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1.  Deep RNA sequencing reveals a high frequency of alternative splicing events in the fungus Trichoderma longibrachiatum 
BMC Genomics  2015;16(1):54.
Alternative splicing is crucial for proteome diversity and functional complexity in higher organisms. However, the alternative splicing landscape in fungi is still elusive.
The transcriptome of the filamentous fungus Trichoderma longibrachiatum was deep sequenced using Illumina Solexa technology. A total of 14305 splice junctions were discovered. Analyses of alternative splicing events revealed that the number of all alternative splicing events (10034), intron retentions (IR, 9369), alternative 5’ splice sites (A5SS, 167), and alternative 3’ splice sites (A3SS, 302) is 7.3, 7.4, 5.1, and 5.9-fold higher, respectively, than those observed in the fungus Aspergillus oryzae using Illumina Solexa technology. This unexpectedly high ratio of alternative splicing suggests that alternative splicing is important to the transcriptome diversity of T. longibrachiatum. Alternatively spliced introns had longer lengths, higher GC contents, and lower splice site scores than constitutive introns. Further analysis demonstrated that the isoform relative frequencies were correlated with the splice site scores of the isoforms. Moreover, comparative transcriptomics determined that most enzymes related to glycolysis and the citrate cycle and glyoxylate cycle as well as a few carbohydrate-active enzymes are transcriptionally regulated.
This study, consisting of a comprehensive analysis of the alternative splicing landscape in the filamentous fungus T. longibrachiatum, revealed an unexpectedly high ratio of alternative splicing events and provided new insights into transcriptome diversity in fungi.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1251-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4324775  PMID: 25652134
Alternative splicing; Fungi; RNA-Seq; Intron retention; Transcriptome; Trichoderma longibrachiatum
2.  Overweight trajectories and psychosocial adjustment among adolescents 
Preventive medicine  2013;57(6):837-843.
Few studies have investigated overweight trajectories and psychosocial adjustment among adolescents. We conducted analyses with data from the multisite Study of Early Child Care and Youth Development (SECCYD).
Sample included 1,350 youth born in 1991. Data consisted of repeated measures of weight, height, and multiple subscales of internalizing and externalizing behavioral problems measured by the Child Behavior Checklist (CBCL) from age nine to age 15.
Three trajectory patterns were identified: never/rarely overweight/obese (59.5%), late start/light overweight/obese (12.1%), and chronically/heavy overweight/obese (28.4%). Youths with chronically/heavy overweight/obese trajectory pattern had significantly higher scores of internalizing problems over time, as well as syndrome subscales of somatic complaints, social problems and social withdrawal over time than youths with the never/rare overweight/obese trajectory pattern. There was no significant difference in either broad-band behavioral problems or narrow-band syndrome subscales between youths with the never/rare overweight/obese trajectory pattern and those with the late start/light overweight/obesity trajectory pattern.
Study findings may advance knowledge on the distinct developmental trajectory patterns of overweight youth and their linkages to the psychosocial adjustment during the period of pubertal transition. The results highlight the need for future prevention research to improve the physical development and mental well-being of adolescents.
PMCID: PMC3903344  PMID: 24075819
Overweight Trajectories; Psychosocial Adjustment; Adolescents
3.  A Proposed Genus Boundary for the Prokaryotes Based on Genomic Insights 
Journal of Bacteriology  2014;196(12):2210-2215.
Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values. A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%. Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation.
PMCID: PMC4054180  PMID: 24706738
4.  Differentiation of Retinal Ganglion Cells and Photoreceptor Precursors from Mouse Induced Pluripotent Stem Cells Carrying an Atoh7/Math5 Lineage Reporter 
PLoS ONE  2014;9(11):e112175.
The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs). The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.
