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1.  Weichang’an and 5-fluorouracil suppresses colorectal cancer in a mouse model 
AIM: To examine the effect of Weichang’an (WCA) and 5-fluorouracil (5-FU) on colorectal tumor and hepatic metastasis in a mouse model.
METHODS: Quantitative determination of hesperidin, the effective component in WCA decoction, was performed using HPLC. In vitro cytotoxicity of WCA was determined by treating HCT-116 cells with WCA diluents or serum extracted from rats that received WCA by oral gavage for 1 wk and MTT assays. Forty-eight nude mice received cecum implantation with tumor blocks subcutaneously amplified from human colon cancer cell line HCT-116. Mice were randomly divided into four treatment groups: control (CON), WCA, 5-FU and combination (WCA + 5-FU). Pathological examination of tumors consisted of tissue sectioning and hematoxylin and eosin staining. Tumor weight and size were measured, and the number of metastatic lesions was counted. Serum carcinoembryonic antigen (CEA) level was determined by ELISA. The expression levels of tumor genesis and metastasis-related proteins β-catenin and matrix metalloproteinase (MMP)-7 were measured by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry and immunoblotting. Cell fractionation was used to investigate intracellular distribution of β-catenin.
RESULTS: Parenchymal tumors were palpable in the abdomens of nude mice 2 wk post-implantation and orthotopic tumor formation rate was 100% in all groups. 5-FU treatment alone significantly decreased tumor weight compared to the CON group (1.203 ± 0.284 g vs 1.804 ± 0.649 g, P < 0.01). WCA treatment alone reduced the rate of metastasis (50% vs 100%, P < 0.05). Combination treatment of WCA + 5-FU was the most effective, reducing the tumor weight (1.140 ± 0.464 g vs 1.804 ± 0.649 g, P < 0.01) and size (1493.438 ± 740.906 mm3 vs 2180.259 ± 816.556 mm3, P < 0.05), the rate of metastases (40% vs 100%, P < 0.01), and serum CEA levels (31.263 ± 7.421 μg/L vs 43.040 ± 11.273 μg/L, P < 0.05). Expression of β-catenin and MMP-7 was decreased in drug-treated groups compared to controls, as detected using real-time quantitative RT-PCR, immunohistochemistry and immunoblotting, respectively. Cell fractionation assays revealed that nuclear translocation of β-catenin was reduced by WCA and/or 5-FU treatments.
CONCLUSION: Combination of WCA with 5-FU significantly inhibited colon tumor growth and hepatic metastases. Decreased expression of β-catenin and MMP-7 may be important.
PMCID: PMC4306156  PMID: 25632185
Colorectal cancer; Hepatic metastasis; Weichang’an formula; Orthotopic transplant nude mouse model; Chemotherapeutics 5-fluorouracil
2.  Performance Characteristics of Seven Bilevel Mechanical Ventilators in Pressure-Support Mode with Different Cycling Criteria: A Comparative Bench Study 
Pressure support ventilation from a bilevel device is a standard technique for non-invasive home ventilation. A bench study was designed to compare the performance and patient-ventilator synchronization of 7 bilevel ventilators, in the presence of system leaks.
Ventilators were connected to a Hans Rudolph Series 1101 lung simulator (compliance, 50 mL/cmH2O; expiratory resistance, 20 cmH2O/L/s; respiratory rate, 15 breaths/min; inspiratory time, 1.0 s). All ventilators were set at 15 cmH2O pressure support and 5 cmH2O positive end-expiratory pressure. Tests were conducted at 2 system leaks (12–15 and 25–28 L/min). The performance characteristics and patient-ventilator asynchrony were assessed, including flow, airway pressure, time, and workload.
The Breas Vivo30 could not synchronize with the simulator (frequent auto-triggering) at a leak of 25–28 L/min, but provided stable assisted ventilation when the leak was 12–15 L/min. Missed efforts and back-up ventilation occurred for the Weinmann VENTImotion and Airox Smartair Plus, requiring adjustment of trigger effort. All ventilators had a short trigger delay time (<200 ms), but significant differences between devices were found in triggering workload, pressurization appearance, tidal volume, and peak inspiratory flow. Premature cycling was frequent when the inspiratory termination criteria were at the highest sensitivity. Cycling synchronization was considerably improved by modifying expiratory triggering sensitivity settings, when available.
Performance and triggering workload varied significantly between bilevel ventilators, possibly due to software algorithm differences. Adjusting the cycling criteria settings can alter the shape of the inspiratory phase and peak expiratory flow, and improve patient-ventilator synchrony.
