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1.  Inhibition of HDAC3 promotes ligand-independent PPARγ activation by protein acetylation 
PPARγ (peroxisome proliferator-activated receptor gamma) is a nuclear receptor whose activation is dependent on a ligand. PPARγ activation by exogenous ligands, such as thiazolidinediones (TZDs), is a strategy in the treatment of type 2 diabetes for the improvement of insulin sensitivity. In addition to a ligand, PPARγ function is also regulated by posttranslational modifications, such as phosphorylation, sumoylation, and ubiquitination. Here, we report that PPARγ protein is modified by acetylation, which induces the PPARγ function in the absence of an external ligand. We observed that histone deacetylase 3 (HDAC3) interacted with PPARγ to deacetylate the protein. In immunoprecipitation, the HDAC3 protein was associated with the PPARγ protein. Inhibition of HDAC3 using RNAi-mediated knockdown or HDAC3 inhibitor increased acetylation of the PPARγ protein. Furthermore, inhibition of HDAC3 enhanced expression of PPARγ target genes such as adiponectin and aP2. The expression was associated with an increase in glucose uptake and insulin signaling in adipocytes. HDAC3 inhibition enhanced lipid accumulation during differentiation of adipocytes. PPARγ acetylation was also induced by pioglitazone and acetylation is required for PPARγ activation. In the absence of TZDs, the acetylation from HDAC3 inhibition was sufficient to induce the transcriptional activity of PPARγ. Treating the Dio mice with HDAC3 inhibitor or pioglitazone for 2 weeks significantly improved high fat diet induced–insulin resistance. Our data suggest that acetylation of PPARγ is a ligand-independent mechanism of PPARγ activation. HDAC3 inhibitor is a potential PPARγ activator for improvement of insulin sensitivity.
PMCID: PMC4391273  PMID: 24982244
type 2 diabetes; insulin sensitivity; metabolic syndrome; adipocytes; adipogenesis; PPARγ; posttranslational modifications; histone deacetylase; HDAC inhibitors; acetylation
2.  The Genus Letrouitia (Letrouitiaceae: Lichenized Ascomycota) New to Cambodia 
Mycobiology  2015;43(2):163-165.
The genus Letrouitia is newly recorded for Cambodia, including the four species as L. domingensis, L. leprolytoides, L. sayeri, and L. subvulpina. A brief description and illustrations are provided.
PMCID: PMC4505005  PMID: 26190924
Cambodia; Letrouitia
3.  Induction of Energy Expenditure by Sitagliptin Is Dependent on GLP-1 Receptor 
PLoS ONE  2015;10(5):e0126177.
Sitagliptin (SG) increases serum GLP-1 (Glucagon-like peptide-1) through inhibition of the hormone degradation. Resistant starch (RS) induces GLP-1 expression by stimulating L-cells in the intestine. Sitagliptin and resistant starch may have a synergistic interaction in the induction of GLP-1. This possibility was tested in current study in a mouse model of type 2 diabetes. Hyperglycemia was induced in the diet-induced obese mice by a signal injection of streptozotocin (STZ). Sitagliptin (0.4g/100g diet) was tested in the mice (n = 55) with dietary RS (HAM-RS2) at three dosages (0, 15, or 28g/100g diet). Energy and glucose metabolism were monitored in the evaluation of synergistic activity, and GLP-1 activity was determined in the GLP-1 receptor knockout (KO) mice. In the wild type mice, body weight and adiposity were reduced by sitagliptin, which was enhanced by RS (28g). Serum GLP-1 was induced and energy expenditure was enhanced by sitagliptin. Fasting glucose, insulin, and leptin levels were decreased by sitagliptin. The sitagliptin effects were lost in the KO mice (n = 25) although induction of serum GLP-1 by sitagliptin was even stronger in KO mice. The data suggests that sitagliptin is able to reduce adiposity and insulin resistance through induction of energy expenditure. The effect of sitagliptin is partially enhanced by RS. GLP-1 receptor may regulate serum GLP-1 by facilitating the hormone clearance.
PMCID: PMC4418617  PMID: 25938560
4.  Altered expression of connexin43 and phosphorylation connexin43 in glioma tumors 
In this study, we aim to evaluate the connexin (Cx43) and phosphorylation Cx43 (p-Cx43) expression of human glioma tumors and correlate their expression with degrees of malignancy and proliferation, apoptosis, and migration activity of tumors. Cx43 and p-Cx43 expression were examined by Western blot analysis and immunohistochemical staining. The U251 cell viability was measured by MTT analysis. The apoptosis and migration were also evaluated by flow cytometric analysis and fluoroblok transwell chambers, respectively. We found that the Cx43 expression were significantly downregulated in in malignant glioma (WHO grade III and IV), compared to the malignant glioma (WHO grade I and II) and the p-Cx43 expression levels of malignant glioma (WHO grade III and IV) were significantly increased (P<0.05), compared to the malignant glioma (WHO grade I and II) at immunohistochemical analysis. After treatment of cells with a specific inhibitor of PKC, MAPK, and PTK inhibitors, the cell viability and migration were significantly decreased, while the apoptosis was slightly induced. In conclusion, the Cx43 expression level is inversely correlated with the tumor grade and proliferation and migration activity of tumor. Higher p-Cx43 expression level in high tumor grade suggests that a complex mechanism is involved in the suppression of tumor growth by connexins.
PMCID: PMC4502994  PMID: 26191122
Connexin43; phosphorylation; malignant glioma; U251
5.  Hematology research output from Chinese authors and other countries: a 10-year survey of the literature 
Hematologic disease affects people of all ages worldwide. In the past decade, researchers have made great progress in the field of hematology. In the present study we compared the hematology research output from China and other countries (USA, Germany, UK, Japan and South Korea) over the past 10 years and 5 years.
