets from human HEK293T cell line and mouse midbrain cells and was shown to be able to recapitulate known m6A distribution in transcripts and identify novel m6A sites in long non-coding RNAs.
Conclusions
In this paper, a novel HMM-based peak calling algorithm, HEPeak, was developed for peak calling for MeRIP-seq data. HEPeak is written in R and is publicly available.
doi:10.1186/1471-2164-16-S4-S2
PMCID: PMC4416174  PMID: 25917296
25.  MTHFR gene C677T polymorphism and type 2 diabetic nephropathy in Asian populations: a meta-analysis 
Background: Many studies have suggested a correlation between the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene and diabetic nephropathy (DN), but their results are inconclusive. Methods: To confirm this correlation, we performed a meta-analysis of 15 studies. The dichotomous data are presented as odds ratios (OR) with 95% confidence intervals (CI). Results: The results of this study suggested that the MTHFR 677 T allele was more likely to increase the risk of DN in Asian (OR = 1.466, 95% CI = 1.143-1.880, P = 0.003), West Asian (OR = 1.750, 95% CI = 1.150-2.664, P = 0.009), and Chinese populations (OR = 2.162, 95% CI = 1.719-2.719, P = 0.001), but not in East Asian or Japanese populations. The carriers of the MTHFR 677 T allele were associated with progression of DN in the “5-10 year duration” group, but not in the “> 10 year duration” group (OR = 2.187, 95% CI = 1.787-2.677, P = 0.001). Conclusion: Development of DN is associated with MTHFR C677T polymorphisms in Asian populations, especially in early type 2 diabetes.
PMCID: PMC4443095  PMID: 26064261
Diabetic nephropathy; MTHFR polymorphisms; type 2 diabetes

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