PMCID: PMC4315647  PMID: 25619202
Air Pressure; Continuous Positive Airway Pressure; Lung; Respiratory System; Ventilators, Mechanical
3.  Oxidative Stress Activates SIRT2 to Deacetylate and Stimulate Phosphoglycerate Mutase 
Cancer research  2014;74(13):3630-3642.
Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of nucleotide precursors and amino acids. Inhibition of PGAM by small RNAi or small molecule attenuates cell proliferation and tumor growth. PGAM activity is commonly upregulated in tumor cells, but how PGAM activity is regulated in vivo remains poorly understood. Here we report that PGAM is acetylated at lysine 100 (K100), an active site residue that is invariably conserved from bacteria, to yeast, plant, and mammals. K100 acetylation is detected in fly, mouse, and human cells and in multiple tissues and decreases PGAM2 activity. The cytosolic protein deacetylase sirtuin 2 (SIRT2) deacetylates and activates PGAM2. Increased levels of reactive oxygen species stimulate PGAM2 deacetylation and activity by promoting its interaction with SIRT2. Substitution of endogenous PGAM2 with an acetylation mimetic mutant K100Q reduces cellular NADPH production and inhibits cell proliferation and tumor growth. These results reveal a mechanism of PGAM2 regulation and NADPH homeostasis in response to oxidative stress that impacts cell proliferation and tumor growth.
PMCID: PMC4303242  PMID: 24786789
4.  Education influences the association between genetic variants and refractive error: a meta-analysis of five Singapore studies 
Human Molecular Genetics  2013;23(2):546-554.
Refractive error is a complex ocular trait governed by both genetic and environmental factors and possibly their interplay. Thus far, data on the interaction between genetic variants and environmental risk factors for refractive errors are largely lacking. By using findings from recent genome-wide association studies, we investigated whether the main environmental factor, education, modifies the effect of 40 single nucleotide polymorphisms on refractive error among 8461 adults from five studies including ethnic Chinese, Malay and Indian residents of Singapore. Three genetic loci SHISA6-DNAH9, GJD2 and ZMAT4-SFRP1 exhibited a strong association with myopic refractive error in individuals with higher secondary or university education (SHISA6-DNAH9: rs2969180 A allele, β = −0.33 D, P = 3.6 × 10–6; GJD2: rs524952 A allele, β = −0.31 D, P = 1.68 × 10−5; ZMAT4-SFRP1: rs2137277 A allele, β = −0.47 D, P = 1.68 × 10−4), whereas the association at these loci was non-significant or of borderline significance in those with lower secondary education or below (P for interaction: 3.82 × 10−3–4.78 × 10−4). The evidence for interaction was strengthened when combining the genetic effects of these three loci (P for interaction = 4.40 × 10−8), and significant interactions with education were also observed for axial length and myopia. Our study shows that low level of education may attenuate the effect of risk alleles on myopia. These findings further underline the role of gene–environment interactions in the pathophysiology of myopia.
PMCID: PMC3869359  PMID: 24014484
5.  Role of CYP2A13 in the bioactivation and lung tumorigenicity of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: in vivo studies using a CYP2A13-humanized mouse model 
Carcinogenesis  2013;35(1):131-137.
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is abundant in tobacco smoke, is a potent lung procarcinogen. The present study was aimed to prove that transgenic expression of human cytochrome P450 2A13 (CYP2A13), known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, will enhance NNK bioactivation and NNK-induced tumorigenesis in the mouse lung. Kinetic parameters of NNK bioactivation in vitro and incidence of NNK-induced lung tumors in vivo were determined for wild-type, Cyp2a5-null and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. As expected, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in Michaelis constant (K m) values for the formation of 4-oxo-4-(3-pyridyl)-butanal, representing the reactive intermediate that can lead to the formation of O6-methylguanine (O6-mG) DNA adducts; however, the gain of CYP2A13 at a fraction of the level of mouse lung CYP2A5 led to recovery of the activity in the lung, but not in the liver. The levels of O6-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of CYP2A13-humanized mice than in Cyp2a5-null mice. Moreover, incidences of lung tumorigenesis were significantly greater in CYP2A13-humanized mice than in Cyp2a5-null mice, and the magnitude of the differences in incidence was greater at low (30mg/kg) than at high (200mg/kg) NNK doses. These results indicate that CYP2A13 is a low K m enzyme in catalyzing NNK bioactivation in vivo and support the notion that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans.
PMCID: PMC3871935  PMID: 23917075
6.  Development of the fetal bone marrow niche and regulation of HSC quiescence and homing ability by emerging osteolineage cells 
Cell reports  2014;9(2):581-590.
