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1.  Low Level of Low-Density Lipoprotein Receptor-Related Protein 1 Predicts an Unfavorable Prognosis of Hepatocellular Carcinoma after Curative Resection 
PLoS ONE  2012;7(3):e32775.
Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC).
LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens.
Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set.
LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.
PMCID: PMC3299691  PMID: 22427881
2.  Cardiac arrhythmia and heart failure: From bench to bedside 
PMCID: PMC3390068  PMID: 22783298
cardiac arrhythmia; heart failure; aging
3.  Cell and gene therapy for arrhythmias: Repair of cardiac conduction damage 
Action potentials generated in the sinoatrial node (SAN) dominate the rhythm and rate of a healthy human heart. Subsequently, these action potentials propagate to the whole heart via its conduction system. Abnormalities of impulse generation and/or propagation in a heart can cause arrhythmias. For example, SAN dysfunction or conduction block of the atrioventricular node can lead to serious bradycardia which is currently treated with an implanted electronic pacemaker. On the other hand, conduction damage may cause reentrant tachyarrhythmias which are primarily treated pharmacologically or by medical device-based therapies, including defibrillation and tissue ablation. However, drug therapies sometimes may not be effective or are associated with serious side effects. Device-based therapies for cardiac arrhythmias, even with well developed technology, still face inadequacies, limitations, hardware complications, and other challenges. Therefore, scientists are actively seeking other alternatives for antiarrhythmic therapy. In particular, cells and genes used for repairing cardiac conduction damage/defect have been investigated in various studies both in vitro and in vivo. Despite the complexities of the excitation and conduction systems of the heart, cell and gene-based strategies provide novel alternatives for treatment or cure of cardiac arrhythmias. This review summarizes some highlights of recent research progress in this field.
PMCID: PMC3390069  PMID: 22783301
cell therapy; gene therapy; conduction repair; arrhythmia
4.  Meta-Analysis of the Adverse Effects of Long-Term Azithromycin Use in Patients with Chronic Lung Diseases 
The adverse effects of azithromycin on the treatment of patients with chronic lung diseases (CLD) were evaluated in the present study. MEDLINE and other databases were searched for relevant articles published until August 2013. Randomized controlled trials that enrolled patients with chronic lung diseases who received long-term azithromycin treatment were selected, and data on microbiological studies and azithromycin-related adverse events were abstracted from articles and analyzed. Six studies were included in the meta-analysis. The risk of bacterial resistance in patients receiving long-term azithromycin treatment was increased 2.7-fold (risk ratio [RR], 2.69 [95% confidence interval {95% CI}, 1.249, 5.211]) compared with the risk in patients receiving placebo treatment. On the other hand, the risk of bacterial colonization decreased in patients receiving azithromycin treatment (RR, 0.551 [95% CI, 0.460, 0.658]). Patients receiving long-term azithromycin therapy were at risk of increased impairment of hearing (RR, 1.168 [95% CI, 1.030, 1.325]). This analysis provides evidence supporting the idea that bacterial resistance can develop with long-term azithromycin treatment. Besides the increasingly recognized anti-inflammatory role of azithromycin used in treating chronic lung diseases, we should be aware of the potential for adverse events with its long-term use.
PMCID: PMC3910718  PMID: 24189261
5.  Combination of Gefitinib and DNA Methylation Inhibitor Decitabine Exerts Synergistic Anti-Cancer Activity in Colon Cancer Cells 
PLoS ONE  2014;9(5):e97719.
Despite recent advances in the treatment of human colon cancer, the chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the epidermal growth factor receptor (EGFR) and DNA methyltransferase were examined in human colon cancer cells. We demonstrated that decitabine (a DNA methyltransferase inhibitor) synergized with gefitinib (an EGFR inhibitor) to reduce cell viability and colony formation in SW1116 and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM460 cells, a normal human colon mucosal epithelial cell line. The combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor. Concomitantly, cell apoptosis resulting from the co-treatment of gefitinib and decitabine was accompanied by induction of BAX, cleaved caspase 3 and cleaved PARP, along with reduction of Bcl-2 compared to treatment with either drug alone. Interestingly, combined treatment with these two drugs increased the expression of XIAP-associated factor 1 (XAF1) which play an important role in cell apoptosis. Moreover, small interfering RNA (siRNA) depletion of XAF1 significantly attenuated colon cancer cells apoptosis induced by the combination of the two drugs. Our findings suggested that gefitinib in combination with decitabine exerted enhanced cell apoptosis in colon cancer cells were involved in mitochondrial-mediated pathway and induction of XAF1 expression. In conclusion, based on the observations from our study, we suggested that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating colon cancer.