The related articles were extracted based on the PubMed database. We recorded the number of publications, clinical trials, randomized controlled trials, meta-analyses, case reports, reviews, citations, impact factors, articles in the top 10 journals and most published journals to assess the quantity and quality of research output in each region.
A total of 120,641 hematology-related articles were published from 2004 to 2013. The USA accounted for 27.13% (32,732/120,641) of the publications, followed by Germany (7,479/120,641; 6.20%), Japan (6,347/120,641; 5.26%), the UK (5,453/120,641; 4.52%), China (2,924/120,641; 2.42%) and South Korea (1,413/120,641; 1.17%). The ranking for cumulative impact factors was as follows: USA; Germany; UK; Japan; China and South Korea. The median impact factors in the UK, USA, and Germany were higher than Japan, South Korea, and China. Interestingly, the median impact factors in the three Asia countries were similar both in 2004–2013 and 2009–2013. The UK had the highest percentage of publications in the top 25% of journals, while China lagged behind and ranked last. When comparing the number of articles in the top 10 journals, the results were similar to the IF findings. Germany had the highest number of average citations, while China had the lowest number of average citation. The status of hematology research output from the 6 countries in 2009–2013 had little difference from 2004–2013.
Thus, the USA has had a dominant role in hematologic research in the past 10 years. Overall, the quality of publications in European countries was better than Asia countries. Although China has made considerable progress in hematology research, the quality of research needs improvement.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-014-0103-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4332745  PMID: 25653049
Hematology; Publications; Impact factor; Citations; Science Citation Index Expanded (SCIE)
6.  The Role of IL-23/Th17 Pathway in Patients with Primary Immune Thrombocytopenia 
PLoS ONE  2015;10(1):e0117704.
Primary immune thrombocytopenia (ITP) is an autoimmune bleeding disorder with an unclear etiology. This study aims to investigate the role of IL-23/Th17 pathway in patients with ITP.
The gene expressions of IL-17, IL-23 and their receptors in ITP patients and healthy controls were analyzed by quantitative real-time PCR. ELISA was used to test the IL-17 and IL-23 levels in plasma. Flow cytometry was used to detect the frequency of Th17 cells. The correlation between plasma IL-23 and IL-17 levels, Th17 cells, platelets were analyzed. The level of Th17-related cytokines was measured by ELISA following stimulation with IL-23. Subsequently, the IL-23 and IL-17 levels were measured in patients post-treatment.
The PBMCs of ITP patients showed increased mRNA expression levels in each of the following: IL-23p19, IL-12p40, IL-23R, IL-12Rβ1, IL-17A, IL-17F, and RORC. In addition, elevated Th17 cells and plasma IL-17, IL-23 levels were also observed in these ITP patients. Furthermore, it was found that IL-23 levels in plasma are positively correlated with IL-17 levels and Th17 cells, yet negatively correlated with platelet count. Following IL-23 stimulation in vitro, IL-17 levels showed significant elevation. Furthermore, both IL-23 and IL-17 levels decreased after effective treatment.
The IL-23/Th17 pathway may be involved in the pathogenesis of ITP through enhancement of the Th17 response. Moreover, our results suggest that the IL-23/Th17 pathway is a potential therapeutic target in future attempts of ITP treatment.
PMCID: PMC4306550  PMID: 25621490
7.  Molecular characteristics of the HSP70 gene and its differential expression in female and male golden apple snails (Pomacea canaliculata) under temperature stimulation 
Cell Stress & Chaperones  2013;19(4):579-589.
Heat-shock protein 70 (HSP70) is one of the most important heat-shock proteins that helps organisms to modulate stress response via over-expression. The HSP70 gene from Pomacea canaliculata was cloned using the RACE approach; the gene is 2,767 bp in length and contains an open reading frame of 1,932 bp, which is encoded by a polypeptide of 643 amino acids. BLAST analysis showed that the predicted amino acid sequence of the P. canaliculata HSP70 gene shared a relatively high similarity with that of other known eukaryotic species that display conserved HSP characteristics. The phylogeny demonstrated a separate clustering of the apple snail HSP70 with other constitutive members from other mollusk species. Quantitative real-time RT-PCR was used to detect the differential expression of HSP70 in both sexes of P. canaliculata at different temperature conditions. These results showed that HSP70 transcript levels decreased slightly under cold shock and increased significantly under heat-shock conditions in both sexes compared to normal temperatures (26 °C). Under cold-shock treatment, the sex effect was not significant. With heat treatment, HSP70 expression could be induced at 36 °C in both females and males, and it peaked at 42 and 39 °C in females and males, respectively. In addition, a clear time-dependent HSP70 expression pattern of the apple snail exposed to the same high temperature (36 °C) was observed at different time points. The maximal induction of HSP70 expression appeared at 12 and 48 h in males and females after heat shock, respectively. The maximal induction in females was significantly higher compared to males under heat stimulus. Taken together, these results strongly suggested that males were more susceptible to heat than females and provided useful molecular information for the ecological adaptability of P. canaliculata against extreme environmental stress.
PMCID: PMC4041941  PMID: 24368711
Pomacea canaliculata; HSP70; Gene expression; Temperature stress
8.  A more consistent intraluminal rhesus monkey model of ischemic stroke 
Neural Regeneration Research  2014;9(23):2087-2094.