Hematopoietic stem cells (HSC) reside within a specialized niche where interactions with vasculature, osteoblasts and stromal components regulate their self-renewal and differentiation. Little is known about bone marrow niche formation or the role of its cellular components in HSC development; therefore, we established the timing of murine fetal long bone vascularization and ossification relative to the onset of HSC activity. Adult-repopulating HSC emerged at E16.5, coincident with marrow vascularization, and were contained within the c-Kit+Sca-1+Lin− (KSL) population. We used Osterix-null (Osx−/−) mice that form vascularized marrow, but lack osteolineage cells to dissect the role(s) of these cellular components in HSC development. Osx−/− fetal bone marrow cells formed multi-lineage colonies in vitro, but were hyper-proliferative and failed to home to and/or engraft transplant recipients. Thus, in developing bone marrow, the vasculature can sustain multi-lineage progenitors, but interactions with osteolineage cells are needed to regulate LT-HSC proliferation and potential.
PMCID: PMC4266564  PMID: 25310984
7.  A Model of Metabolic Syndrome and Related Diseases with Intestinal Endotoxemia in Rats Fed a High Fat and High Sucrose Diet 
PLoS ONE  2014;9(12):e115148.
We sought develop and characterize a diet-induced model of metabolic syndrome and its related diseases.
The experimental animals (Spague-Dawley rats) were randomly divided into two groups, and each group was fed a different feed for 48 weeks as follows: 1) standard control diet (SC), and 2) a high sucrose and high fat diet (HSHF). The blood, small intestine, liver, pancreas, and adipose tissues were sampled for analysis and characterization.
Typical metabolic syndrome (MS), non-alcoholic fatty liver disease (NAFLD), and type II diabetes (T2DM) were common in the HSHF group after a 48 week feeding period. The rats fed HSHF exhibited signs of obesity, dyslipidemia, hyperglycaemia, glucose intolerance, and insulin resistance (IR). At the same time, these animals had significantly increased levels of circulating LPS, TNFα, and IL-6 and increased ALP in their intestinal tissue homogenates. These animals also showed a significant reduction in the expression of occluding protein. The HSHF rats showed fatty degeneration, inflammation, fibrosis, cirrhosis, and lipid accumulation when their liver pathologies were examined. The HSHF rats also displayed increased islet diameters from 12 to 24 weeks, while reduced islet diameters occurred from 36 to 48 weeks with inflammatory cell infiltration and islet fat deposition. The morphometry of adipocytes in HSHF rats showed hypertrophy and inflammatory cell infiltration. HSHF CD68 analysis showed macrophage infiltration and significant increases in fat and pancreas size. HSHF Tunel analysis showed significant increases in liver and pancreas cell apoptosis.
This work demonstrated the following: 1) a characteristic rat model of metabolic syndrome (MS) can be induced by a high sucrose and high fat diet, 2) this model can be used to research metabolic syndrome and its related diseases, such as NAFLD and T2DM, and 3) intestinal endotoxemia (IETM) may play an important role in the pathogenesis of MS and related diseases, such as NAFLD and T2DM.
PMCID: PMC4263741  PMID: 25502558
8.  Chondrocytes Transdifferentiate into Osteoblasts in Endochondral Bone during Development, Postnatal Growth and Fracture Healing in Mice 
PLoS Genetics  2014;10(12):e1004820.
One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.
Author Summary
During endochondral bone formation, which is responsible for the generation of most bones in mammals and many other species, osteoblasts deposit a bone-specific matrix on the surface of cartilage scaffolds made by chondrocytes and hypertrophic chondrocytes. It has long been thought that the terminally differentiated chondrocytes in this cartilage scaffold undergo cell death. Here we demonstrate that chondrocytes can transdifferentiate into osteoblasts and that these transdifferentiated osteoblasts represent a substantial fraction of the bone forming cells in mice, We also provide evidence that chondrocytes can transdifferentiate into osteoblasts during bone fracture repair, a process similar to endochondral bone formation.
PMCID: PMC4256265  PMID: 25474590
9.  Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation 
Genome Biology  2014;15(12):521.
The brown planthopper, Nilaparvata lugens, the most destructive pest of rice, is a typical monophagous herbivore that feeds exclusively on rice sap, which migrates over long distances. Outbreaks of it have re-occurred approximately every three years in Asia. It has also been used as a model system for ecological studies and for developing effective pest management. To better understand how a monophagous sap-sucking arthropod herbivore has adapted to its exclusive host selection and to provide insights to improve pest control, we analyzed the genomes of the brown planthopper and its two endosymbionts.