PMCID: PMC4038521  PMID: 24874286
6.  Competitive Exclusion among Fig Wasps Achieved via Entrainment of Host Plant Flowering Phenology 
PLoS ONE  2014;9(5):e97783.
Molecular techniques are revealing increasing numbers of morphologically similar but co-existing cryptic species, challenging the niche theory. To understand the co-existence mechanism, we studied phenologies of morphologically similar species of fig wasps that pollinate the creeping fig (F. pumila) in eastern China. We compared phenologies of fig wasp emergence and host flowering at sites where one or both pollinators were present. At the site where both pollinators were present, we used sticky traps to capture the emerged fig wasps and identified species identity using mitochondrial DNA COI gene. We also genotyped F. pumila individuals of the three sites using polymorphic microsatellites to detect whether the host populations were differentiated. Male F. pumila produced two major crops annually, with figs receptive in spring and summer. A small partial third crop of receptive figs occurred in the autumn, but few of the second crop figs matured at that time. Hence, few pollinators were available to enter third crop figs and they mostly aborted, resulting in two generations of pollinating wasps each year, plus a partial third generation. Receptive figs were produced on male plants in spring and summer, timed to coincide with the release of short-lived adult pollinators from the same individual plants. Most plants were pollinated by a single species. Plants pollinated by Wiebesia sp. 1 released wasps earlier than those pollinated by Wiebesia sp. 3, with little overlap. Plants occupied by different pollinators were not spatially separated, nor genetically distinct. Our findings show that these differences created mismatches with the flight periods of the other Wiebesia species, largely ‘reserving’ individual plants for the resident pollinator species. This pre-emptive competitive displacement may prevent long term co-existence of the two pollinators.
PMCID: PMC4029822  PMID: 24849458
7.  Comprehensive Multiple Molecular Profile of Epithelial Mesenchymal Transition in Intrahepatic Cholangiocarcinoma Patients 
PLoS ONE  2014;9(5):e96860.
The aim of this study is to investigate the expression profile of multiple epithelial mesenchymal transition (EMT)-related molecules in intrahepatic cholangiocarcinoma (ICC) and the related prognostic significance.
Immunohistochemistry was performed to determine the expression of E-cadherin, Vimentin, Snail, slug and β-catenin in a tissue microarray consisting of tumor tissues of 140 ICC patients undergoing curative resection. The correlation between the expression of these molecules and the clinicopathological characteristics of ICC patients was analyzed, and their prognostic implication was evaluated.
Reduced E-cadherin and increased Vimentin expression, the characteristic changes of EMT, identified in 55.0% and 55.7% of primary ICCs, respectively, were correlated with lymphatic metastasis and poorer overall survival (OS) and disease-free survival (DFS) of ICCs. The overexpression of snail and nonmembranous β-catenin, which are the major regulators of the EMT, were identified in 49.2% and 45.7% of primary ICCs, while little slug expression was detected in ICCs. Cytoplasmic/nuclear β-catenin did not significantly predict worse DFS and was not related with E-cadherin loss. The overexpression of snail predicted worse OS and DFS. Snail overexpression correlated with the down-regulation of E-cadherin and the up-regulation of Vimentin. Inhibition of snail in an ICC cell line decreased the expression of E-cadherin, enhanced the expression of Vimentin and impaired the invasion and migration ability of ICC cells.
These data support the hypothesis that EMT plays vital roles in ICC progression and suggest that snail but not slug and β-catenin plays a crucial role in the EMT induction of ICC.
PMCID: PMC4016113  PMID: 24816558
8.  Haemoproteus iwa in Great Frigatebirds (Fregata minor) in the Islands of the Western Indian Ocean 
PLoS ONE  2014;9(5):e97185.