Endovascular surgery is advantageous in experimentally induced ischemic stroke because it causes fewer cranial traumatic lesions than invasive surgery and can closely mimic the pathophysiology in stroke patients. However, the outcomes are highly variable, which limits the accuracy of evaluations of ischemic stroke studies. In this study, eight healthy adult rhesus monkeys were randomized into two groups with four monkeys in each group: middle cerebral artery occlusion at origin segment (M1) and middle cerebral artery occlusion at M2 segment. The blood flow in the middle cerebral artery was blocked completely for 2 hours using the endovascular microcoil placement technique (1 mm × 10 cm) (undetachable), to establish a model of cerebral ischemia. The microcoil was withdrawn and the middle cerebral artery blood flow was restored. A reversible middle cerebral artery occlusion model was identified by hematoxylin-eosin staining, digital subtraction angiography, magnetic resonance angiography, magnetic resonance imaging, and neurological evaluation. The results showed that the middle cerebral artery occlusion model was successfully established in eight adult healthy rhesus monkeys, and ischemic lesions were apparent in the brain tissue of rhesus monkeys at 24 hours after occlusion. The rhesus monkeys had symptoms of neurological deficits. Compared with the M1 occlusion group, the M2 occlusion group had lower infarction volume and higher neurological scores. These experimental findings indicate that reversible middle cerebral artery occlusion can be produced with the endovascular microcoil technique in rhesus monkeys. The M2 occluded model had less infarction and less neurological impairment, which offers the potential for application in the field of brain injury research.
PMCID: PMC4316474  PMID: 25657726
nerve regeneration; brain injury; rhesus monkeys; model middle cerebral artery; microcoil; infarction; stroke; interventional therapy; digital subtraction angiography; magnetic resonance image; neuroimaging; neuroregeneration
9.  cDNA-AFLP analysis reveals the adaptive responses of citrus to long-term boron-toxicity 
BMC Plant Biology  2014;14:284.
Boron (B)-toxicity is an important disorder in agricultural regions across the world. Seedlings of ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) were fertigated every other day until drip with 10 μM (control) or 400 μM (B-toxic) H3BO3 in a complete nutrient solution for 15 weeks. The aims of this study were to elucidate the adaptive mechanisms of citrus plants to B-toxicity and to identify B-tolerant genes.
B-toxicity-induced changes in seedlings growth, leaf CO2 assimilation, pigments, total soluble protein, malondialdehyde (MDA) and phosphorus were less pronounced in C. sinensis than in C. grandis. B concentration was higher in B-toxic C. sinensis leaves than in B-toxic C. grandis ones. Here we successfully used cDNA-AFLP to isolate 67 up-regulated and 65 down-regulated transcript-derived fragments (TDFs) from B-toxic C. grandis leaves, whilst only 31 up-regulated and 37 down-regulated TDFs from B-toxic C. sinensis ones, demonstrating that gene expression is less affected in B-toxic C. sinensis leaves than in B-toxic C. grandis ones. These differentially expressed TDFs were related to signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein and amino acid metabolism, lipid metabolism, cell wall and cytoskeleton modification, stress responses and cell transport. The higher B-tolerance of C. sinensis might be related to the findings that B-toxic C. sinensis leaves had higher expression levels of genes involved in photosynthesis, which might contribute to the higher photosyntheis and light utilization and less excess light energy, and in reactive oxygen species (ROS) scavenging compared to B-toxic C. grandis leaves, thus preventing them from photo-oxidative damage. In addition, B-toxicity-induced alteration in the expression levels of genes encoding inorganic pyrophosphatase 1, AT4G01850 and methionine synthase differed between the two species, which might play a role in the B-tolerance of C. sinensis.
C. sinensis leaves could tolerate higher level of B than C. grandis ones, thus improving the B-tolerance of C. sinensis plants. Our findings reveal some novel mechanisms on the tolerance of plants to B-toxicity at the gene expression level.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0284-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4219002  PMID: 25348611
Boron-tolerance; Boron-toxicity; cDNA-AFLP; Citrus grandis; Citrus sinensis; Photosynthesis
10.  Clinical and Economic Benefits Associated With the Achievement of Both HbA1c and LDL Cholesterol Goals in Veterans With Type 2 Diabetes 
Diabetes Care  2013;36(10):3297-3304.
This study compared the clinical and economic benefits associated with dual-goal achievement, glycated hemoglobin (HbA1c) <7% (53 mmol/mol) and LDL cholesterol (LDL-C) <100 mg/dL, with achievement of only the LDL-C goal or only the HbA1c goal in veterans with type 2 diabetes mellitus (T2DM).
This retrospective cohort analysis evaluated electronic medical records (Veterans Integrated Service Network 16) in adult T2DM patients with two or more measurements of LDL-C and HbA1c between 1 January 2004 and 30 June 2010 (N = 75,646). Cox proportional hazards models were used to compare microvascular and cardiovascular outcomes by goal achievement status; generalized linear regression models were used to assess diabetes-related resource utilization (hospitalization days and number of outpatient visits) and medical service costs.
Relative to achievement of only the LDL-C goal, dual-goal achievement was associated with lower risk of microvascular complications (adjusted hazard ratio [aHR] 0.79), acute coronary syndrome (0.88), percutaneous coronary intervention (0.78), and coronary artery bypass graft (CABG) (0.74); it was also associated with fewer hospitalization days (adjusted incidence rate ratio [aIRR] 0.93) and outpatient visits (0.88), as well as lower diabetes-related annual medical costs (−$130.89). Compared with achievement of only the HbA1c goal, dual-goal achievement was associated with lower risk of the composite cardiovascular-related end point (aHR 0.87) and CABG (aHR 0.62), as well as fewer outpatient visits (aIRR 0.98).
Achieving both HbA1c and LDL-C goals in diabetes care is associated with additional clinical and economic benefits, as compared with the achievement of either goal alone.