We describe the 1.14 gigabase planthopper draft genome and the genomes of two microbial endosymbionts that permit the planthopper to forage exclusively on rice fields. Only 40.8% of the 27,571 identified Nilaparvata protein coding genes have detectable shared homology with the proteomes of the other 14 arthropods included in this study, reflecting large-scale gene losses including in evolutionarily conserved gene families and biochemical pathways. These unique genomic features are functionally associated with the animal’s exclusive plant host selection. Genes missing from the insect in conserved biochemical pathways that are essential for its survival on the nutritionally imbalanced sap diet are present in the genomes of its microbial endosymbionts, which have evolved to complement the mutualistic nutritional needs of the host.
Our study reveals a series of complex adaptations of the brown planthopper involving a variety of biological processes, that result in its highly destructive impact on the exclusive host rice. All these findings highlight potential directions for effective pest control of the planthopper.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0521-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4269174  PMID: 25609551
11.  Association between serum free fatty acid levels and possible related factors in patients with type 2 diabetes mellitus and acute myocardial infarction 
Free fatty acids (FFAs) play importance roles in the development of diabetes and cardiovascular diseases. We measured serum FFA levels from type 2 diabetes mellitus (T2DM) and acute myocardial infarction (AMI) patients and assay the correlation between serum FFA levels and related factors. The present study was undertaken to investigate a possible relation between the changes in serum free fatty acid concentration with acute myocardial infarction and type 2 diabetes mellitus.
The study population consisted of 540 healthy individuals and 103 patients with T2DM, 59 patients with AMI and 21 volunteers. Serum FFAs were measured with high pressure liquid chromatography. Blood urea nitrogen and uric acid were measured in clinical laboratory, as were glycemic, lipid and blood routine parameters. We selected 242 individuals with age over 60 years, 143 healthy individuals and 52 patients with T2DM, 47 patients with AMI were incorporated into three groups as control group, T2DM group and AMI group. Associations were analyzed with stepwise regression analysis with adjusted for age, sex, body mass index.
Serum FFA levels were significantly higher in the age over 60 years individuals compared to 20 ~ 50 years (logFFA μmmol/L:2.60 ± 0.16 vs. 2.73 ± 0.18, P < .001) in the healthy group. We found lower FFA levels in the AMI compared to the T2DM and control group (2.64 ± 0.22 vs. 2.72 ± 0.13&2.72 ± 0.16, respectively, P < .05&P < 0.01) in the age over 60, fasting blood glucose level higher in the AMI and T2DM (5.78 ± 1.32&7.75 ± 2.93 mmol/L vs. 4.90 ± 0.47 mmol/L, P < .01&P < .001) compared with the normal group, HDL level (1.01 ± 0.22&0.98 ± 0.18 mmol/L vs.1.30 ± 0.22 mmol/L, P < .001&P < .001). With stepwise regression analysis, the serum FFA levels was positively associated with the HDL in the control group (YlogFFA = 2.32 + 0.33XHDL, R = 0.26, P < .01) and T2MD (YlogFFA = 2.46 + 0.27XHDL, R = 0.36, P < .05), AST in AMI (YlogFFA =2.24 + 0. 015XAST, R = 0.49, P < .01).
Compared to control group, serum FFA levels were decreased only in AMI group, while HDL level was increased in both AMI and T2DM group. The serum FFA levels were positive association with the HDL level in both T2DM and control group, FFA levels were positive association with AST in AMI.
PMCID: PMC4236447  PMID: 25399059
Acute myocardial infarction (AMI); Type 2 diabetes mellitus (T2DM); Free fatty acid (FFA); High density lipoprotein cholesterol (HDL-c); Aspartate aminotransferase (AST)
12.  A Three-Dimensional Computational Model of Collagen Network Mechanics 
PLoS ONE  2014;9(11):e111896.
Extracellular matrix (ECM) strongly influences cellular behaviors, including cell proliferation, adhesion, and particularly migration. In cancer, the rigidity of the stromal collagen environment is thought to control tumor aggressiveness, and collagen alignment has been linked to tumor cell invasion. While the mechanical properties of collagen at both the single fiber scale and the bulk gel scale are quite well studied, how the fiber network responds to local stress or deformation, both structurally and mechanically, is poorly understood. This intermediate scale knowledge is important to understanding cell-ECM interactions and is the focus of this study. We have developed a three-dimensional elastic collagen fiber network model (bead-and-spring model) and studied fiber network behaviors for various biophysical conditions: collagen density, crosslinker strength, crosslinker density, and fiber orientation (random vs. prealigned). We found the best-fit crosslinker parameter values using shear simulation tests in a small strain region. Using this calibrated collagen model, we simulated both shear and tensile tests in a large linear strain region for different network geometry conditions. The results suggest that network geometry is a key determinant of the mechanical properties of the fiber network. We further demonstrated how the fiber network structure and mechanics evolves with a local formation, mimicking the effect of pulling by a pseudopod during cell migration. Our computational fiber network model is a step toward a full biomechanical model of cellular behaviors in various ECM conditions.