Blood parasites of the sub-genus Haemoproteus have been reported in seabirds, in particular in species in the Suliformes order. These parasites are transmitted by hippoboscid flies of the genus Olfersia; strong specificity has been suggested between the vector and its vertebrate host. We investigated the prevalence of Haemoproteus infection in Suliformes and hippoboscid flies in two oceanic islands of the Western Indian Ocean: Europa and Tromelin. In total, 209 blood samples were collected from great frigatebirds (Fregata minor), masked boobies (Sula dactylatra) and red-footed boobies (Sula sula). Forty-one hippoboscid flies were also collected from birds. Seventeen frigatebirds and one fly collected on Europa tested positive for the presence of Haemoproteus parasites by polymerase chain reaction. Phylogenetic analyses based on partial sequences of the Cytochrome b gene showed that parasites were closely related to Haemoproteus iwa reported from frigatebirds in the Pacific Ocean and in the Caribbean. Plasmodium was also detected in a frigatebird on Europa; however, its placement on the phylogenetic tree could not be resolved. We provide strong support for transmission of blood parasites in seabirds in the Western Indian Ocean and suggest that migrations between the Pacific and the Indian oceans could favor the large-scale distribution of Haemoproteus iwa in frigatebird populations.
PMCID: PMC4014603  PMID: 24810172
9.  Cell-surface processing of extracellular human immunodeficiency virus type 1 Vpr by proprotein convertases 
Virology  2007;372(2):384-397.
Increasing evidence suggests that extracellular Vpr could contribute to HIV pathogenesis through its effect on bystander cells. Soluble forms of Vpr have been detected in the sera and cerebrospinal fluids of HIV-1-infected patients, and in vitro studies have implicated extracellular Vpr as an effector of cellular responses, including G2 arrest, apoptosis and induction of cytokines and chemokines production, presumably through its ability to transduce into multiple cell types. However, the mechanism underlying Vpr release from HIV-1-producing cells remains undefined and the biological modifications that the extracellular protein may undergo are largely unknown. We provide evidence indicating that soluble forms of Vpr are present in the extracellular medium of HIV-1-producing cells. Release of Vpr in the extracellular medium did not originate from decaying or disrupted HIV-1 virions that package Vpr but rather appeared associated with HIV-1-mediated cytopathicity. Interestingly, Vpr was found to undergo proteolytic processing at a very well conserved proprotein convertase (PC) cleavage site, R85QRR88↓, located within the functionally important C-terminal arginine-rich domain of the protein. Vpr processing occurred extracellularly upon close contact to cells and most likely involved a cell surface-associated PC. Consistently, PC inhibitors suppressed Vpr processing, while expression of extracellular matrix-associated PC5 and PACE4 enhanced Vpr cleavage. PC-mediated processing of extracellular Vpr led to the production of a truncated Vpr product that was defective for the induction of cell cycle arrest and apoptosis when expressed in human cells. Collectively, these results suggest that cell surface processing of extracellular Vpr by PCs might regulate the levels of active soluble Vpr.
PMCID: PMC3955186  PMID: 18061232 CAMSID: cams4183
HIV-1; Vpr; Extracellular processing; Proprotein convertase; Protease; Extracellular matrix
10.  A Network Pharmacology Approach to Understanding the Mechanisms of Action of Traditional Medicine: Bushenhuoxue Formula for Treatment of Chronic Kidney Disease 
PLoS ONE  2014;9(3):e89123.
Traditional Chinese medicine (TCM) has unique therapeutic effects for complex chronic diseases. However, for the lack of an effective systematic approach, the research progress on the effective substances and pharmacological mechanism of action has been very slow. In this paper, by incorporating network biology, bioinformatics and chemoinformatics methods, an integrated approach was proposed to systematically investigate and explain the pharmacological mechanism of action and effective substances of TCM. This approach includes the following main steps: First, based on the known drug targets, network biology was used to screen out putative drug targets; Second, the molecular docking method was used to calculate whether the molecules from TCM and drug targets related to chronic kidney diseases (CKD) interact or not; Third, according to the result of molecular docking, natural product-target network, main component-target network and compound-target network were constructed; Finally, through analysis of network characteristics and literature mining, potential effective multi-components and their synergistic mechanism were putatively identified and uncovered. Bu-shen-Huo-xue formula (BSHX) which was frequently used for treating CKD, was used as the case to demonstrate reliability of our proposed approach. The results show that BSHX has the therapeutic effect by using multi-channel network regulation, such as regulating the coagulation and fibrinolytic balance, and the expression of inflammatory factors, inhibiting abnormal ECM accumulation. Tanshinone IIA, rhein, curcumin, calycosin and quercetin may be potential effective ingredients of BSHX. This research shows that the integration approach can be an effective means for discovering active substances and revealing their pharmacological mechanisms of TCM.
