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1.  Kruppel-like factor 5 Controls Villus Formation and Initiation of Cytodifferentiation in the Embryonic Intestinal Epithelium 
Developmental biology  2012;375(2):128-139.
Kruppel-like factor 5 (Klf5) is a transcription factor expressed by embryonic endodermal progenitors that form the lining of the gastrointestinal tract. A Klf5 floxed allele was efficiently deleted from the intestinal epithelium by a Cre transgene under control of the Shh promoter resulting in the inhibition of villus morphogenesis and epithelial differentiation. Although proliferation of the intestinal epithelium was maintained, the expression of Elf3, Pparγ, Atoh1, Ascl2, Neurog3, Hnf4α, Cdx1, and other genes associated with epithelial cell differentiation was inhibited in the Klf5-deficient intestines. At E18.5, Klf5Δ/Δ fetuses lacked the apical brush border characteristic of enterocytes, and a loss of goblet and enteroendocrine cells was observed. The failure to form villi was not attributable to the absence of HH or PDGF signaling, known mediators of this developmental process. Klf5-deletion blocked the decrease in FoxA1 and Sox9 expression that accompanies normal villus morphogenesis. KLF5 directly inhibited activity of the FoxA1 promoter, and in turn FOXA1 inhibited Elf3 gene expression in vitro, linking the observed loss of Elf3 with the persistent expression of FoxA1 observed in Klf5-deficient mice. Genetic network analysis identified KLF5 as a key transcription factor regulating intestinal cell differentiation and cell adhesion. These studies indicate a novel requirement for KLF5 to initiate morphogenesis of the early endoderm into a compartmentalized intestinal epithelium comprised of villi and terminally differentiated cells.
PMCID: PMC3582784  PMID: 23266329
Elf3; FoxA1; intestine development; cell adhesion
2.  Design and Exploration of Novel Boronic Acid Inhibitors Reveals Important Interactions with a Clavulanic Acid-Resistant Sulfhydryl-Variable (SHV) β-Lactamase 
Journal of medicinal chemistry  2013;56(3):1084-1097.
Inhibitor resistant (IR) class A β-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV β-lactamase, from Klebsiella pneumoniae, results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting β-lactamases revealed that only –Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM Ki for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR β-lactamases.
PMCID: PMC3943433  PMID: 23252553
3.  Survival Benefit of Traditional Chinese Herbal Medicine (A Herbal Formula for Invigorating Spleen) in Gastric Cancer Patients with Peritoneal Metastasis 
Objective. We evaluated the efficiency of traditional Chinese herbal medicine (a compound herbal formula for invigorating spleen) as a complementary and alternative therapy for gastric cancer patients with peritoneal metastasis. Methods. Between 2001 and 2012, 93 gastric cancer patients with peritoneal metastasis were enrolled in this study. The effect of traditional Chinese herbal medicine on their long-term outcome was investigated. Kaplan-Meier method was used to assess the difference in survival time, and Cox proportional hazards regression analysis was performed to identify independent prognostic factors. Result. First-line palliative chemotherapy plus traditional Chinese herbal medicine was performed in 47 patients and the other 46 patients received chemotherapy alone. The overall survival was different between patients with and without traditional Chinese herbal medicine (12.0 versus 10.5 months; P = 0.046). According to the Cox proportional hazard model, first-line chemotherapy cycle (hazards ratio [HR] = 0.527; 95% CI = 0.323~0.860) and TCHM (hazards ratio [HR] = 0.644; 95% CI = 0.481~0.992) were selected as independent prognostic factors for survival. Conclusion. The results suggest that traditional Chinese herbal medicine could improve the prognosis of the gastric cancer patients with peritoneal metastasis.
PMCID: PMC3958679  PMID: 24723961
4.  Inhibition of metastasis by HEXIM1 through effects on cell invasion and angiogenesis 
Oncogene  2012;32(33):3829-3839.