PMCID: PMC3781519  PMID: 23801723
11.  Abdominal compartment syndrome: Incidence and prognostic factors influencing survival in Singapore 
Aim of Study:
Abdominal compartment syndrome (ACS) is a distinct clinical entity in the critically ill-patient, which leads to end-organ dysfunction. However, data on the incidence of ACS is scarce, and this is also likely contributed by under-diagnosis of this clinical condition. This study reports all cases of ACS in a tertiary institution in Singapore over 10 years, and evaluates prognostic factors affecting survival.
Materials and Methods:
This retrospective clinical study included 17 patients with ACS, of which 13 underwent decompressive laparotomy, over a 10 years period. Univariate and multivariate analyses of prognostic factors predicting mortality was performed using Chi-square or Fisher-exact test as appropriate.
Mean arterial pressure was significantly improved postoperatively, and intra-abdominal pressure and positive end-expiratory pressure significantly decreased. Overall mortality was 47.1%. Advanced age of more than 65 years, gender, large volume resuscitation of more than 3.5 L over 24 h, three or more co-morbidities, requirement of inotropes, usage of mechanical ventilation, and the presence of concurrent lung and renal dysfunction were not adverse prognostic indicators of poorer outcome. The occurrence of multiple relook laparotomies was shown to be the only independent prognostic factor predicting a favorable outcome among these patients on univariate and multivariate analyses. The incidence of ACS accounts for only 0.1% of all Intensive Care Unit admissions during the study period of 10 years, likely due to under-diagnosis.
We believe that a protocol for a focused measurement in high-risk groups will increase the diagnostic yield of this condition. Multiple laparotomies for abdominal decompression can lead to improved survival.
PMCID: PMC4195194  PMID: 25316974
Abdominal compartment syndrome; incidence; prognostic factors
12.  Hepatic Resection Is Safe and Effective for Patients with Hepatocellular Carcinoma and Portal Hypertension 
PLoS ONE  2014;9(9):e108755.
Background & Aims
Official guidelines do not recommend hepatic resection (HR) for patients with hepatocellular carcinoma (HCC) and portal hypertension (PHT). This study aims to investigate the safety and efficacy of HR for patients with HCC and PHT.
Mortality and survival after HR were analyzed retrospectively in a consecutive sample of 1738 HCC patients with PHT (n = 386) or without it (n = 1352). To assess the robustness of findings, we repeated the analysis using propensity score-matched analysis. We also comprehensively searched the PubMed database for studies evaluating the efficacy and safety of HR for patients with HCC and PHT.
The 90-day mortality rate was 6.7% among those with PHT and 2.1% among those without it (P<.001). Patients without PHT had a survival benefit over those with PHT at 1, 3, and 5 years (96% vs 90%, 75% vs 67%, 54% vs 45%, respectively; P = .001). In contrast, PHT was not associated with worse short- or long-term survival when only propensity score-matched pairs of patients and those with early-stage HCC or those who underwent minor hepatectomy were included in the analysis (all P>.05). Moreover, the recurrence rates were similar between the two groups. Consistent with our findings, all 9 studies identified in our literature search reported HR to be safe and effective for patients with HCC and PHT.
HR is safe and effective in HCC patients with PHT and preserved liver function. This is especially true for patients who have early-stage HCC or who undergo minor hepatectomy.
PMCID: PMC4182657  PMID: 25268959
13.  Investigations of heme ligation and ligand switching in cytochromes P450 and P420 
Biochemistry  2013;52(34):5941-5951.
It is generally accepted that the inactive P420 form of cytochrome P450 (CYP) involves the protonation of the native cysteine thiolate to form a neutral thiol heme ligand. On the other hand, it has also been suggested that recruitment of a histidine to replace the native cysteine thiolate ligand might underlie the P450→P420 transition. Here we discuss resonance Raman investigations of the H93G myoglobin (Mb) mutant in the presence of tetrahydrothiophene (THT) or cyclopentathiol (CPSH), and on pressure-induced cytochrome P420cam (CYP101), that show a histidine becomes the heme ligand upon CO binding. The Raman mode near 220 cm−1, normally associated with the Fe-histidine vibration in heme proteins, is not observed in either reduced P420cam or the reduced H93G Mb samples, indicating that histidine is not the ligand in the reduced state. The absence of a mode near 220 cm−1 is also inconsistent with a generalization of the suggestion that the 221 cm−1 Raman mode, observed in the P420-CO photoproduct of inducible nitric oxide synthase (iNOS), arises from a thiol-bound ferrous heme. This leads us to assign the 218 cm−1 mode observed in the 10 ns P420cam-CO photoproduct Raman spectrum to a Fe-histidine vibration, in analogy to many other histidine bound heme systems. Additionally, the inverse correlation plots of the νFe-His and νCO frequencies for the CO adducts of P420cam and the H93G analogs provide supporting evidence that histidine is the heme ligand in the P420-CO bound state. We conclude that, when CO binds to the ferrous P420 state, a histidine ligand is recruited as the heme ligand. The common existence of a HXC-Fe motif in many CYP systems allows the C→H ligand switch to occur with only minor conformational changes. One suggested conformation of P420-CO involves the addition of another turn in the proximal L helix so that, when the protonated Cys ligand is dissociated from the heme, it can become part of the helix and the heme is ligated by the His residue from the adjoining loop region. In other systems, such as iNOS and CYP3A4 (where the HXC-Fe motif is not found) a somewhat larger conformational change would be necessary to recuit a nearby histidine.