PMCID: PMC4234374  PMID: 25401462
5.  Relative income inequality and selected health outcomes in urban Chinese youth 
Self reported cross-sectional data gathered in 2002 from 12,449 middle and high school students from seven major cities in China were examined to explore the association of self-perceived relative income inequality (SPRII) with general health status, depression, stress, and cigarette smoking. Two types of self-perceived relative income were evaluated: household income relative to peers (SPRII-S) and relative to their own past (SPRII-P). SPRII-S and SPRII-P were coded as three-level categorical variables: lower, equal, and higher. As hypothesized, the youth in the “Lower” SPRII-S or SPRII-P groups reported the worst general health and the highest levels of depression and stress; the youth in the “Higher” groups reported the best general health. Unexpectedly, the youth in the “Higher” groups did not report the lowest levels of depression and stress, and the relationship between SPRII and cigarette smoking was even less straightforward. The expected positive relationship between SPRII and the general health status is consistent with previous research, but the relationships between SPRII and depression, stress, and cigarette smoking behavior are not. Further studies are needed to elucidate the complex associations between SPRII and health outcomes in rapidly transforming economies such as China.
PMCID: PMC4184428  PMID: 22137733
Income inequality; Relative deprivation; Affluence; Health behavior; Youth; Mental health; China; Self-reported health
6.  Developmental Trajectories of Cigarette Use and Associations With Multilayered Risk Factors Among Chinese Adolescents 
Nicotine & Tobacco Research  2013;15(10):1673-1681.
We aimed to identify developmental trajectories of cigarette use and risk factors associated with the distinct developmental courses of smoking in Chinese early adolescents from age 12 to 16 years.
Analysis was conducted with secondary data from a longitudinal, prospective cohort of 3,521 Chinese adolescents randomly selected from 4 rural and 7 urban middle schools in Wuhan, China. A group-based growth mixture modeling approach was adopted to identify developmental trajectories of cigarette use. Multilayered intrapersonal (e.g., attitudes toward smoking) and interpersonal (e.g., parental smoking and perceived parental disapproval of smoking) risk factors selected from an ecological perspective were prospectively linked to the identified patterns of smoking trajectory.
Three trajectory patterns were identified from the whole cohort: nonsmokers (48.7%), stable light/occasional smokers (48.6%), and accelerating smokers (2.7%). After adjustments for gender, urban residence, and family socioeconomic status, adolescents with higher levels of problems in parent–child relationships and family disharmony, higher perceived norms of peer smoking, higher proportion of good friend smoking, having more troubles with teachers, poorer academic performance, and reporting more frequent depressive symptoms were significantly more likely to be in the trajectory group of either stable light/occasional smokers or accelerating smokers than in the group of nonsmokers. The probability of being in the accelerating smoking trajectory group was positively and significantly related to parental smoking and lack of school bonding.
Study findings help to advance knowledge of the distinct developmental courses of smoking behavior and their associations with multilayered risk factors among Chinese early adolescents.
PMCID: PMC3768331  PMID: 23525597
7.  HBx Inhibits CYP2E1 Gene Expression via Downregulating HNF4α in Human Hepatoma Cells 
PLoS ONE  2014;9(9):e107913.
CYP2E1, one of the cytochrome P450 mixed-function oxidases located predominantly in liver, plays a key role in metabolism of xenobiotics including ethanol and procarcinogens. Recently, down-expression of CYP2E1 was found in hepatocellular carcinoma (HCC) with the majority to be chronic hepatitis B virus (HBV) carriers. In this study, we tested a hypothesis that HBx may inhibit CYP2E1 gene expression via hepatocyte nuclear factor 4α (HNF4α). By enforced HBx gene expression in cultured HepG2 cells, we determined the effect of HBx on CYP2E1 mRNA and protein expression. With a bioinformatics analysis, we found a consensus HNF-4α binding sequence located on −318 to −294 bp upstream of human CYP2E1 promoter. Using reporter gene assay and site-directed mutagenesis, we have shown that mutation of this site dramatically decreased CYP2E1 promoter activity. By silencing endogenous HNF-4α, we have further validated knockdown of HNF-4α significantly decreased CYP2E1expression. Ectopic overexpression of HBx in HepG2 cells inhibits HNF-4α expression, and HNF-4α levels were inversely correlated with viral proteins both in HBV-infected HepG2215 cells and as well as HBV positive HCC liver tissues. Moreover, the HBx-induced CYP2E1 reduction could be rescued by ectopic supplement of HNF4α protein expression. Furthermore, human hepatoma cells C34, which do not express CYP2E1, shows enhanced cell growth rate compared to E47, which constitutively expresses CYP2E1. In addition, the significantly altered liver proteins in CYP2E1 knockout mice were detected with proteomics analysis. Together, HBx inhibits human CYP2E1 gene expression via downregulating HNF4α which contributes to promotion of human hepatoma cell growth. The elucidation of a HBx-HNF4α-CYP2E1 pathway provides novel insight into the molecular mechanism underlining chronic HBV infection associated hepatocarcinogenesis.