We report on the role of Hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) as an inhibitor of metastasis. HEXIM1 expression is decreased in human metastatic breast cancers when compared to matched primary breast tumors. Similarly we observed decreased expression of HEXIM1 in lung metastasis when compared to primary mammary tumors in a mouse model of metastatic breast cancer, the Polyoma Middle-T antigen (PyMT) transgenic mouse. Re-expression of HEXIM1 (through transgene expression or localized delivery of a small molecule inducer of HEXIM1 expression, Hexamethylene-bis-acetamide) in PyMT mice resulted in inhibition of metastasis to the lung. Our present studies indicate that HEXIM1 downregulation of HIF-1α protein allows not only for inhibition of VEGF-regulated angiogenesis, but also inhibition of compensatory pro-angiogenic pathways and recruitment of bone marrow derived cells (BMDCs). Another novel finding is that HEXIM1 inhibits cell migration and invasion, that can be partly attributed to decreased membrane localization of the 67kDa laminin receptor, 67LR, and inhibition of the functional interaction of 67LR with laminin. Thus HEXIM1 re-expression in breast cancer has therapeutic advantages by simultaneously targeting more than one pathway involved in angiogenesis and metastasis. Our results also support the potential for HEXIM1 to indirectly act on multiple cell types to suppress metastatic cancer.
PMCID: PMC3525740  PMID: 22964639
HEXIM1; breast cancer; metastasis; angiogenesis
5.  Design, Synthesis and Characterization of Novel Small Molecular Inhibitors of Ephrin-B2 Binding to EphB4 
Biochemical pharmacology  2012;85(4):10.1016/j.bcp.2012.12.005.
EphB4 is a member of the large Eph receptor tyrosine kinase family. By interacting with its preferred ligand ephrin-B2, which is also a transmembrane protein, EphB4 plays a role in a variety of physiological and pathological processes ranging from bone remodeling to cancer malignancy. EphB4-ephrin-B2 binding occurs at sites of contact between cells. Ephrin-B2 causes EphB4 clustering and increased kinase activity to generate downstream signals that affect cell behavior. Previous work identified a high-affinity antagonistic peptide that targets EphB4, named TNYL-RAW. This peptide is 15 amino acid long, has a molecular weight of ~1,700 Da and binds to the ephrin-binding pocket of EphB4. Here we report the structure-based design and chemical synthesis of two novel small molecules of ~600–700 Da, which were designed starting from the small and functionally critical C-terminal portion of the TNYL-RAW peptide. These compounds inhibit ephrin-B2 binding to EphB4 at low micromolar concentrations. Additionally, although the ephrin-B2 ligand can interact with multiple other Eph receptors besides EphB4, the two compounds retain the high selectivity of the TNYL-RAW peptide in targeting EphB4. TNYL-RAW peptide displacement experiments using the more potent of the two compounds, compound 5, suggest a competitive mode of inhibition. These EphB4 antagonistic compounds can serve as promising templates for the further development of small molecule drugs targeting EphB4.