PMCID: PMC4227658  PMID: 25386649
13.  Hydroxyapatite-Coated Sillicone Rubber Enhanced Cell Adhesion and It May Be through the Interaction of EF1β and γ-Actin 
PLoS ONE  2014;9(11):e111503.
Silicone rubber (SR) is a common soft tissue filler material used in plastic surgery. However, it presents a poor surface for cellular adhesion and suffers from poor biocompatibility. In contrast, hydroxyapatite (HA), a prominent component of animal bone and teeth, can promote improved cell compatibility, but HA is an unsuitable filler material because of the brittleness in mechanism. In this study, using a simple and economical method, two sizes of HA was applied to coat on SR to counteract the poor biocompatibility of SR. Surface and mechanical properties of SR and HA/SRs confirmed that coating with HA changes the surface topology and material properties. Analysis of cell proliferation and adhesion as well as measurement of the expression levels of adhesion related molecules indicated that HA-coated SR significantly increased cell compatibility. Furthermore, mass spectrometry proved that the biocompatibility improvement may be related to elongation factor 1-beta (EF1β)/γ-actin adjusted cytoskeletal rearrangement.
PMCID: PMC4227678  PMID: 25386892
14.  An ER-associated miRNA signature predicts prognosis in ER-positive breast cancer 
Breast cancer patients with positive estrogen receptor (ER) have a better prognosis. However, no prognostic miRNA signature was reported in the ER-positive breast cancer. The aim of the study was to identify and assess the prognostic significance of a miRNA signature in ER-positive breast cancer.
Two cohorts from The Cancer Genome Atlas (TCGA) dataset were used as training (n =596) and testing set (n =319). Differential expression profiling was identified in the training set. And the prognostic value of the miRNA signature was then assessed in the two cohorts.
A total of 14 miRNAs were observed to be associated with the status of ER by significance analysis of microarrays (SAM) in the training set. Patients were characterized as high score or low score group according to the calculated risk scores from each miRNA. And patients in high score group had worse overall survival compared with those in low score group both in the training and testing set.
Our study revealed a miRNA signature including 14 miRNAs associated with ER status which could act as a prognostic marker in ER-positive breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-014-0094-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4232612  PMID: 25373603
Breast cancer; ER status; miRNA signature; prognosis
15.  The MluI Cell Cycle Box (MCB) Motifs, but Not Damage-Responsive Elements (DREs), Are Responsible for the Transcriptional Induction of the rhp51+ Gene in Response to DNA Replication Stress 
PLoS ONE  2014;9(11):e111936.
DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.
PMCID: PMC4221157  PMID: 25372384
16.  Do we really need closed-suction drainage in total hip arthroplasty? A meta-analysis 
International Orthopaedics  2013;37(11):2109-2118.
The clinical use of closed-suction drainage, which aims to reduce postoperative wound haematomas and infection, is common. This study was performed to determine whether closed-suction drainage is safe and effective in promoting wound healing and reducing blood loss and other complications compared with no-drainage in total hip arthroplasty.
The literature search was based on PubMed, the Cochrane Library, MEDLINE, and EMBASE. The data were evaluated using the generic evaluation tool designed by the Cochrane Bone, Joint and Muscle Trauma Group, and then analysed using RevMan 5.0. Twenty randomised controlled trials involving 3,186 patients were included in our analysis.
The results of our meta-analysis indicate that closed-suction drainage reduces the requirement for dressing reinforcement, but increases the rate of homologous blood transfusion. No significant difference was observed in the incidence of infection, blood loss, changes in haemoglobin and haematocrit, functional assessment, or other complications when the drainage group was compared with the no-drainage group.
Our results of the comparison between closed-suction drainage and no drainage in THA have indicated that the routine use of closed-suction drainage for elective total hip arthroplasty may be of more harm than benefit.
PMCID: PMC3824906  PMID: 23982636
Closed-suction drainage; Total hip arthroplasty; Blood loss; Transfusion; Meta-analysis
17.  Elevated Cardiac Markers in Chronic Kidney Disease as a Consequence of Hyperphosphatemia-Induced Cardiac Myocyte Injury 
Elevated cardiac markers (CMs) and hyperphosphatemia are commonly encountered in patients with chronic kidney diseases (CKD), but the causal relationship between them has not been established.