PMCID: PMC4169590  PMID: 25238230
8.  Inter-hospital transfers and outcomes of critically ill patients with severe acute kidney injury: a multicenter cohort study 
Critical Care  2014;18(5):513.
Patients with severe acute kidney injury (AKI) who are hospitalized at centers that do not provide renal replacement therapy (RRT) are frequently subjected to inter-hospital transfer for the provision of RRT. It is unclear whether such transfers are associated with worse patient outcomes as compared with the receipt of initial care in a center that provides RRT. This study examined the relationship between inter-hospital transfer and 30-day mortality among critically ill patients with AKI who received RRT.
We conducted a retrospective cohort study of all critically ill patients who commenced RRT for AKI at two academic hospitals in Toronto, Canada. The exposure of interest was inter-hospital transfer for the administration of RRT. We evaluated the relationship between transfer status and 30-day mortality (primary outcome) and RRT dependence at 30 days following RRT initiation (secondary outcome), by using multivariate logistic regression with adjustment for patient demographics, clinical factors, biochemical indices, and severity of illness.
Of 370 patients who underwent RRT for AKI, 82 (22.2%) were transferred for this purpose from another hospital. Compared with non-transferred patients who started RRT, transferred patients were younger (61 ± 15 versus 65 ± 15 years, P = 0.03) and had a higher serum creatinine concentration at RRT initiation (474 ± 295 versus 365 ± 169 μmol/L, P = 0.002). Inter-hospital transfer was not associated with mortality (adjusted odds ratio 0.61, 95% confidence interval 0.33 to 1.12) or RRT-dependence (adjusted odds ratio 1.64, 95% confidence interval 0.70 to 3.81) at 30 days.
Within the limitations of this observational study and the potential for residual confounding, inter-hospital transfer of critically ill patients with AKI was not associated with a higher risk of death or dialysis dependence 30 days after the initiation of acute RRT.
Electronic supplementary material
The online version of this article (doi:10.1186/s13054-014-0513-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4189586  PMID: 25228166
9.  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
10.  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
11.  Psychiatrists’ attitudes toward the procedure of involuntary admission to mental hospitals in China 
In order to protect the rights of the mentally ill, legislation on the standards and procedures of compulsory detention has been made at the local and national level in China.
This study aims to examine psychiatrists’ attitude to seeking involuntary admission in China mainland.
Three hundred and fourteen qualified members of Chinese Psychiatrist Association (CPA) were surveyed using a questionnaire to assess their attitudes about the procedure of involuntary admission to mental hospitals. Data were analyzed using chi-squares and logistic regression.
Some psychiatrists in CPA had several arbitrary attitudes in the process of admission. Female, age under 35, low education level and low position in institution are associated with stricter attitudes in the procedure of involuntary admission. Areas with mental health legislation showed significant positive relations with stricter attitudes.
Every effort needs to be made to minimize these arbitrary attitudes to prevent negative outcomes. Protecting the rights of people diagnosed with mental illness still has a long way to go.
PMCID: PMC4108612  PMID: 21807812
Attitude; Involuntary admission; Informed consent; Proxy consent; Legislation
12.  Epidermal Growth Factor-Like Domain-Containing Protein 7 (EGFL7) Enhances EGF Receptor−AKT Signaling, Epithelial−Mesenchymal Transition, and Metastasis of Gastric Cancer Cells 
PLoS ONE  2014;9(6):e99922.