PMCID: PMC3807243  PMID: 23253822
Small molecular inhibitors; Eph receptors; structure-based drug design; tumor angiogenesis; protein-protein interactions
6.  Asymmetric Ligand Binding Facilitates Conformational Transitions in Pentameric Ligand-Gated Ion Channels 
The anesthetic propofol inhibits the currents of the homo-pentameric ligand-gated ion channel GLIC, yet the crystal structure of GLIC with five propofol molecules bound symmetrically shows an open-channel conformation. To address this dilemma and determine if symmetry of propofol binding sites affects the channel conformational transition, we performed a total of 1.5 As of molecular dynamics simulations for different GLIC systems with propofol occupancies of 0, 1, 2, 3, and 5. GLIC without propofol binding or with five propofol molecules bound symmetrically showed similar channel conformation and hydration status over multiple replicates of 100-ns simulations. In contrast, asymmetric binding to one, two or three equivalent sites in different subunits accelerated the channel dehydration, which was accompanied by increased conformational heterogeneity of the pore and shifted the lateral and radial tilting angles of the pore-lining TM2 towards a closed-channel conformation. The results differentiate two groups of systems based on the propofol binding symmetry. The difference between symmetric and asymmetric groups is correlated with the variance in the propofol-binding cavity adjacent to the hydrophobic gate and the force imposed by the bound propofol. Asymmetrically bound propofol produced greater variance in the cavity size that could further elevate the conformation heterogeneity. The force trajectory generated by propofol in each subunit over the course of a simulation exhibits an ellipsoidal shape, which has the larger component tangential to the pore. Asymmetric propofol binding creates an unbalanced force that expedites the channel conformation transitions. The findings from this study not only suggest that asymmetric binding underlies the propofol functional inhibition of GLIC, but also advocate for the role of symmetry breaking in facilitating channel conformational transitions.
PMCID: PMC3582375  PMID: 23339564
anesthetics; propofol; GLIC; pLGICs; ion channels; molecular dynamics
7.  Posterior short segment pedicle screw fixation and TLIF for the treatment of unstable thoracolumbar/lumbar fracture 
Currently, Posterior Short Segment Pedicle Screw Fixation is a popular procedure for treating unstable thoracolumbar/lumbar burst fracture. But progressive kyphosis and a high rate of hardware failure because of lack of the anterior column support remains a concern. The efficacy of different methods remains debatable and each technique has its advantages and disadvantages.
A consecutive series of 20 patients with isolated thoracolumbar/lumbar burst fractures were treated by posterior short segment pedicle screw fixation and transforaminal thoracolumbar/lumbar interbody fusion (TLIF) between January 2005 and December 2007. All patients were followed up for a minimum of 2 years. Demographic data, neurologic status, anterior vertebral body heights, segmental Cobb angle and treatment-related complications were evaluated.
The mean operative time was 167 minutes (range, 150–220). Blood loss was 450 ~ 1200 ml, an average of 820 ml. All patients recovered with solid fusion of the intervertebral bone graft, without main complications like misplacement of the pedicle screw, nerve or vessel lesion or hard ware failure. The post-operative radiographs demonstrated a good fracture reduction and it was well maintained until the bone graft fusion. Neurological recovery of one to three Frankel grade was seen in 14 patients with partial neurological deficit, three grades of improvement was seen in one patient, two grades of improvement was observed in 6 patients and one grade of improvement was found in 6 patients. All the 6 patients with no paraplegia on admission remained neurological intact, and in one patient with Frankel D on admission no improvement was observed.
Posterior short-segment pedicle fixation in conjunction with TLIF seems to be a feasible option in the management of selected thoracolumbar/lumbar burst fractures, thereby addressing all the three columns through a single approach with less trauma and good results.
PMCID: PMC3930337  PMID: 24517217
Short segment fixation; Thoracic vertebrae; Lumbar vertebrae; Unstable burst fractures; Pedicle screw; TLIF
8.  Prevalence and Correlates of Cigarette Smoking among Chinese Schizophrenia Inpatients Receiving Antipsychotic Mono-Therapy 
PLoS ONE  2014;9(2):e88478.
To investigate the prevalence rate of cigarette smoking and its socio-demographic and clinical correlates in Chinese schizophrenia inpatients receiving antipsychotic mono-therapy.
This study was a cross-sectional, two-site, hospital-based survey. Four hundred and twenty-nine schizophrenia patients (male/female: 66.9% vs. 33.1%) were consecutively recruited from psychosis inpatient wards of two large specialty psychiatric hospitals in mainland China. Patients were assessed using a cigarette smoking questionnaire, the Positive and Negative Symptom Scale, the Simpson Angus Scale, the Barnes Akathisia Rating Scale, and the Abnormal Involuntary Movement Scale. Socio-demographic and other clinical data were also collected. We calculated the prevalence of current smoking in our sample as well as its indirectly standardized prevalence ratio (ISPR) using data from the 2010 Global Adult Tobacco Survey in China.