PMCID: PMC3777526  PMID: 23905516
Cytochrome P450; cytochrome P420; H93G; myoglobin; resonance Raman; ligand switch
14.  Increased prevalence of hepatitis C virus subtype 6a in China: a comparison between 2004–2007 and 2008–2011 
Archives of Virology  2014;159(12):3231-3237.
Different hepatitis C virus (HCV) genotypes exhibit differences in disease pathogenesis and progression, as well as disease outcomes and response to therapy. Tracking the change of HCV genotypes in various epidemiological settings is critical for both disease surveillance and the development of improved antiviral treatment. Here, we tracked the changes in the prevalence of the HCV genotypes in China between 2004-2007 and 2008-2011. HCV-RNA-positive sera were collected from volunteer blood donors during the period 2008-2011. The genotypes were determined by phylogenic analysis using the NS5B and E1 sequences. Geographical and demographic distribution patterns related to the HCV genotypes obtained in 2008-2011 were compared with our previous study, which recorded data in the period 2004-2007. Pearson chi-square test and t-test were used to statistically analyze the results. In 2008-2011, HCV subtypes 1b and 6a were detected in 43.8 % (184/420) and 34.3 % (144/420), respectively. The male/female ratio was found to be higher for HCV genotype 6 than for genotypes 1 and 2. When compared with the period of 2004-2007, although no significant difference was found in gender or age for genotypes 1, 2, 3 and 6, the subtype 6a frequency was significantly increased from 11 % to 26.5 % in the blood donors from outside of Guangdong Province in 2008-2011. A pattern of increase in HCV subtype 6a was found in blood donors outside of Guangdong Province, indicating that HCV subtype 6a has rapidly spread from Guangdong to other regions of China over the past 10 years.
Electronic supplementary material
The online version of this article (doi:10.1007/s00705-014-2185-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4221604  PMID: 25085624
15.  Association between Dairy Intake and Gastric Cancer: A Meta-Analysis of Observational Studies 
PLoS ONE  2014;9(7):e101728.
Observational studies have given inconsistent findings on the relationship between intake of dairy products and gastric cancer. We therefore conducted a systematic review with a meta-analysis of observational studies to summarize available evidence on this point.
We searched the electronic literature databases of PubMed (Medline), EMBASE and the Chinese Biomedical Literature Database up until August 30, 2013. All studies were limited to the English language. Random-effects models were used to pool study results between dairy products consumption and the risk of gastric cancer. We also performed subgroup, publication bias and sensitivity analysis.
Eight prospective studies and 18 case-control studies were included in our analysis, with a total number of 7272 gastric cancer cases and 223,355 controls. Pooled relative risks of all studies showed no significant association between dairy intake and gastric cancer (odds ratio [OR]: 1.09, 95% confidence interval [CI]: 0.96–1.25). When study design was separately analyzed, population-based case-control studies showed a positive association between dairy intake and gastric cancer risk (OR: 1.36; 95% CI: 1.07–1.74), whereas no associations were shown by hospital-based case-control studies (OR: 0.86, 95% CI: 0.72–1.02) or cohort studies (OR = 1.01, 95% CI = 0.91–1.13).
The meta-analysis shows that no clear association apparently exists between consumption of dairy products and gastric cancer risk. Further well-designed cohort and intervention studies should be conducted to verify this lack of association.
PMCID: PMC4090187  PMID: 25006674
16.  Hepatitis B Virus Inhibits Apoptosis of Hepatoma Cells by Sponging the MicroRNA 15a/16 Cluster 
Journal of Virology  2013;87(24):13370-13378.
Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. In this study, we found that HBV inhibited the chemotherapy drug etoposide-induced apoptosis of hepatoma cells. Further analysis revealed that HBV mRNAs possess a microRNA 15a/16 (miR-15a/16)-complementary site (HBV nucleotides [nt] 1362 to 1383) that acts as a sponge to bind and sequester endogenous miR-15a/16. Consequently, Bcl-2, known as the target of miR-15a/16, was upregulated in HBV-infected cells. The data from HBV-transgenic mice further confirmed that HBV transcripts cause the reduction of miR-15a/16 and increase of Bcl-2. More importantly, we examined the levels of HBV transcripts and miR-15a/16 in HBV-infected HCC from patients and found that the amount of HBV mRNA and the level of miR-15a/16 were negatively correlated. Consistently, the level of Bcl-2 mRNA was upregulated in HBV-infected patients. In conclusion, we identified a novel HBV mRNA–miR-15a/16–Bcl-2 regulatory pathway that is involved in inhibiting etoposide-induced apoptosis of hepatoma cells, which may contribute to facilitating chronic HBV infection and hepatoma development.
PMCID: PMC3838258  PMID: 24089558
17.  Regulation of Insulin Degrading Enzyme Activity by Obesity-Associated Factors and Pioglitazone in Liver of Diet-Induced Obese Mice 
PLoS ONE  2014;9(4):e95399.
Insulin degrading enzyme (IDE) is a potential drug target in the treatment of type 2 diabetes (T2D). IDE controls circulating insulin through a degradation-dependent clearance mechanism in multiple tissues. However, there is not sufficient information about IDE regulation in obesity. In this study, we test obesity-associated factors and pioglitazone in the regulation of IDE in diet-induced obese (DIO) C57BL/6 mice. The enzyme activity and protein level of IDE were increased in the liver of DIO mice. Pioglitazone (10 mg/kg/day) administration for 2 months significantly enhanced the enzyme activity (75%), protein (180%) and mRNA (100%) of IDE in DIO mice. The pioglitazone-induced changes were coupled with 50% reduction in fasting insulin and 20% reduction in fasting blood glucose. The mechanism of IDE regulation in liver was investigated in the mouse hepatoma cell line (Hepa 1c1c7 cells), in which pioglitazone (5 µM) increased IDE protein and mRNA in a time-dependent manner in an 8 h study. Free fatty acid (palmitate 300 µM) induced IDE protein, but reduced the mRNA. Glucagon induced, and TNF-α decreased IDE protein. Insulin did not exhibit any activity in the same condition. In summary, pioglitazone, FFA and glucagon directly increased, but TNF-α decreased the IDE activity in hepatocytes. The results suggest that IDE activity is regulated in liver by multiple factors in obesity and pioglitazone may induce IDE activity in the control of T2D.