Epidermal growth factor-like domain-containing protein 7 (EGFL7) is upregulated in human epithelial tumors and so is a potential biomarker for malignancy. Indeed, previous studies have shown that high EGFL7 expression promotes infiltration and metastasis of gastric carcinoma. The epithelial–mesenchymal transition (EMT) initiates the metastatic cascade and endows cancer cells with invasive and migratory capacity; however, it is not known if EGFL7 promotes metastasis by triggering EMT. We found that EGFL7 was overexpressed in multiple human gastric cancer (GC) cell lines and that overexpression promoted cell invasion and migration as revealed by scratch wound and transwell migration assays. Conversely, shRNA-mediated EGFL7 knockdown reduced invasion and migration. Furthermore, EGFL7-overexpressing cells grew into larger tumors and were more likely to metastasize to the liver compared to underexpressing CG cells following subcutaneous injection in mice. EGFL7 overexpression protected GC cell lines against anoikis, providing a plausible mechanism for this enhanced metastatic capacity. In excised human gastric tumors, expression of EGFL7 was positively correlated with expression levels of the mesenchymal marker vimentin and the EMT-associated transcription repressor Snail, and negatively correlated with expression of the epithelial cell marker E-cadherin. In GC cell lines, EGFL7 knockdown reversed morphological signs of EMT and decreased both vimentin and Snail expression. In addition, EGFL7 overexpression promoted EGF receptor (EGFR) and protein kinase B (AKT) phospho-activation, effects markedly suppressed by the EGFR tyrosine kinase inhibitor AG1478. Moreover, AG1478 also reduced the elevated invasive and migratory capacity of GC cell lines overexpressing EGFL7. Collectively, these results strongly suggest that EGFL7 promotes metastasis by activating EMT through an EGFR−AKT−Snail signaling pathway. Disruption of EGFL7−EGFR−AKT−Snail signaling may a promising therapeutic strategy for gastric cancer.
PMCID: PMC4063792  PMID: 24945379
13.  Sling Exercise for Chronic Low Back Pain: A Systematic Review and Meta-Analysis 
PLoS ONE  2014;9(6):e99307.
Trials on sling exercise (SE), commonly performed to manage chronic low back pain (LBP), yield conflicting results. This study aimed to review the effects of SE on chronic LBP.
The randomized controlled trials comparing SE with other treatments or no treatment, published up to August 2013, were identified by electronic searches. Primary outcomes were pain, function, and return to work. The weighted mean difference (WMD) and 95% confidence interval (CI) were calculated, using a random-effects model.
Risk of bias was rated as high in 9 included trials, where some important quality components such as blinding were absent and sample sizes were generally small. We found no clinically relevant differences in pain or function between SE and other forms of exercise, traditional Chinese medical therapy, or in addition to acupuncture. Based on two trials, SE was more effective than thermomagnetic therapy at reducing pain (short-term: WMD –13.90, 95% CI –22.19 to –5.62; long-term: WMD –26.20, 95% CI –31.32 to –21.08) and improving function (short-term: WMD –10.54, 95% CI –14.32 to –6.75; long-term: WMD –25.75, 95% CI –30.79 to –20.71). In one trial we found statistically significant differences between SE and physical agents combined with drug therapy (meloxicam combined with eperisone hydrochloride) but of borderline clinical relevance for pain (short-term: WMD –15.00, 95% CI –19.64 to −10.36) and function (short-term: WMD −10.00; 95% CI −13.70 to −6.30). There was substantial heterogeneity among the two trials comparing SE and thermomagnetic therapy; both these trials and the trial comparing SE with physical agents combined with drug therapy had serious methodological limitations.
Based on limited evidence from 2 trials, SE was more effective for LBP than thermomagnetic therapy. Clinically relevant differences in effects between SE and other forms of exercise, physical agents combined with drug therapy, traditional Chinese medical therapy, or in addition to acupuncture could not be found. More high-quality randomized trials on the topic are warranted.
PMCID: PMC4053356  PMID: 24919119
14.  Identification of IFN-γ-producing innate B cells 
Cell Research  2013;24(2):161-176.
Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be further investigated. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus and Toll-like receptor ligands, we identified an inducible CD11ahiFcγRIIIhi B cell subpopulation that is significantly expanded and produces high levels of IFN-γ during the early stage of the immune response. This subpopulation of B cells can promote macrophage activation via generating IFN-γ, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-γ-producing innate B cells. Dendritic cells were essential for the inducible generation of these innate B cells from the follicular B cells via CD40L-CD40 ligation. Increased Bruton's tyrosine kinase activation was found to be responsible for the increased activation of non-canonical NF-κB pathway in these innate B cells after CD40 ligation, with the consequent induction of additional IFN-γ production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.
PMCID: PMC3915900  PMID: 24296781
B cells; IFN-γ; innate response; dendritic cells; CD40
15.  Comparative Genomics Provide Insights into Evolution of Trichoderma Nutrition Style 
Genome Biology and Evolution  2014;6(2):379-390.
Saprotrophy on plant biomass is a recently developed nutrition strategy for Trichoderma. However, the physiology and evolution of this new nutrition strategy is still elusive. We report the deep sequencing and analysis of the genome of Trichoderma longibrachiatum, an efficient cellulase producer. The 31.7-Mb genome, smallest among the sequenced Trichoderma species, encodes fewer nutrition-related genes than saprotrophic T. reesei (Tr), including glycoside hydrolases and nonribosomal peptide synthetase–polyketide synthase. Homology and phylogenetic analyses suggest that a large number of nutrition-related genes, including GH18 chitinases, β-1,3/1,6-glucanases, cellulolytic enzymes, and hemicellulolytic enzymes, were lost in the common ancestor of T. longibrachiatum (Tl) and Tr. dN/dS (ω) calculation indicates that all the nutrition-related genes analyzed are under purifying selection. Cellulolytic enzymes, the key enzymes for saprotrophy on plant biomass, are under stronger purifying selection pressure in Tl and Tr than in mycoparasitic species, suggesting that development of the nutrition strategy of saprotrophy on plant biomass has increased the selection pressure. In addition, aspartic proteases, serine proteases, and metalloproteases are subject to stronger purifying selection pressure in Tl and Tr, suggesting that these enzymes may also play important roles in the nutrition. This study provides insights into the physiology and evolution of the nutrition strategy of Trichoderma.
PMCID: PMC3942035  PMID: 24482532
Trichoderma longibrachiatum; cellulolytic enzymes; carbohydrate-active enzymes; proteases; purifying selection; dN/dS
16.  Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913 
Pseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. Although genome sequence analysis of Pseudoalteromonas has revealed some specific features associated with adaptation to the extreme deep-sea environment, it is still difficult to study how Pseudoalteromonas adapt to the deep-sea environment due to the lack of a genetic manipulation system. The aim of this study is to develop a genetic system in the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913, making it possible to perform gene mutation by homologous recombination.
The sensitivity of Pseudoalteromonas sp. SM9913 to antibiotic was investigated and the erythromycin resistance gene was chosen as the selective marker. A shuttle vector pOriT-4Em was constructed and transferred into Pseudoalteromonas sp. SM9913 through intergeneric conjugation with an efficiency of 1.8 × 10-3, which is high enough to perform the gene knockout assay. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and sacB gene as the counterselective marker. The epsT gene encoding the UDP-glucose lipid carrier transferase was selected as the target gene for inactivation by in-frame deletion. The epsT was in-frame deleted using a two-step integration–segregation strategy after transferring the suicide vector pMT into Pseudoalteromonas sp. SM9913. The ΔepsT mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that epsT is an important gene involved in the EPS production of SM9913.
A conjugal transfer system was constructed in Pseudoalteromonas sp. SM9913 with a wide temperature range for selection and a high transfer efficiency, which will lay the foundation of genetic manipulation in this strain. The epsT gene of SM9913 was successfully deleted with no selective marker left in the chromosome of the host, which thus make it possible to knock out other genes in the same host. The construction of a gene knockout system for Pseudoalteromonas sp. SM9913 will contribute to the understanding of the molecular mechanism of how Pseudoalteromonas adapt to the deep-sea environment.