The prevalence rate of current smoking was 40.6% in our sample, and 57.5% in males and 6.3% in females. The ISPRs of all patients, men and women were 1.11(95%CI: 0.95∼1.29), 1.07(95%CI = 0.91∼1.24) and 4.64(95%CI = 2.12∼8.82), respectively. The overall and male-specific prevalence of current smoking did not differ significantly between patients and the general population. In multiple logistic regression analysis, male sex, older age, poor marital status, alcohol use, use of first-generation antipsychotics, longer duration of illness, more frequent hospitalizations, and more severe negative symptoms were independently associated with current smoking.
Male Chinese inpatients with schizophrenia who received a mono-therapy of antipsychotics were not more likely to smoke than the general population. Cigarette smoking is more common in schizophrenia patients with more severe illness.
PMCID: PMC3919787  PMID: 24520390
9.  Design and Synthesis of Potent Bivalent Peptide Agonists Targeting the EphA2 Receptor 
ACS medicinal chemistry letters  2013;4(3):10.1021/ml3004523.
Designing potent and selective peptides and small molecules that target Eph receptor tyrosine kinases remains a challenge and new strategies are needed for developing novel and potent ligands for these receptors. In this study, we performed a structure-activity relationship study of a previously identified 12 amino acid-long peptide, SWL, by alanine scanning to identify residues important for receptor binding. To further enhance and optimize the receptor binding affinity of the SWL peptide, we applied the concept of bivalent ligand design to synthesize several SWL-derived dimeric peptides as novel ligands capable of binding simultaneously to two EphA2 receptor molecules. The dimeric peptides possess higher receptor binding affinity than the original monomeric SWL peptide, consistent with bivalent binding. The most potent dimeric peptide, a SWL dimer with a 6 carbon linker, has about 13 fold increased potency compared to SWL. Furthermore, similar to SWL, the dimeric peptide is an agonist and can promote EphA2 tyrosine phosphorylation (activation) in cultured cells.
PMCID: PMC3807576  PMID: 24167659
peptide inhibitors; Eph receptors; structure-based drug design; protein-protein interactions
10.  NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR 
Biochimica et biophysica acta  2012;1828(2):398-404.
The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the 2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury, et. al. 2011). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.
PMCID: PMC3534943  PMID: 23000369
halothane; ketamine; general anesthetics; NMR; α4β2 nAChR; protein dynamics
11.  Critical Role in CXCR4 Signaling and Internalization of the Polypeptide Main Chain in the Amino Terminus of SDF-1α Probed by Novel N-Methylated Synthetically and Modularly Modified Chemokine Analogues 
Biochemistry  2012;51(30):5951-5957.
The replication of human immunodeficiency virus type 1 (HIV-1) can be profoundly inhibited by the natural ligands of two major HIV-1 coreceptors, CXCR4 and CCR5. Stromal cell-derived factor-1α (SDF-1α) is a natural ligand of CXCR4. We have recently developed a synthetic biology approach of using synthetically and modularly modified (SMM)-chemokines to dissect various aspects of the structure-function relationship of chemokines and their receptors. Here, we used this approach to design novel SMM-SDF-1α analogues containing unnatural N-methylated residues in the amino terminus to investigate whether the polypeptide main chain amide bonds in the N-terminus of SDF-1α play a role in SDF-1α signaling via CXCR4 and/or receptor internalization. The results show that SDF-1α analogues with a modified N-methylated main chain at position 2, 3, or 5 retain significant CXCR4 binding and yet completely lose signaling activities. Furthermore, a representative N-methylated analogue has been shown to be incapable of causing CXCR4 internalization. These results suggest that the ability of SDF-1α to activate CXCR4 signaling and internalization is dependent upon the main chain amide bonds in the N-terminus of SDF-1α. This study demonstrates the feasibility and value of applying a synthetic biology approach to chemically engineer natural proteins and peptide ligands as probes of important biological functions that are not addressed by other biological techniques.