We enrolled 151 patients with different kidney functions in a cross-sectional study to explore the relationship of serum phosphorus with CMs, including cardiac troponin T (cTnT), myoglobin (MYO), creatine kinase-MB (CK-MB), and brain natriuretic peptide (BNP). Then, the effect of reducing phosphorus levels on CMs by taking phosphate binder for 3 months was prospectively observed in 64 hemodialysis patients. Finally, human cardiomyocytes were exposed to different concentrations of inorganic phosphorus to examine its underlying mechanism.
1) Serum phosphorus and CMs gradually increased as the glomerular filtration rate declined in CKD patients (p<0.01). 2) Elevation of CMs was much greater and cardiac structure and function were worse in CKD patients who had higher serum phosphorus concentrations (p<0.05). 3) Serum phosphorus level positively correlated with cTnT, MYO, and BNP in CKD patients (p<0.001). 4) In hemodialysis patients, the reduction of cTnT, MYO, and CK-MB was synchronous with the pharmacologically-induced decline of serum phosphorus level. However, levels of serum Fibroblast growth factor 23 (FGF23) had no statistical decrease. 5) Simulated hyperphosphatemia inhibited proliferation of human cardiomyocytes in a time- and concentration-dependent manner.
Hyperphosphatemia may induce myocardial damage in CKD patients, possibly through triggering apoptosis of human cardiomyocytes, and this could account for the elevated cardiac markers in CKD patients.
PMCID: PMC4214699  PMID: 25344353
Apoptosis; Biological Markers; Fibroblast Growth Factors; Hyperphosphatemia; Renal Insufficiency, Chronic; Troponin T
18.  In-Depth Transcriptome Analysis of the Red Swamp Crayfish Procambarus clarkii 
PLoS ONE  2014;9(10):e110548.
The red swamp crayfish Procambarus clarkii is a highly adaptable, tolerant, and fecund freshwater crayfish that inhabits a wide range of aquatic environments. It is an important crustacean model organism that is used in many research fields, including animal behavior, environmental stress and toxicity, and studies of viral infection. Despite its widespread use, knowledge of the crayfish genome is very limited and insufficient for meaningful research. This is the use of next-generation sequencing techniques to analyze the crayfish transcriptome. A total of 324.97 million raw reads of 100 base pairs were generated, and a total of 88,463 transcripts were assembled de novo using Trinity software, producing 55,278 non-redundant transcripts. Comparison of digital gene expression between four different tissues revealed differentially expressed genes, in which more overexpressed genes were found in the hepatopancreas than in other tissues, and more underexpressed genes were found in the testis and the ovary than in other tissues. Gene ontology (GO) and KEGG enrichment analysis of differentially expressed genes revealed that metabolite- and immune-related pathway genes were enriched in the hepatopancreas, and DNA replication-related pathway genes were enriched in the ovary and the testis, which is consistent with the important role of the hepatopancreas in metabolism, immunity, and the stress response, and with that of the ovary and the testis in reproduction. It was also found that 14 vitellogenin transcripts were highly expressed specifically in the hepatopancreas, and 6 transcripts were highly expressed specifically in the ovary, but no vitellogenin transcripts were highly expressed in both the hepatopancreas and the ovary. These results provide new insight into the role of vitellogenin in crustaceans. In addition, 243,764 SNP sites and 43,205 microsatellite sequences were identified in the sequencing data. We believe that our results provide an important genome resource for the crayfish.
PMCID: PMC4206422  PMID: 25338101
19.  The Association between Bile Salt Export Pump Single-Nucleotide Polymorphisms and Primary Biliary Cirrhosis Susceptibility and Ursodeoxycholic Acid Response 
Disease Markers  2014;2014:350690.
Background. Primary biliary cirrhosis (PBC) is a chronic and progressive cholestasis liver disease. Bile salt export pump (BSEP) is the predominant bile salt efflux system of hepatocytes. BSEP gene has been attached great importance in the susceptibility of PBC and the response rate of ursodeoxycholic acid (UDCA) treatment of PBC patients. Methods. In this study, TaqMan assay was used to genotype four variants of BSEP, and the Barcelona criteria were used for evaluating the response rate of UDCA treatment. Results. Variant A allele of BSEP rs473351 (dominant model, OR = 2.063; 95% CI, 1.254–3.393; P = 0.004) was highly associated with PBC susceptibility. On the contrary, variant A allele of BSEP rs2287618 (dominant model, OR = 0.617; 95% CI, 0.411–0.928; P = 0.020) provided a protective role and Barcelona evaluation criterion indicated that the frequency of variant allele at BSEP rs2287618 was significantly decreased in UDCA-responsive PBC patients (P = 0.021). Conclusion. These results suggested that BSEP rs473351 was closely associated with the susceptibility of PBC and if people with BSEP rs2287618 were diagnosed as PBC, the UDCA treatment was not satisfactory. Larger studies with mixed ethnicity subjects and stratified by clinical and subclinical characteristics are needed to validate our findings.