PMCID: PMC3930924  PMID: 24450434
17.  Tobacco Smoking, Quitting, and Relapsing Among Adult Males in Mainland China: The China Seven Cities Study 
Nicotine & Tobacco Research  2012;15(1):223-230.
Despite an estimated 1 million tobacco-related deaths annually in China, public health officials face overwhelming barriers to implementing effective tobacco control policies and programs. Models of effective tobacco control can be adapted for Chinese tobacco use and culture based on reliable and valid data regarding predictors of smoking and abstaining.
As part of the China Seven Cities Study to assess the role of rapid social, economic, and cultural change on tobacco use and related health practices and outcomes, 4,072 adult male smokers provided data in 3 annual waves. Measures included current smoking, nicotine dependence, readiness for quitting, perceived stress, hostility, depressive symptoms, as well as covariates (e.g., age, marital status, educational attainment, and family income).
Odds of being abstinent at Wave 3 were increased by: lower nicotine dependence at Wave 1 and becoming less dependent between Waves 1 and 3; progressing beyond the contemplation stage between Waves 1 and 3; perceiving less stress, whether initially at Wave 1 or over time from Wave 1 to Wave 3; and lower hostility scores at Wave 1 and decreased hostility from Wave 1 to Wave 3. Among those who quit, odds of remaining abstinent rather than relapsing by Wave 3 were higher among those who were less dependent at Wave 1 and who became less dependent from Wave 1 to Wave 3; and those who showed decreases in hostility from Wave 1 to Wave 3.
The public health challenge posed by very high prevalence of male smoking in China can be met by policies and programs that lead to successful long-term cessation. This can only be done successfully by designing interventions based on knowledge of the country’s smokers and the current study suggests several elements.
PMCID: PMC3611989  PMID: 22581939
18.  Detecting miRNAs in deep-sequencing data: a software performance comparison and evaluation 
Briefings in Bioinformatics  2012;14(1):36-45.
Deep sequencing has become a popular tool for novel miRNA detection but its data must be viewed carefully as the state of the field is still undeveloped. Using three different programs, miRDeep (v1, 2), miRanalyzer and DSAP, we have analyzed seven data sets (six biological and one simulated) to provide a critical evaluation of the programs performance. We selected these software based on their popularity and overall approach toward the detection of novel and known miRNAs using deep-sequencing data. The program comparisons suggest that, despite differing stringency levels they all identify a similar set of known and novel predictions. Comparisons between the first and second version of miRDeep suggest that the stringency level of each of these programs may, in fact, be a result of the algorithm used to map the reads to the target. Different stringency levels are likely to affect the number of possible novel candidates for functional verification, causing undue strain on resources and time. With that in mind, we propose that an intersection across multiple programs be taken, especially if considering novel candidates that will be targeted for additional analysis. Using this approach, we identify and performed initial validation of 12 novel predictions in our in-house data with real-time PCR, six of which have been previously unreported.
PMCID: PMC3999373  PMID: 23334922
deep sequencing; software; miRNA detection; comparison
19.  Altered dynamics between neural systems sub-serving decisions for unhealthy food 
Using BOLD functional magnetic resonance imaging (fMRI) techniques, we examined the relationships between activities in the neural systems elicited by the decision stage of the Iowa Gambling Task (IGT), and food choices of either vegetables or snacks high in fat and sugar. Twenty-three healthy normal weight adolescents and young adults, ranging in age from 14 to 21, were studied. Neural systems implicated in decision-making and inhibitory control were engaged by having participants perform the IGT during fMRI scanning. The Youth/Adolescent Questionnaire, a food frequency questionnaire, was used to obtain daily food choices. Higher consumption of vegetables correlated with higher activity in prefrontal cortical regions, namely the left superior frontal gyrus (SFG), and lower activity in sub-cortical regions, namely the right insular cortex. In contrast, higher consumption of fatty and sugary snacks correlated with lower activity in the prefrontal regions, combined with higher activity in the sub-cortical, insular cortex. These results provide preliminary support for our hypotheses that unhealthy food choices in real life are reflected by neuronal changes in key neural systems involved in habits, decision-making and self-control processes. These findings have implications for the creation of decision-making based intervention strategies that promote healthier eating.