PMCID: PMC3891921  PMID: 22779681
12.  Synthesis and Anticancer Mechanism Investigation of Dual Hsp27 and Tubulin Inhibitors 
Journal of medicinal chemistry  2013;56(13):10.1021/jm4004736.
Heat shock protein 27 (Hsp27) is a chaperone protein, and its expression is increased in response to various stress stimuli including anticancer chemotherapy, which allows the cells to survive and causes drug resistance. We previously identified lead compounds that bound to Hsp27 and tubulin via proteomic approaches. Systematic ligand based optimization in the current study significantly increased the cell growth inhibition and apoptosis inducing activities of the compounds. Compared to the lead compounds, one of the new derivatives exhibited much better potency to inhibit tubulin polymerization but a decreased activity to inhibit Hsp27 chaperone function, suggesting that the structural modification dissected the dual targeting effects of the compound. The most potent compounds 20 and 22 exhibited strong cell proliferation inhibitory activities at subnanomolar concentration against 60 human cancer cell lines conducted by Developmental Therapeutic Program at the National Cancer Institute and represented promising candidates for anticancer drug development.
PMCID: PMC3855081  PMID: 23767669
13.  Correlation between the overexpression of epidermal growth factor receptor and mesenchymal makers in endometrial carcinoma 
The objective of this study was to evaluate the effect of overexpression of epidermal growth factor receptor (EGFR) on the expression of epithelial cell markers (E-cadherin and α-catenin) and mesenchymal cell markers (N-cadherin and vimentin) in endometrial carcinoma.
The expression of all 4 markers was evaluated in EGFR overexpressing Ishikawa cells, control Ishikawa cells, and KLE cells using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The expression of these 4 markers was also determined in cancerous tissues of patients with endometrial carcinoma using immunohistochemical staining.
Ishikawa cells transfected with EGFR showed decreased expression of E-cadherin and α-catenin and increased expression of N-cadherin and vimentin compared with control Ishikawa cells (p<0.01 for all). The expression of N-cadherin and vimentin was higher and the expression of E-cadherin and α-catenin was lower in stage II-III than stage I and in grade II-III than grade I endometrial carcinoma tissue (p<0.01 for all).
Decreased expression of epithelial markers (E-cadherin and α-catenin) and increased expression of mesenchymal markers (N-cadherin and vimentin) were observed in human endometrial carcinoma tissue. These findings correlate with high EGFR expression in cultured endometrial carcinoma cells.
PMCID: PMC3893673  PMID: 24459579
Endometrial carcinoma cells; Epidermal growth factor receptor; Epithelial-mesenchymal transition
14.  SRC-1 and Twist1 Expression Positively Correlates with a Poor Prognosis in Human Breast Cancer 
To evaluate the possible prognostic value of Steroid Receptor Coactivator-1 (SRC-1) and Twist1 expression in human breast cancer, we examined SRC-1 and Twist1 expression using immunohistochemistry on tissue microarray sections containing 137 breast cancer specimens. All patients were followed up for a median of 5 years following surgery. Survival curves were generated using the Kaplan-Meier method. Multivariate analysis was performed using the Cox proportional hazard regression model to assess the prognostic values. The results showed a positive correlation between SRC-1 and Twist1 expression at protein levels (P < 0.001). Also, SRC-1 expression positively correlated with HER2 expression (P = 0.024). The protein expression of Twist1 positively associated with lymph node metastasis (P < 0.001), but inversely correlated with PR status (P = 0.041). Patients with SRC-1 or Twist1-positive expression exhibited poorer overall survival (OS) and disease-free survival (DFS) than did those with SRC-1 or Twist1-negative expression (P < 0.05 for all). In addition, SRC-1-negativeive/Twist1-negative patients had the best OS and DFS (P < 0.01 for both). In multivariate survival analysis, SRC-1 expression, tumor stage, and PR were found to be independent prognostic factors related to OS (P = 0.019, < 0.001 and 0.02, respectively) and Twist1 expression, lymph node status and PR were independent predictors of DFS (P = 0.006, 0.001 and 0.029, respectively). These results suggest that a combined SRC-1/Twist1 expression status could improve the prognostic judgment for breast cancer patients.