PMCID: PMC3943740  PMID: 24598793
11.  High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded autopsy lung tissue sample from the 1918 influenza pandemic 
The Journal of pathology  2013;229(4):535-545.
Most biopsy and autopsy tissues are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. The RNA genome of the 1918 pandemic influenza virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Here, the full genome of the 1918 virus at 3000× coverage was determined in one high-throughput sequencing run of a library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. Bacterial sequences associated with secondary bacterial pneumonias were also detected. Host transcripts were well represented in the library. Compared to a 2009 pandemic influenza virus FFPE post-mortem library, the 1918 sample showed significant enrichment for host defence and cell death response genes, concordant with prior animal studies. This methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of diseases.
PMCID: PMC3731037  PMID: 23180419
influenza A virus; pandemic; 1918 influenza virus; formalin-fixed; paraffin-embedded tissue; NGS
12.  Efficacy of combination therapy of triazole and echinocandin in treatment of invasive aspergillosis: a systematic review of animal and human studies 
Journal of Thoracic Disease  2014;6(2):99-108.
The effectiveness of the combination therapy of triazole and echinocandin in treatment of invasive aspergillosis (IA) remains controversial. The objective of this systematic review was to assess the efficacy of combination therapy of triazole and echinocandin in treatment of IA.
Relevant articles on the combination therapy of triazole and echinocandin in IA, including the animal studies and clinical studies from January 1966 to October 2013, were searched on Web of Science, PubMed and Cochrane Library. The prolongation of survival of the combination therapy of triazole and echinocandin in IA was performed as risk ratio (RR) with 95% confidence interval (95% CI).
Nine animal studies with a total of 1,582 animals and five clinical trials totaling 872 patients were included. The survival of the included animal studies with combination therapy was significantly prolonged compared with echinocandin alone [RR =2.26, (95% CI, 1.79-2.87; P<0.00001)], but no statistical difference compared with monotherapy of triazole [RR =1.19, (95% CI, 0.98-1.44; P=0.08)]. Of the four human cohort studies, two studies observed that the combination therapy of triazole and echinocandin was associated with a significant reduction in mortality compared with other treatments, and one study might be considered as a preferable therapy [HR =0.58, (95% CI, 0.3-1.14; P=0.117)]. While another study revealed that there was no significant difference among the combination therapy of triazole and echinocandin and either of the monotherapy. In the randomized clinical trial (RCT), of the 135 patients who received the combination therapy, 39 died, while 55 died out of 142 patients who received monotherapy (P=0.08, 95% CI, –21.4, 1.09) by week 12.
The combination therapy of triazole and echinocandin in treating IA results in a trend towards improved overall survival in animals’ studies and clinical studies. Well-designed RCTs and further improved clinical trials are necessary to study the effectiveness of the combination therapy.
PMCID: PMC3944170  PMID: 24605223
Triazole; echinocandin; invasive aspergillosis (IA); systematic review
13.  Decreased Expression of GATA2 Promoted Proliferation, Migration and Invasion of HepG2 In Vitro and Correlated with Poor Prognosis of Hepatocellular Carcinoma 
PLoS ONE  2014;9(1):e87505.
GATA family of transcription factors are critical for organ development and associated with progression of various cancer types. However, their expression patterns and prognostic values for hepatocellular carcinoma (HCC) are still largely unknown.
Expression of GATA transcription factors in HCC cell lines and tissues (n = 240) were evaluated by RT-qPCR, western blot and immunohistochemistry. Cellular proliferation, migration and invasion of HepG2 was evaluated by CCK-8 kit, scratch wound assay and transwell matrigel invasion assay, respectively.
GATA2 expression was decreased in HCC cell lines (p = 0.056 for mRNA, p = 0.040 for protein) and tissues (p = 1.27E-25) compared with normal hepatocytes. Decreased expression of intratumoral GATA2 protein significantly correlated with elevated alpha feto-protein (p = 2.7E-05), tumor size >5 cm (p = 0.049), absence of tumor capsule (p = 0.002), poor differentiation (p = 0.005), presence of tumor thrombi (p = 0.005) and advanced TNM stage (p = 0.001) and was associated with increased recurrence rate and decreased overall survival rate by univariate (p = 1.6E-04 for TTR, p = 1.7E-04 for OS) and multivariate analyses (HR = 0.63, 95% CI = 0.43–0.90, p = 0.012 for TTR; HR = 0.67, 95% CI = 0.47–0.95, p = 0.026 for OS). RNAi-mediated knockdown of GATA2 expression significantly enhanced proliferation, migration and invasion of HepG2 cell in vitro.