PMCID: PMC4220120  PMID: 25414630
Iowa Gambling Task (IGT); food choice; Self-Control; Eating; insula
20.  Operationalizing the involuntary treatment regulations of China's new mental health law 
Shanghai Archives of Psychiatry  2013;25(6):384-386.
PMCID: PMC4054579  PMID: 24991181
21.  DNA promoter methylation in breast tumors: No association with genetic polymorphisms in MTHFR and MTR 
Aberrant promoter methylation is recognized as an important feature of breast carcinogenesis. We hypothesized that genetic variation of genes for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR), two critical enzymes in one-carbon metabolism, may alter DNA methylation levels, and thus influence DNA methylation in breast cancer. We evaluated case-control association of MTHFR C677T, A1298C, and MTR A2756G polymorphisms for cases strata defined by promoter methylation status for each of three genes, E- cadherin, p16, and RAR-β2 in breast cancer; in addition, we evaluated case-case comparisons of likelihood of promoter methylation in relation to genotypes using a population-based case-control study conducted in Western New York State. Methylation was evaluated with real time methylation-specific PCRs for 803 paraffin embedded breast tumor tissues from women with primary, incident breast cancer. We applied unordered polytomous regression and unconditional logistic regression to derive adjusted odds ratios (OR) and 95% confidence intervals (CI). We did not find any association of MTHFR and MTR polymorphisms with breast cancer risk stratified by methylation status nor between polymorphisms and likelihood of promoter methylation of any of the genes. There was no evidence of difference within strata defined by menopausal status, ER status, folate intake and lifetime alcohol consumption. Overall, we found no evidence that these common polymorphisms of the MTHFR and MTR genes are associated with promoter methylation of E- cadherin, p16, and RAR-β2 genes in breast cancer.
PMCID: PMC3837294  PMID: 19240236
promoter methylation; MTHFR; MTR; breast cancer; epidemiology; genetic polymorphisms
22.  Functional Networking of Human Divergently Paired Genes (DPGs) 
PLoS ONE  2013;8(10):e78896.
Divergently paired genes (DPGs), also known as bidirectional (head-to-head positioned) genes, are conserved across species and lineages, and thus deemed to be exceptional in genomic organization and functional regulation. Despite previous investigations on the features of their conservation and gene organization, the functional relationship among DPGs in a given species and lineage has not been thoroughly clarified. Here we report a network-based comprehensive analysis on human DPGs and our results indicate that the two members of the DPGs tend to participate in different biological processes while enforcing related functions as modules. Comparing to randomly paired genes as a control, the DPG pairs have a tendency to be clustered in similar “cellular components” and involved in similar “molecular functions”. The functional network bridged by DPGs consists of three major modules. The largest module includes many house-keeping genes involved in core cellular activities. This module also shows low variation in expression in both CNS (central nervous system) and non-CNS tissues. Based on analyses of disease transcriptome data, we further suggest that this particular module may play crucial roles in HIV infection and its disease mechanism.
PMCID: PMC3815023  PMID: 24205343
23.  Long-Lasting Changes in DNA Methylation Following Short-Term Hypoxic Exposure in Primary Hippocampal Neuronal Cultures 
PLoS ONE  2013;8(10):e77859.