PMCID: PMC3979992  PMID: 24719557
SRC-1; Twist1; Correlation; Breast cancer; Prognosis.
15.  Correction: Klotho Suppresses Cardiomyocyte Apoptosis in Mice with Stress-Induced Cardiac Injury via Downregulation of Endoplasmic Reticulum Stress 
PLoS ONE  2013;8(12):10.1371/annotation/d7fe4fdf-4be4-4aa5-b3f0-d98790fc0b11.
PMCID: PMC3876929
16.  Efficicent (R)-Phenylethanol Production with Enantioselectivity-Alerted (S)-Carbonyl Reductase II and NADPH Regeneration 
PLoS ONE  2013;8(12):e83586.
The NADPH-dependent (S)-carbonyl reductaseII from Candida parapsilosis catalyzes acetophenone to chiral phenylethanol in a very low yield of 3.2%. Site-directed mutagenesis was used to design two mutants Ala220Asp and Glu228Ser, inside or adjacent to the substrate-binding pocket. Both mutations caused a significant enantioselectivity shift toward (R)-phenylethanol in the reduction of acetophenone. The variant E228S produced (R)-phenylethanol with an optical purity above 99%, in 80.2% yield. The E228S mutation resulted in a 4.6-fold decrease in the KM value, but nearly 5-fold and 21-fold increases in the kcat and kcat/KM values with respect to the wild type. For NADPH regeneration, Bacillus sp. YX-1 glucose dehydrogenase was introduced into the (R)-phenylethanol pathway. A coexpression system containing E228S and glucose dehydrogenase was constructed. The system was optimized by altering the coding gene order on the plasmid and using the Shine–Dalgarno sequence and the aligned spacing sequence as a linker between them. The presence of glucose dehydrogenase increased the NADPH concentration slightly and decreased NADP+ pool 2- to 4-fold; the NADPH/NADP+ ratio was improved 2- to 5-fold. The recombinant Escherichia coli/pET-MS-SD-AS-G, with E228S located upstream and glucose dehydrogenase downstream, showed excellent performance, giving (R)-phenylethanol of an optical purity of 99.5 % in 92.2% yield in 12 h in the absence of an external cofactor. When 0.06 mM NADP+ was added at the beginning of the reaction, the reaction duration was reduced to 1 h. Optimization of the coexpression system stimulated an over 30-fold increase in the yield of (R)-phenylethanol, and simultaneously reduced the reaction time 48-fold compared with the wild-type enzyme. This report describes possible mechanisms for alteration of the enantiopreferences of carbonyl reductases by site mutation, and cofactor rebalancing pathways for efficient chiral alcohols production.
PMCID: PMC3866161  PMID: 24358299
17.  Momordica charantia (Bitter Melon) Reduces Obesity-Associated Macrophage and Mast Cell Infiltration as well as Inflammatory Cytokine Expression in Adipose Tissues 
PLoS ONE  2013;8(12):e84075.