Decreased expression of hematopoietic factor GATA2 was associated with poor prognosis of HCC following resection.
PMCID: PMC3907524  PMID: 24498120
14.  The Wall Eclipsing Sign on Pulmonary Artery Computed Tomography Angiography Is Pathognomonic for Pulmonary Artery Sarcoma 
PLoS ONE  2013;8(12):e83200.
The objective of this study was to evaluate the imaging characteristics of pulmonary artery sarcoma (PAS) on pulmonary artery computed tomography angiography (PACTA) that can be used to differentiate between PAS and pulmonary thromboembolic diseases, including chronic thromboembolic pulmonary hypertension (CTEPH) and acute pulmonary embolism (APE).
The clinical data and imaging characteristics of 12 patients with PAS, 156 patients with CTEPH, and 426 patients with APE who were treated at Beijing Anzhen Hospital from January 2007 to August 2013 were retrospectively analyzed. All patients underwent PACTA before treatment, and the diagnoses of PAS and CTEPH were all confirmed by surgical biopsy.
All 12 PAS patients were initially misdiagnosed and received inappropriate thrombolytic and/or anticoagulant therapy before they were referred for surgical intervention. The mean time from PACTA to surgical intervention was 5.5±3.7 months (range 2–11 months). On PACTA, the PAS lesion always eclipsed the wall of the pulmonary artery before infiltrating outside the pulmonary artery, which was termed the wall eclipsing sign. This sign was observed in all PAS patients but was not observed in any CTEPH or APE patients.
PAS is a rare neoplasm with a poor prognosis, and is easily misdiagnosed as thromboembolic disease. The wall eclipsing sign on PACTA is pathognomonic for PAS, and patients with this sign should be investigated to confirm the diagnosis and should undergo surgical intervention as soon as possible, rather than receiving thrombolytic or anticoagulant therapy.
PMCID: PMC3877030  PMID: 24391746
15.  Enhanced Expression of Cystathionine β-Synthase and Cystathionine γ-Lyase During Acute Cholecystitis-Induced Gallbladder Inflammation 
PLoS ONE  2013;8(12):e82711.
Hydrogen sulfide (H2S) has recently been shown to play an important role in the digestive system, but the role of endogenous H2S produced locally in the gallbladder is unknown. The aim of this study was to investigate whether gallbladder possesses the enzymatic machinery to synthesize H2S, and whether H2S synthesis is changed in gallbladder inflammation during acute acalculous cholecystitis (AC).
Adult male guinea pigs underwent either a sham operation or common bile duct ligation (CBDL). One, two, or three days after CBDL, the animals were sacrificed separately. Hematoxylin and eosin-stained slides of gallbladder samples were scored for inflammation. H2S production rate in gallbladder tissue from each group was determined; immunohistochemistry and western blotting were used to determine expression levels of the H2S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in gallbladder.
There was a progressive inflammatory response after CBDL. Immunohistochemistry analysis showed that CBS and CSE were expressed in the gallbladder epithelium, muscular layer, and blood vessels and that the expression increased progressively with increasing inflammation following CBDL. The expression of CBS protein as well as the H2S-production rate was significantly increased in the animals that underwent CBDL, compared to those that underwent the sham operation.
Both CBS and CSE are expressed in gallbladder tissues. The expression of these enzymes, as well as H2S synthesis, was up-regulated in the context of inflammation during AC.
PMCID: PMC3857271  PMID: 24349344
16.  Efficacy of the Combination of Voriconazole and Caspofungin in Experimental Pulmonary Aspergillosis by Different Aspergillus Species 
Mycopathologia  2013;177:11-18.
Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus, Aspergillus flavus, or Aspergillus niger is associated with high mortality. We evaluated the efficacy and compared the therapeutic effect differences of voriconazole (VRC) in combination with caspofungin (CAS) in transiently neutropenic rats infected by A. fumigatus, A. flavus, or A. niger.
Treatment groups consisted of VRC (10 mg/kg q12 h) monotherapy, CAS (1 mg/kg/day) monotherapy, combination of VRC (10 mg/kg q12 h) + CAS (1 mg/kg/day), and no drug for 10 consecutive days. The efficacy and the difference in the treatments were evaluated through prolongation of survival, reduction in serum galactomannan levels and residual fungal burden, and histological studies.