While the effects of hypoxia on gene expression have been investigated in the CNS to some extent, we currently do not know what role epigenetics plays in the transcription of many genes during such hypoxic stress. To start understanding the role of epigenetic changes during hypoxia, we investigated the long-term effect of hypoxia on gene expression and DNA methylation in hippocampal neuronal cells. Primary murine hippocampal neuronal cells were cultured for 7 days. Hypoxic stress of 1% O2, 5% CO2 for 24 hours was applied on Day 3, conditions we found to maximize cellular hypoxic stress response without inducing cell death. Cells were returned to normoxia for 4 days following the period of hypoxic stress. On Day 7, Methyl-Sensitive Cut Counting (MSCC) was used to identify a genome-wide methylation profile of the hippocampal cell lines to assess methylation changes resulting from hypoxia. RNA-Seq was also done on Day 7 to analyze changes in gene transcription. Phenotypic analysis showed that neuronal processes were significantly shorter after 1 day of hypoxia, but there was a catch-up growth of these processes after return to normoxia. Transcriptome profiling using RNA-Seq revealed 369 differentially expressed genes with 225 being upregulated, many of which form networks shown to affect CNS development and function. Importantly, the expression level of 59 genes could be correlated to the changes in DNA methylation in their promoter regions. CpG islands, in particular, had a strong tendency to remain hypomethylated long after hypoxic stress was removed. From this study, we conclude that short-term, sub-lethal hypoxia results in long-lasting changes to genome wide DNA methylation status and that some of these changes can be highly correlated with transcriptional modulation in a number of genes involved in functional pathways that have been previously implicated in neural growth and development.
PMCID: PMC3808424  PMID: 24205000
24.  Number and characteristics of medical professionals working in Chinese mental health facilities 
Shanghai Archives of Psychiatry  2013;25(5):277-285.
Mental disorders account for a substantial proportion of the total disease burden in China but the number, distribution and characteristics of the mental health professionals available to provide services for mentally ill individuals is unknown.
Describe the distribution and characteristics of medical professionals working in mental health facilities around China.
Information on the numbers and characteristics of health professionals working in mental health facilities in China in 2010 was obtained from the Statistical Information Center of the Ministry of Health and the population-adjusted ratios and characteristics of these mental health professionals were compared across the seven geographic regions of the country.
Among the 757 specialized mental health facilities identified, 649 (86%) were psychiatric hospitals. A total of 68,796 medical professionals (5.16/100,000 population) were working in these facilities, including 20,480 psychiatrists (1.54/100,000) and 35,337 registered nurses (2.65/100,000). Over 98% of these medical professionals worked in psychiatric hospitals. Among the psychiatrists, 29% only had a technical school degree and 14% had no academic degree at all; among the nurses, 46% had no academic qualifications. The duration of employment as a psychiatrist or as a psychiatric nurse was longer among medical professionals working in the economically less dynamic northern parts of the country. The population ratios of licensed physicians and registered nurses working in mental health facilities were significantly higher than average in the relatively wealthy eastern and northeastern parts of the country.
Almost all mental health professionals in China work in specialty psychiatric hospitals. The numbers and geographic distribution of trained mental health professionals in China are grossly inadequate to meet the mental health needs of the population. China has a much smaller mental health workforce per 100,000 residents than other upper-middle-income countries, and the range of professionals who provide mental health services is much narrower.
PMCID: PMC4054572  PMID: 24991167
25.  Heat localization for targeted tumor treatment with nanoscale near-infrared radiation absorbers 
Physics in medicine and biology  2012;57(18):5765-5775.
Focusing heat delivery while minimizing collateral damage to normal tissues is essential for successful nanoparticle-mediated laser-induced thermal cancer therapy. We present thermal maps obtained via magnetic resonance imaging (MRI) characterizing laser heating of a phantom tissue containing a multiwalled carbon nanotube inclusion. The data demonstrate that heating continuously over tens of seconds leads to poor localization (~ 0.5 cm) of the elevated temperature region. By contrast, for the same energy input, heat localization can be reduced to the millimeter rather than centimeter range by increasing the laser power and shortening the pulse duration. The experimental data can be well understood within a simple diffusive heat conduction model. Analysis of the model indicates that to achieve 1 mm or better resolution, heating pulses of ~ 2s or less need to be used with appropriately higher heating power. Modeling these data using a diffusive heat conduction analysis predicts parameters for optimal targeted delivery of heat for ablative therapy.
PMCID: PMC3468294  PMID: 22948207
carbon nanotube; MR thermometry; photothermal therapy; heat localization; diffusive heat model; MR Imaging; proton resonance shift

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