Obesity is a world-wide epidemic disease that correlates closely with type 2 diabetes and cardiovascular diseases. Obesity-induced chronic adipose tissue inflammation is now considered as a critical contributor to the above complications. Momordica charantia (bitter melon, BM) is a traditional Chinese food and well known for its function of reducing body weight gain and insulin resistance. However, it is unclear whether BM could alleviate adipose tissue inflammation caused by obesity. In this study, C57BL/6 mice were fed high fat diet (HFD) with or without BM for 12 weeks. BM-contained diets ameliorated HFD-induced obesity and insulin resistance. Histological and real-time PCR analysis demonstrated BM not only reduced macrophage infiltration into epididymal adipose tissues (EAT) and brown adipose tissues (BAT). Flow cytometry show that BM could modify the M1/M2 phenotype ratio of macrophages in EAT. Further study showed that BM lowered mast cell recruitments in EAT, and depressed pro-inflammatory cytokine monocyte chemotactic protein-1 (MCP-1) expression in EAT and BAT as well as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression in EAT. Finally, ELISA analysis showed BM-contained diets also normalized serum levels of the cytokines. In summary, in concert with ameliorated insulin resistance and fat deposition, BM reduced adipose tissue inflammation in diet-induced obese (DIO) mice.
PMCID: PMC3866167  PMID: 24358329
18.  Correction: Building Large Collections of Chinese and English Medical Terms from Semi-Structured and Encyclopedia Websites 
PLoS ONE  2013;8(12):10.1371/annotation/fab3bd89-3ae3-445f-a418-207bbd7d4377.
PMCID: PMC3865328
20.  The diaphragms of fenestrated endothelia – gatekeepers of vascular permeability and blood composition 
Developmental cell  2012;23(6):1203-1218.
Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries causing a major leak of plasma proteins. This disruption results in early death of animals due to severe non-inflammatory protein loosing enteropathy. Deletion of PV1 in endothelium, but not the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
PMCID: PMC3525343  PMID: 23237953
21.  Klotho Suppresses Cardiomyocyte Apoptosis in Mice with Stress-Induced Cardiac Injury via Downregulation of Endoplasmic Reticulum Stress 
PLoS ONE  2013;8(12):e82968.
Cardiomyocyte apoptosis is a common pathological alteration in heart disease which results in systolic dysfunction or sudden death. Klotho is a novel anti-aging hormone. We tested the effects of klotho on cell apoptosis in isoproterenol-treated cardiomyocytes. In BALB/c mice, cardiac injury was induced by subcutaneous injection of isoproterenol (5mg/kg, for 9days, sc). Klotho (0.01 mg/kg, every other day for 4days, ip) was administered to determine the changes in isoproterenol-induced apoptosis. Mouse heart was harvested at day 2, day 5, and day 9 after isoproterenol injection. Isoproterenol induced cardiac apoptosis and endoplasmic reticulum (ER) stress in a time-dependent manner. However, klotho partly reversed isoproterenol-induced cardiac apoptosis and ER stress. These same effects were observed in cultured cardiomyocytes. Furthermore, the results also showed that SB203580, a p38 inhibitor, and SP600125, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduced cardiomyocyte apoptosis and ER stress, however, klotho suppressed isoproterenol-induced activation of p38 and JNK. Taken together, these results indicated that cardioprotection by klotho was related to the attenuation of ER stress and ER stress-induced apoptosis, at least partly, through suppressing activation of the p38 and JNK pathway.
PMCID: PMC3858310  PMID: 24340070
22.  The Effect of Conditioned Media of Adipose-Derived Stem Cells on Wound Healing after Ablative Fractional Carbon Dioxide Laser Resurfacing 
BioMed Research International  2013;2013:519126.
Objective. To evaluate the benefits of conditioned medium of Adipose-derived stem cells (ADSC-CM) on wound healing after fractional carbon dioxide laser resurfacing (FxCR) on human skin. Materials and Methods. Nineteen subjects were treated with FxCR on the bilateral inner arms. ADSC-CM was applied on FxCR site of one randomly selected arm. Transepidermal water loss (TEWL), skin color, and gross-elasticity of FxCR site on both arms were measured. Skin samples were taken by biopsy from three subjects 3 weeks after treatment for histopathological manifestations and mRNA expressions of procollagen types I and III, elastin genes were noted. Results. The index of erythema, melanin, and TEWL of the ADSC-CM-treated skin were significantly lower than those of the control side. The mRNA expression of type III procollagen in ADSC-CM-treated group at 3 weeks posttreatment was 2.6 times of that of the control group. Conclusion. Application of allograft ADSC-CM is an effective method for enhancing wound healing after FxCR, by reducing transient adverse effects such as erythema, hyperpigmentation, and increased TEWL.