PMCID: PMC4216684  PMID: 25392597
20.  Meta-Analysis of the Prognostic Value of Smad4 Immunohistochemistry in Various Cancers 
PLoS ONE  2014;9(10):e110182.
Accumulating evidence indicates that Smad4 (DPC4) plays a fundamental role in the development and prognosis of several types of cancer. The objective of this study was to conduct a meta-analysis to evaluate whether the loss of Smad4 staining could serve as a prognostic marker.
A comprehensive meta-analysis was conducted using major useful databases to determine the relationship between the immunohistochemical detection of Smad4 and the survival of patients with various cancers. We used hazard ratios (HRs) with 95% confidence interval (CIs) as the effect estimation to evaluate the association of Smad4 with overall survival (OS), cancer-specific survival (CSS) or recurrence-free survival (RFS). The relationship between the clinical characteristics of patients and Smad4 was also evaluated using the odds ratio (OR).
A total of 7570 patients from 26 studies were included in the analysis. The pooled results showed that loss of Smad4 staining was a negative predictor of OS with an HR of 1.97 (95% CI: 1.55–2.51; Pheterogeneity<0.001) and CSS/RFS (HR = 1.81; 95% CI: 1.30–2.54; Pheterogeneity<0.001). In addition, loss of Smad4 staining was more likely to be found in older (OR = 1.69, 95% CI: 1.09–2.61; Pheterogeneity = 0.648) colorectal cancer patients with a late tumor stage (OR = 2.31, 95% CI: 1.71–3.10; Pheterogeneity = 0.218) and in gastric cancer patients with lymph node metastasis (OR = 2.11, 95% CI: 1.03–4.34; Pheterogeneity = 0.038).
Based on these results, our meta-analysis provided evidence that loss of Smad4 staining could act as an unfavorable biomarker in the prognosis of various cancers and should be used as a powerful tool in future clinical trials.
PMCID: PMC4198206  PMID: 25333693
21.  Impact of ozone exposure on the response to glucocorticoid in a mouse model of asthma: involvements of p38 MAPK and MKP-1 
Respiratory Research  2014;15(1):126.
Molecular mechanisms involved in the oxidative stress induced glucocorticoids insensitivity remain elusive. The mitogen-activated protein kinase phosphatase (MKP) 1 mediates a part of glucocorticoids action and can be modified by exogenous oxidants. Whether oxidant ozone (O3) can affect the function of MKP-1 and hence blunt the response to corticotherapy is not clear.
Here we employed a murine model of asthma established with ovalbumin (OVA) sensitization and challenge to evaluate the influence of O3 on the inhibitory effect of dexamethasone on AHR and airway inflammation, and by administration of SB239063, a selective p38 MAPK inhibitor, to explore the underlying involvements of the activation of p38 MAPK and the expression of MKP-1.
Ozone exposure not only aggravated the pulmonary inflammation and AHR, but also decreased the inhibitory effects of dexamethasone, accompanied by the elevated oxidative stress, airway neutrophilia, enhanced phosphorylation of p38 MAPK, and upregulated expression of IL-17. Administration of SB239063 caused significant inhibition of the p38 MAPK phosphorylation, alleviation of the airway neutrophilia, and decrement of the ozone-induced IL-17 expression, and partly restored the ozone-impaired effects of dexamethasone. Ozone exposure not only decreased the protein expression of MKP-1, but also diminished the dexamethasone-mediated induction process of MKP-1 mRNA and protein expression.
The glucocorticoids insensitivity elicited by ozone exposure on current asthma model may involve the enhanced phosphorylation of p38 MAPK and disturbed expression of MKP-1.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-014-0126-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4196074  PMID: 25287866
Ozone; Glucocorticoids insensitivity; p38 MAPK; MKP-1; IL-17; Asthma
22.  Multiplex sequencing of pooled mitochondrial genomes—a crucial step toward biodiversity analysis using mito-metagenomics 
Nucleic Acids Research  2014;42(22):e166.