For all the strains, the combination of VRC and CAS led to significant prolongation in survival (P < 0.05) and reduction in residual fungal burden (P < 0.05) compared with CAS alone, and decrease in serum galactomannan levels (P < 0.05) compared with either agent alone. The survival in the combined therapy groups was significantly improved compared to VRC monotherapy for the strains of A. flavus and A. niger (P < 0.05), but no significant difference for the strains of A. fumigatus (P > 0.05).
Combination of VRC and CAS was synergistic in IPA by A. flavus and A. niger, but small efficacy benefits in IPA by A. fumigatus.
PMCID: PMC3915080  PMID: 24306184
Voriconazole; Caspofungin; Combination; Aspergillus fumigatus; Aspergillus flavus; Aspergillus niger
17.  Mycobacterial MazG Safeguards Genetic Stability via Housecleaning of 5-OH-dCTP 
PLoS Pathogens  2013;9(12):e1003814.
Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo.
Author Summary
The cellular nucleotide pool is a significant target for oxidation by reactive oxygen species and reactive nitrogen species. Misincorporation of these oxidized non-canonical nucleotides into DNA is known to cause mutations, and may be related to carcinogenesis, aging and neurodegeneration. Cells have evolved a group of bio-degradation housecleaning enzymes that may specifically eliminate certain non-canonical nucleotide from the nucleotide pool and thus prevent their incorporation into DNA. The most well-characterized housecleaning enzymes are the MutT-like proteins which specifically hydrolyze the oxidized purine nucleotides, such as 8-oxo-dGTP and 2-OH-dATP. Lack of MutT activity in cells leads to significant increase of AT-CG mutation and genetic instability. However, housecleaning enzymes specific for oxidized pyrimidine nucleotides are yet to be identified. Here we show that the dNTP pyrophosphohydrolase MazG from mycobacteria is a 5-OH-dCTP-specific housecleaning enzyme. Deletion of mazG in mycobacteria results in increased CG to TA mutation under oxidative stress and in the stationary phase of growth. Both biochemical and chemical genetic analyses demonstrate that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mtb leads to reduced survival in activated macrophages and in the spleen of infected mice. These results reveal a novel housecleaning pathway for mycobacteria to maintain genetic stability and survival in vivo.
PMCID: PMC3855555  PMID: 24339782
18.  Xylazine-induced reduction of tissue sensitivity to insulin leads to acute hyperglycemia in diabetic and normoglycemic monkeys 
BMC Anesthesiology  2013;13:33.
The α2-adrenoceptor agonist xylazine as an anesthetic has been widely used either alone or in combination with other anesthetics, such as ketamine, in veterinary clinic and research. In the last decade xylazine has been used in drug abusers in certain geographic area. This study investigated the effects of xylazine on blood glucose level and insulin secretion in normoglycemic and insulin-dependent diabetic monkeys.
Both adult cynomolgus (n = 10) and rhesus (n = 8) monkeys with either sex were used in the study. Xylazine (1–2 mg/kg) was administrated intramuscularly. Blood glucose, insulin, glucagon and glucagon-like peptide 1 in overnight-fasted monkeys were measured immediately before and after xylazine administration. The hyperinsulinemic-euglycemic clamp method was used in the study for assessing the potential mechanism of xylazine-induced hyperglycemia.
Xylazine administration increased the blood glucose levels from 58 ± 3 to 108 ± 12 mg/dL in normoglycemic (n = 5, p < 0.01) and from 158 ± 9 to 221 ± 13 mg/dL in insulin-dependent diabetic (n = 5, p < 0.01) monkeys and was not accompanied by any significant changes in blood insulin, glucagon, and glucagon-like peptide-1. Xylazine-induced hyperglycemia occurred within 10 min and reached the peak at 35 min after injection. Xylazine-induced hyperglycemia declined slowly in diabetic animals. The α2-adrenoceptor antagonist yohimbine was administrated to bring down the elevated glucose level to the pre-xylazine one in 4 out of 5 diabetic animals. To assess the potential mechanism, the hyperinsulinemic-euglycemic clamp was used to maintain a nearly saturated and constant insulin level for minimizing endogenous insulin glucoregulation. Xylazine administration decreased glucose infusion rate, from 14.3 ± 1.4 to 8.3 ± 0.8 mg/min/kg (n = 6, p < 0.01) in normoglycemic rhesus monkeys, which indicates that the glucose metabolic rate (M rate) was decreased by xylazine. In addition, after clamping blood glucose level in a range of 55 to 75 mg/dL for 40 min with constant glucose infusion, xylazine administration still increased blood glucose concentration.