PMCID: PMC3867954  PMID: 24381938
23.  Polymorphisms of Genes in Neurotransmitter Systems Were Associated with Alcohol Use Disorders in a Tibetan Population 
PLoS ONE  2013;8(11):e80206.
Studies of linkage and association in various ethnic populations have revealed many predisposing genes of multiple neurotransmitter systems for alcohol use disorders (AUD). However, evidence often is contradictory regarding the contribution of most candidate genes to the susceptibility of AUD. We, therefore, performed a case-control study to investigate the possible associations of genes selected from multiple neurotransmitter systems with AUD in a homogeneous Tibetan community population in China. AUD cases (N = 281) with an alcohol use disorder identification test (AUDIT) score ≥10, as well as healthy controls (N = 277) with an AUDIT score ≤5, were recruited. All participants were genotyped for 366 single nucleotide polymorphisms (SNPs) of 34 genes selected from those involved in neurotransmitter systems. Association analyses were performed using PLINK version 1.07 software. Allelic analyses before adjustment for multiple tests showed that 15 polymorphisms within seven genes were associated with AUD (p<0.05). After adjustment for the number of SNPs genotyped within each gene, only the association of a single marker (rs10044881) in HTR4 remained statistically significant. Haplotype analysis for two SNPs in HTR4 (rs17777298 and rs10044881) showed that the haplotype AG was significantly associated with the protective effect for AUD. In conclusion, the present study discovered that the HTR4 gene may play a marked role in the pathogenesis of AUD. In addition, this Tibetan population sample marginally replicated previous evidence regarding the associations of six genes in AUD.
PMCID: PMC3842251  PMID: 24312204
24.  Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance 
PLoS ONE  2013;8(11):e82062.
Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR.
PMCID: PMC3841137  PMID: 24303078
25.  Elevated urinary level of vitamin D-binding protein as a novel biomarker for diabetic nephropathy 
Improving the early prediction and detection of diabetic nephropathy (DN) remains a great challenge in disease management. The aim of this study was to evaluate the early detection power of urinary vitamin D-binding protein (VDBP) for the diagnosis of DN. Urine samples were obtained from 45 healthy volunteers and 105 diabetic patients with normoalbuminuria (DM group), microalbuminuria (DN1 group) and macroalbuminuria (DN2 group) (n=35 per group). The VDBP expression patterns in urine from patients and controls were quantified by western blot analysis. The excretion levels of urinary VDBP were quantified with enzyme-linked immunosorbent assay. The quantification results were obtained by correcting for creatinine expression and showed that urinary VDBP levels were significantly elevated in the patients of the DN1 and DN2 groups compared with those of the DM group and normal controls (1,011.33±325.30 and 1,406.34±239.66 compared with 466.54±213.63 and 125.48±98.27 ng/mg, respectively) (P<0.001). Receiver operating characteristic analysis of urinary VDBP levels for the diagnosis of DN rendered an optimum cut-off value of 552.243 ng/mg corresponding to 92.86% sensitivity and 85.00% specificity, which also showed an area under the ROC curve of 0.966. In conclusion, the findings of the present study suggest that urinary VDBP may be a potential biomarker for the early detection and prevention of DN. Further studies are required to examine the pathogenic mechanisms of elevated VDBP levels and their role in the diagnosis of DN.
PMCID: PMC3881051  PMID: 24396416
vitamin D binding protein; urine; diabetic nephropathy

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