The advent in high-throughput-sequencing (HTS) technologies has revolutionized conventional biodiversity research by enabling parallel capture of DNA sequences possessing species-level diagnosis. However, polymerase chain reaction (PCR)-based implementation is biased by the efficiency of primer binding across lineages of organisms. A PCR-free HTS approach will alleviate this artefact and significantly improve upon the multi-locus method utilizing full mitogenomes. Here we developed a novel multiplex sequencing and assembly pipeline allowing for simultaneous acquisition of full mitogenomes from pooled animals without DNA enrichment or amplification. By concatenating assemblies from three de novo assemblers, we obtained high-quality mitogenomes for all 49 pooled taxa, with 36 species >15 kb and the remaining >10 kb, including 20 complete mitogenomes and nearly all protein coding genes (99.6%). The assembly quality was carefully validated with Sanger sequences, reference genomes and conservativeness of protein coding genes across taxa. The new method was effective even for closely related taxa, e.g. three Drosophila spp., demonstrating its broad utility for biodiversity research and mito-phylogenomics. Finally, the in silico simulation showed that by recruiting multiple mito-loci, taxon detection was improved at a fixed sequencing depth. Combined, these results demonstrate the plausibility of a multi-locus mito-metagenomics approach as the next phase of the current single-locus metabarcoding method.
PMCID: PMC4267667  PMID: 25294837
23.  Mitogenic and Oncogenic Stimulation of K433 Acetylation Promotes PKM2 Protein Kinase Activity and Nuclear Localization 
Molecular cell  2013;52(3):340-352.
Alternative splicing of the PKM2 gene produces two isoforms, M1 and M2, which are preferentially expressed in adult and embryonic tissues, respectively. The M2 isoform is reexpressed in human cancer and has nonmetabolic functions in the nucleus as a protein kinase. Here, we report that PKM2 is acetylated by p300 acetyltransferase at K433, which is unique to PKM2 and directly contacts its allosteric activator, fructose 1,6-bisphosphate (FBP). Acetylation prevents PKM2 activation by interfering with FBP binding and promotes the nuclear accumulation and protein kinase activity of PKM2. Acetylationmimetic PKM2(K433) mutant promotes cell proliferation and tumorigenesis. K433 acetylation is decreased by serum starvation and cell-cell contact, increased by cell cycle stimulation, epidermal growth factor (EGF), and oncoprotein E7, and enriched in breast cancers. Hence, K433 acetylation links cell proliferation and transformation to the switch of PKM2 from a cytoplasmic metabolite kinase to a nuclear protein kinase.
PMCID: PMC4183148  PMID: 24120661
24.  Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth 
Molecular cell  2013;51(4):506-518.
Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.
PMCID: PMC4180208  PMID: 23932781
25.  High-resolution melting analysis reveals genetic polymorphisms in MicroRNAs confer hepatocellular carcinoma risk in Chinese patients 
BMC Cancer  2014;14(1):643.
Although several single-nucleotide polymorphisms in microRNA (miRNA) genes have been associated with primary hepatocellular carcinoma, published findings regarding this relationship are inconsistent and inconclusive.
The high-resolution melting (HRM) analysis was used to determine whether the occurrence of the SNPs of miR-146a C > G (rs2910164), miR-196a2 C > T (rs11614913), miR-301b A > G (rs384262), and miR-499 C > T (rs3746444) differs in frequency-matched 314 HCC patients and 407 controls by age and sex.
The groups’ genotype distributions of miR-196a2 C > T and miR-499 C > T differed significantly (P < 0.01), both of them increased the risk of HCC in different dominant genetic models (P < 0.01); compared with individuals carrying one or neither of the unfavorable genotypes, individuals carrying both unfavorable genotypes (CT + CC) had a 3.11-fold higher HCC risk (95% confidence interval (CI), 1.89–5.09; P = 7.18 × 10−6). Moreover, the allele frequency of miR-499 C > T was significantly different between the two groups, and the HCC risk of carriers of the C allele was higher than that of carriers of the T allele (odds ratio, 1.53; 95% CI, 1.15-2.03; P = 0.003). Further, we found that the activated partial thromboplastin time (APTT) in HCC patients with miR-196a2 CC genotype was longer than patients with TT genotypes (P < 0.05), and HCC patients with miR-499 C allele had higher serum levels of direct bilirubin, globulin, γ-glutamyltranspeptidase, alkaline phosphatase, and lower serum cholinesterase (P < 0.05).
Our findings suggest that the SNPs in miR-196a2 C > T and miR-499 C > T confer HCC risk and that affect the clinical laboratory characteristics of HCC patients.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-643) contains supplementary material, which is available to authorized users.
PMCID: PMC4161871  PMID: 25176041
Hepatocellular carcinoma; MicroRNA; High-resolution melting; Single-nucleotide polymorphisms

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