We conclude that xylazine administration induces hyperglycemia in normoglycemic and insulin-dependent diabetic monkeys potentially via stimulation of α2-adrenoceptors and then reducing tissue sensitivity to insulin and glucose uptake.
PMCID: PMC4016475  PMID: 24138083
Xylazine; Hyperglycemia; Monkey; Insulin; Glucose
19.  Long-term results of clear lens extraction combined with piggyback intraocular lens implantation to correct high hyperopia 
To assess the refractive outcome of clear lensectomy combined with piggyback intraocular lens implantation in highly hyperopic patients.
This case review included 19 eyes of 10 patients with high hyperopia and axial length less than 21mm. Intraocular lens power was calculated for emmetropia using the Holladay II formula in 17 eyes, and SRK/T formula in 2 eyes following clear lens extraction and piggyback intraocular lens implantation. Patients were examined periodically over 24 months for visual acuity and spherical equivalent (SE).
The mean postoperative SE at 24 months was 0.20±1.39D (range, -3.00 to 2.50D), better than preoperative 9.81±2.62D (range, +6.00 to +14.50D) (P<0.001). Five eyes had SE within ±0.5D of emmetropia and 11 eyes within ±1.00D at postoperative 24 months. The mean postoperative uncorrected visual acuity (UCVA) at 24 months was 0.60±0.36, significantly improved compared to preoperative 1.39±0.33 (P<0.001). The mean best-corrected visual acuity (BCVA) at 24 months was 0.49±0.35, not statistically different compared to preoperative 0.38±0.30 (P=0.34). Twelve eyes maintained and 1 gained 1 or more Snellen line of BCVA, 4 eyes lost 1 line, and 2 eyes lost 2 lines at 24 postoperative months. Twelve eyes best-corrected near visual acuity (BCNVA) achieved J1 at postoperative 24 months compared to preoperative 7 eyes and the other 7 eyes better than J3.
Clear lens extraction combined piggyback intraocular lens implantation appears to be an effective procedure to correct high hyperopia but mild overcorrection and intralenticular opacification may require secondary procedure.
PMCID: PMC3808914  PMID: 24195042
piggyback; intraocular lens; high hyperopia
20.  A High-Confidence Interaction Map Identifies SIRT1 as a Mediator of Acetylation of USP22 and the SAGA Coactivator Complex 
Molecular and Cellular Biology  2013;33(8):1487-1502.
Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex.
PMCID: PMC3624249  PMID: 23382074
21.  Diversity of individual mobility patterns and emergence of aggregated scaling laws 
Scientific Reports  2013;3:2678.
Uncovering human mobility patterns is of fundamental importance to the understanding of epidemic spreading, urban transportation and other socioeconomic dynamics embodying spatiality and human travel. According to the direct travel diaries of volunteers, we show the absence of scaling properties in the displacement distribution at the individual level,while the aggregated displacement distribution follows a power law with an exponential cutoff. Given the constraint on total travelling cost, this aggregated scaling law can be analytically predicted by the mixture nature of human travel under the principle of maximum entropy. A direct corollary of such theory is that the displacement distribution of a single mode of transportation should follow an exponential law, which also gets supportive evidences in known data. We thus conclude that the travelling cost shapes the displacement distribution at the aggregated level.
PMCID: PMC3776193  PMID: 24045416
22.  Extensive Divergence of Transcription Factor Binding in Drosophila Embryos with Highly Conserved Gene Expression 
PLoS Genetics  2013;9(9):e1003748.
To better characterize how variation in regulatory sequences drives divergence in gene expression, we undertook a systematic study of transcription factor binding and gene expression in blastoderm embryos of four species, which sample much of the diversity in the 40 million-year old genus Drosophila: D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. We compared gene expression, measured by mRNA-seq, to the genome-wide binding, measured by ChIP-seq, of four transcription factors involved in early anterior-posterior patterning. We found that mRNA levels are much better conserved than individual transcription factor binding events, and that changes in a gene's expression were poorly explained by changes in adjacent transcription factor binding. However, highly bound sites, sites in regions bound by multiple factors and sites near genes are conserved more frequently than other binding, suggesting that a considerable amount of transcription factor binding is weakly or non-functional and not subject to purifying selection.