PMCID: PMC3989328  PMID: 24740421
18.  Coxsackievirus-Induced miR-21 Disrupts Cardiomyocyte Interactions via the Downregulation of Intercalated Disk Components 
PLoS Pathogens  2014;10(4):e1004070.
Intercalated disks (ICDs) are substantial connections maintaining cardiac structures and mediating signal communications among cardiomyocytes. Deficiency in ICD components such as desmosomes, fascia adherens and gap junctions leads to heart dysfunction. Coxsackievirus B3 (CVB3) infection induces cardiac failure but its pathogenic effect on ICDs is unclear. Here we show that CVB3-induced miR-21 expression affects ICD structure, i.e., upregulated miR-21 targets YOD1, a deubiquitinating enzyme, to enhance the K48-linked ubiquitination and degradation of desmin, resulting in disruption of desmosomes. Inhibition of miR-21 preserves desmin during CVB3 infection. Treatment with proteasome inhibitors blocks miR-21-mediated desmin degradation. Transfection of miR-21 or knockdown of YOD1 triggers co-localization of desmin with proteasomes. We also identified K108 and K406 as important sites for desmin ubiquintination and degradation. In addition, miR-21 directly targets vinculin, leading to disturbed fascia adherens evidenced by the suppression and disorientation of pan-cadherin and α-E-catenin proteins, two fascia adherens-components. Our findings suggest a new mechanism of miR-21 in modulating cell-cell interactions of cardiomyocytes during CVB3 infection.
Author Summary
Coxsackievirus B3 (CVB3) is one of most common causes of heart inflammation and failure. However, the mechanism by which CVB3 induces cardiac damage has not been fully elucidated. Particularly, the involvement of microRNAs (miRNAs), a family of small RNAs controlling the progression of a wide range of diseases, in CVB3 infection is still unclear. These small RNAs are essential to understand the CVB3-caused heart muscle cell injury and have great potential to serve therapeutic purposes. Here, we systematically analyzed the miRNA changes during CVB3 infection and found that miR-21 is increased by viral infection. We further demonstrated that the CVB3-induced miR-21 triggers heart muscle cell damage by interfering with the cell-cell interactions. miR-21 suppresses the levels of components in cell-cell interactions by either promoting the degradation of those proteins or directly inhibiting the protein production. Inhibition of miR-21 can reduce the host injury caused by CVB3 infection. Our findings will shed new lights on the pathogenesis of CVB3-induced heart failure.
PMCID: PMC3983067  PMID: 24722419
19.  Laparoscopic-endoscopic cooperative surgery for gastric submucosal tumors 
AIM: To assess the feasibility, safety, and advantages of minimally invasive laparoscopic-endoscopic cooperative surgery (LECS) for gastric submucosal tumors (SMT).
METHODS: We retrospectively analyzed 101 consecutive patients, who had undergone partial, proximal, or distal gastrectomy using LECS for gastric SMT at Peking Union Medical College Hospital from June 2006 to April 2013. All patients were followed up by visit or telephone. Clinical data, surgical approach, pathological features such as the size, location, and pathological type of each tumor; and follow-up results were analyzed. The feasibility, safety and effectiveness of LECS for gastric SMT were evaluated, especially for patients with tumors located near the cardia or pylorus.
RESULTS: The 101 patients included 43 (42.6%) men and 58 (57.4%) women, with mean age of 51.2 ± 13.1 years (range, 14-76 years). The most common symptom was belching. Almost all (n = 97) patients underwent surgery with preservation of the cardia and pylorus, with the other four patients undergoing proximal or distal gastrectomy. The mean distance from the lesion to the cardia or pylorus was 3.4 ± 1.3 cm, and the minimum distance from the tumor edge to the cardia was 1.5 cm. Tumor pathology included gastrointestinal stromal tumor in 78 patients, leiomyoma in 13, carcinoid tumors in three, ectopic pancreas in three, lipoma in two, glomus tumor in one, and inflammatory pseudotumor in one. Tumor size ranged from 1 to 8.2 cm, with 65 (64.4%) lesions < 2 cm, 32 (31.7%) > 2 cm, and four > 5 cm. Sixty-six lesions (65.3%) were located in the fundus, 21 (20.8%) in the body, 10 (9.9%) in the antrum, three (3.0%) in the cardia, and one (1.0%) in the pylorus. During a median follow-up of 28 mo (range, 1-69 mo), none of these patients experienced recurrence or metastasis. The three patients who underwent proximal gastrectomy experienced symptoms of regurgitation and belching.
CONCLUSION: Laparoscopic-endoscopic cooperative surgery is feasible and safe for patients with gastric submucosal tumor. Endoscopic intraoperative localization and support can help preserve the cardia and pylorus during surgery.
PMCID: PMC3769911  PMID: 24039367
Laparoscopic-endoscopic cooperative surgery; Gastric submucosal tumor; Minimally invasive surgery; Laparoscopy; Endoscopy
20.  Leaf cDNA-AFLP analysis of two citrus species differing in manganese tolerance in response to long-term manganese-toxicity 
BMC Genomics  2013;14:621.