Author Summary
Inter-species differences in gene expression during development are a major source of phenotypic diversity, yet the molecular origins of such differences are poorly understood. In this study we use a combination of biochemical and genomic methods to explore how an important component of the machinery of gene regulation varies between species. We mapped the genomic sites bound by four specific proteins regulating gene expression along the head to tail axis of young embryos of four diverse species of fruit flies. We were surprised at the extent of variation we observed, especially as we found far less variation in gene expression between these same species' embryos. We conclude, based on various analyses of our binding divergence data, that most of the time these regulators bind to DNA they have no effect on gene expression, and therefore natural selection does not act to preserve these interactions between species.
PMCID: PMC3772039  PMID: 24068946
23.  In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma 
To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.
Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy.
HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay.
Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.
PMCID: PMC3755297  PMID: 23991372
amniotic epithelial cells; cornea; tissue engineering; keratin
24.  Characterization and Evolution of Conserved MicroRNA through Duplication Events in Date Palm (Phoenix dactylifera) 
PLoS ONE  2013;8(8):e71435.
MicroRNAs (miRNAs) are important regulators of gene expression at the post-transcriptional level in a wide range of species. Highly conserved miRNAs regulate ancestral transcription factors common to all plants, and control important basic processes such as cell division and meristem function. We selected 21 conserved miRNA families to analyze the distribution and maintenance of miRNAs. Recently, the first genome sequence in Palmaceae was released: date palm (Phoenix dactylifera). We conducted a systematic miRNA analysis in date palm, computationally identifying and characterizing the distribution and duplication of conserved miRNAs in this species compared to other published plant genomes. A total of 81 miRNAs belonging to 18 miRNA families were identified in date palm. The majority of miRNAs in date palm and seven other well-studied plant species were located in intergenic regions and located 4 to 5 kb away from the nearest protein-coding genes. Sequence comparison showed that 67% of date palm miRNA members were present in duplicated segments, and that 135 pairs of miRNA-containing segments were duplicated in Arabidopsis, tomato, orange, rice, apple, poplar and soybean with a high similarity of non coding sequences between duplicated segments, indicating genomic duplication was a major force for expansion of conserved miRNAs. Duplicated miRNA pairs in date palm showed divergence in pre-miRNA sequence and in number of promoters, implying that these duplicated pairs may have undergone divergent evolution. Comparisons between date palm and the seven other plant species for the gain/loss of miR167 loci in an ancient segment shared between monocots and dicots suggested that these conserved miRNAs were highly influenced by and diverged as a result of genomic duplication events.
PMCID: PMC3738527  PMID: 23951162
25.  Combination of Intratumoral Invariant Natural Killer T Cells and Interferon-Gamma Is Associated with Prognosis of Hepatocellular Carcinoma after Curative Resection 
PLoS ONE  2013;8(8):e70345.
To investigate the prognostic value of intratumoral invariant natural killer T (iNKT) cells and interferon-gamma (IFN-γ) in hepatocellular carcinoma (HCC) after curative resection.
Experimental Design
Expression of TRAV10, encoding the Vα24 domain of iNKT cells, and IFN-γ mRNA were assessed by quantitative real-time polymerase chain reaction in tumor from 224 HCC patients undergoing curative resection. The prognostic value of these two and other clinicopathologic factors was evaluated.
Either intratumoral iNKT cells and IFN-γ alone or their combination was an independent prognostic factor for OS (P = 0.001) and RFS (P = 0.001) by multivariate Cox proportional hazards analysis. Patients with concurrent low levels of iNKT cells and IFN-γ had a hazard ratio (HR) of 2.784 for OS and 2.673 for RFS. The areas under the curve of iNKT cells, IFN-γand their combination were 0.618 vs 0.608 vs 0.654 for death and 0.591 vs 0.604 vs 0.633 for recurrence respectively by receiver operating characteristic curve analysis. The prognosis was the worst for HCC patients with concurrent low levels of iNKT cells and IFN-γ, which might be related with more advanced pTNM stage and more vascular invasion.
Combination of intratumoral iNKT cells and IFN-γ is a promising independent predictor for recurrence and survival in HCC, which has a better power to predict HCC patients’ outcome compared with intratumoral iNKT cells or IFN-γ alone.
PMCID: PMC3734128  PMID: 23940564

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