Very little is known about manganese (Mn)-toxicity-responsive genes in citrus plants. Seedlings of ‘Xuegan’ (Citrus sinensis) and ‘Sour pummelo’ (Citrus grandis) were irrigated for 17 weeks with nutrient solution containing 2 μM (control) or 600 μM (Mn-toxicity) MnSO4. The objectives of this study were to understand the mechanisms of citrus Mn-tolerance and to identify differentially expressed genes, which might be involved in Mn-tolerance.
Under Mn-toxicity, the majority of Mn in seedlings was retained in the roots; C. sinensis seedlings accumulated more Mn in roots and less Mn in shoots (leaves) than C. grandis ones and Mn concentration was lower in Mn-toxicity C. sinensis leaves compared to Mn-toxicity C. grandis ones. Mn-toxicity affected C. grandis seedling growth, leaf CO2 assimilation, total soluble concentration, phosphorus (P) and magenisum (Mg) more than C. sinensis. Using cDNA-AFLP, we isolated 42 up-regulated and 80 down-regulated genes in Mn-toxicity C. grandis leaves. They were grouped into the following functional categories: biological regulation and signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, lipid metabolism, cell wall metabolism, stress responses and cell transport. However, only 7 up-regulated and 8 down-regulated genes were identified in Mn-toxicity C. sinensis ones. The responses of C. grandis leaves to Mn-toxicity might include following several aspects: (1) accelerating leaf senescence; (2) activating the metabolic pathway related to ATPase synthesis and reducing power production; (3) decreasing cell transport; (4) inhibiting protein and nucleic acid metabolisms; (5) impairing the formation of cell wall; and (6) triggering multiple signal transduction pathways. We also identified many new Mn-toxicity-responsive genes involved in biological and signal transduction, carbohydrate and protein metabolisms, stress responses and cell transport.
Our results demonstrated that C. sinensis was more tolerant to Mn-toxicity than C. grandis, and that Mn-toxicity affected gene expression far less in C. sinensis leaves. This might be associated with more Mn accumulation in roots and less Mn accumulation in leaves of Mn-toxicity C. sinensis seedlings than those of C. grandis seedlings. Our findings increase our understanding of the molecular mechanisms involved in the responses of plants to Mn-toxicity.
PMCID: PMC3847489  PMID: 24034812
cDNA-AFLP; Citrus grandis; Citrus sinensis; Leaves; Manganese
21.  Bone Marrow Injury Induced via Oxidative Stress in Mice by Inhalation Exposure to Formaldehyde 
PLoS ONE  2013;8(9):e74974.
Formaldehyde, a ubiquitous environmental pollutant has been classified as a human leukemogen. However, toxicity of formaldehyde in bone marrow, the target site of leukemia induction, is still poorly understood.
Methodology/Principal Findings
To investigate bone marrow toxicity (bone marrow pathology, hematotoxicity) and underlying mechanisms (oxidative stress, inflammation, apoptosis) in formaldehyde-exposed mice. Male Balb/c mice were exposed to formaldehyde (0, 0.5, and 3.0 mg/m3) by nose-only inhalation for 8 hours/day, over a two week period designed to simulate a factory work schedule, with an exposure-free “weekend” on days 6 and 7, and were sacrificed on the morning of day 13. Counts of white blood cells, red blood cells and lymphocytes were significantly (p<0.05) decreased at 0.5 mg/m3 (43%, 7%, and 39%, respectively) and 3.0 mg/m3 (52%, 27%, and 43%, respectively) formaldehyde exposure, while platelet counts were significantly increased by 109% (0.5 mg/m3) and 67% (3.0 mg/m3). Biomarkers of oxidative stress (reactive oxygen species, glutathione depletion, cytochrome P450 1A1 and glutathione s-transferase theta 1 expression), inflammation (nuclear factor kappa-B, tomour necrosis factor alpha, interleukin-1 beta), and apoptosis (activity of cysteine-aspartic acid protease 3) in bone marrow tissues were induced at one or both formaldehyde doses mentioned above.
Exposure of mice to formaldehyde by inhalation induced bone marrow toxicity, and that oxidative stress, inflammation and the consequential apoptosis jointly constitute potential mechanisms of such induced toxicity.
PMCID: PMC3770590  PMID: 24040369
22.  Increased rate of sporadic and recurrent rare genic copy number variants in Parkinson's disease among Ashkenazi Jews 
To date, only one genome-wide study has assessed the contribution of copy number variants (CNVs) to Parkinson's disease (PD). We conducted a genome-wide scan for CNVs in a case–control dataset of Ashkenazi Jewish (AJ) origin (268 PD cases and 178 controls). Using high-confidence CNVs, we examined the global genome wide burden of large (≥100 kb) and rare (≤1% in the dataset) CNVs between cases and controls. A total of 986 such CNVs were observed in our dataset of 432 subjects. Overall global burden analyses did not reveal significant differences between cases and controls in CNV rate, distribution of deletions or duplications or number of genes affected by CNVs. Overall deletions (total CNV size and ≥2× frequency) were found 1.4 times more often in cases than in controls (P = 0.019). The large CNVs (≥500 kb) were also significantly associated with PD (P = 0.046, 1.24-fold higher in cases than in controls). Global burden was elevated for rare CNV regions. Specifically, for OVOS2 on Chr12p11.21, CNVs were observed only in PD cases (n = 7) but not in controls (P = 0.028) and this was experimentally validated. A total of 81 PD cases carried a rare genic CNV that was absent in controls. Ingenuity pathway analysis (IPA) identified ATXN3, FBXW7, CHCHD3, HSF1, KLC1, and MBD3 in the same disease pathway with known PD genes.
PMCID: PMC3782064  PMID: 24073418
Ashkenazi Jews; candidate genes; case–control study; CNV; Parkinson's disease
23.  Polo-Like Kinase 1 Inhibits the Activity of Positive Transcription Elongation Factor of RNA Pol II b (P-TEFb) 
PLoS ONE  2013;8(8):e72289.
Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase in eukaryotes and plays a critical role in various aspects of the cell cycle. Plk1 exerts its multiple functions by phosphorylating its substrates. In this study, we found that Plk1 can interact with cyclin T1/Cdk9 complex-the main form of the positive transcription elongation complex b (P-TEFb), and its C-terminal polo-box domain is responsible for the binding. Further analysis indicated that Plk1 could phosphorylate cyclin T1 at Ser564 and inhibit the kinase activity of cyclin T1/Cdk9 complex on phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. By taking the approach of luciferase assay, we demonstrated that over-expression of both wild type Plk1 and constitutively active form of Plk1 inhibits the P-TEFb dependent HIV-1 LTR transcription, while knockdown of Plk1 increases the HIV-1 LTR transcription. Consistently, the data from the HIV-1 pseudovirus reporter assay indicated that Plk1 blocks the gene expression of HIV-1 pseudovirus. Taken together, our results revealed that Plk1 negatively regulates the RNA polymerase II-dependent transcription through inhibiting the activity of cyclin T1/Cdk9 complex.
PMCID: PMC3745420  PMID: 23977272
24.  Portal for Families Overcoming Neurodevelopmental Disorders (PFOND): Implementation of a Software Framework for Facilitated Community Website Creation by Nontechnical Volunteers 
JMIR Research Protocols  2013;2(2):e25.
The Portal for Families Overcoming Neurodevelopmental Disorders (PFOND) provides a structured Internet interface for the sharing of information with individuals struggling with the consequences of rare developmental disorders. Large disease-impacted communities can support fundraising organizations that disseminate Web-based information through elegant websites run by professional staff. Such quality resources for families challenged by rare disorders are infrequently produced and, when available, are often dependent upon the continued efforts of a single individual.
The project endeavors to create an intuitive Web-based software system that allows a volunteer with limited technical computer skills to produce a useful rare disease website in a short time period. Such a system should provide access to emerging news and research findings, facilitate community participation, present summary information about the disorder, and allow for transient management by volunteers who are likely to change periodically.
The prototype portal was implemented using the WordPress software system with both existing and customized supplementary plug-in software modules. Gamification scoring features were implemented in a module, allowing editors to measure progress. The system was installed on a Linux-based computer server, accessible across the Internet through standard Web browsers.
A prototype PFOND system was implemented and tested. The prototype system features a structured organization with distinct partitions for background information, recent publications, and community discussions. The software design allows volunteer editors to create a themed website, implement a limited set of topic pages, and connect the software to dynamic RSS feeds providing information about recent news or advances. The prototype was assessed by a fraction of the disease sites developed (8 out of 27), including Aarskog-Scott syndrome, Aniridia, Adams-Oliver syndrome, Cat Eye syndrome, Kabuki syndrome, Leigh syndrome, Peters anomaly, and Rothmund-Thomson syndrome. The editor progress score was used to measure performance for a portion of sites.
The PFOND system provides a convenient and structured Internet resource for the facilitated creation of information resources for families confronted by rare disorders. The system empowers volunteers to participate in the creation of quality content, while allowing for the inevitable turnover of contributors over time. The next phase of PFOND development will focus on volunteer participation in system development and community engagement.
PMCID: PMC3742411  PMID: 23920006
medical informatics; medical genetics; inborn genetic disease; rare disease; social media; consumer participation
25.  Hepatitis B Virus mRNA-Mediated miR-122 Inhibition Upregulates PTTG1-Binding Protein, Which Promotes Hepatocellular Carcinoma Tumor Growth and Cell Invasion 
Journal of Virology  2013;87(4):2193-2205.
As the most abundant liver-specific microRNA, miR-122 is involved in diverse aspects of hepatic function and neoplastic transformation. Our previous study showed that miR-122 levels are significantly decreased in hepatitis B virus (HBV)-infected patients, which may facilitate viral replication and persistence (S. Wang, L. Qiu, X. Yan, W. Jin, Y. Wang, L. Chen, E. Wu, X. Ye, G. F. Gao, F. Wang, Y. Chen, Z. Duan, and S. Meng, Hepatology 55:730–741, 2012). Loss of miR-122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity.). In this study, we provide evidence that all HBV mRNAs harboring an miR-122 complementary site act as sponges to bind and sequester endogenous miR-122, indicating that the highly redundant HBV transcripts are involved in HBV-mediated miR-122 suppression. We next identified pituitary tumor-transforming gene 1 (PTTG1) binding factor (PBF) as a target of miR-122 and demonstrated that HBV replication causes an obvious increase in PBF levels. Furthermore, we observed that the miR-122 levels were decreased and PBF was upregulated in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). Overexpression and knockdown studies both revealed that PBF enhances proliferation and invasion of HCC cells, and silencing PBF resulted in a dramatic reduction of HCC tumor growth in vivo. Mechanistic analysis demonstrated that PBF interacts with PTTG1 and facilitates PTTG1 nuclear translocation, subsequently increasing its transcriptional activities. Therefore, we identified a novel HBV mRNA-miR-122-PBF regulatory pathway that facilitates malignant hepatocyte growth and invasion in CHB which may contribute to CHB-induced HCC development and progression. Our work underscores the reciprocal interplay of host miRNA sequestration and depletion by viral mRNAs, which may contribute to chronic-infection-related cancer.
PMCID: PMC3571498  PMID: 23221562

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