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1.  Targeted deletion of Rad9 in mouse skin keratinocytes enhances genotoxin-induced tumor development 
Cancer research  2008;68(14):5552-5561.
The Rad9 gene is evolutionarily conserved from yeast to human, and plays crucial roles in genomic maintenance, DNA repair and cell cycle checkpoint controls. However, the function of this gene with respect to tumorigenesis is not well understood. A Rad9-null mutation in mice causes embryonic lethality. In this study, we created mice in which mouse Rad9, Mrad9, was deleted only in keratinocytes to permit examination of the potential function of the gene in tumor development. Mice with Mrad9+/− or Mrad9−/− keratinocytes demonstrated no overt, spontaneous morphological defects and appeared similar to wild-type controls. Painting the carcinogen 7,12-dimethylbenzanthracene (DMBA) onto the skin of the animals caused earlier onset and more frequent formation of tumors and senile skin plaques in Mrad9−/− mice, compared to Mrad9+/− and Mrad9+/+ littermates. DNA damage response genes p21, p53 and Mrad9B were expressed at higher levels in Mrad9−/− relative to Mrad9+/+ skin. Keratinocytes isolated from Mrad9−/− skin had more spontaneous and DMBA-induced DNA double strand breaks than Mrad9+/+ keratinocytes, and the levels were reduced by incubation with the antioxidant EGCG. These data suggest that Mrad9 plays an important role in maintaining genomic stability and preventing tumor development in keratinocytes.
PMCID: PMC4331354  PMID: 18632607
Rad9; conditional gene knockout; genomic stability; skin tumorigenesis; skin ageing
2.  miR-101 Suppresses Vascular Endothelial Growth Factor C That Inhibits Migration and Invasion and Enhances Cisplatin Chemosensitivity of Bladder Cancer Cells 
PLoS ONE  2015;10(2):e0117809.
The microRNA miR-101 is downregulated in several cancers, including bladder cancer. However, miR-101’s role in the invasion, metastasis, and chemosensitivity of bladder cancer cells remains unclear. This study was conducted to determine miR-101’s role on the lymphangiogenic molecule vascular endothelial growth factor C (VEGF-C) and their effects upon bladder cancer cell migration, invasion, and chemosensitivity to cisplatin.
Two bladder cancer cell lines (T24 and 5637) and the tool cell line 293T were employed here. Bladder cancer cells were transfected with either a miR-101 overexpression vector or a scrambled-sequence lentivirus, both of which exhibited a high transfection efficiency. Non-transfection was used as a mock negative control. Wound healing and Transwell assays were performed to measure cell migration and invasiveness. A luciferase reporter assay was performed to validate miR-101’s interaction with VEGF-C’s 3′ untranslated region followed by RT-PCR and Western blot confirmation. An MTS assay was used to evaluate the cisplatin sensitivity of the cell lines.
miR-101 overexpression significantly inhibited the migration and invasiveness while significantly enhancing cisplatin sensitivity. miR-101 negatively regulated VEGF-C protein expression, and VEGF-C overexpression rescued the effects of miR-101 overexpression, indicating that miR-101 negatively regulates VEGF-C protein expression post-transcriptionally. miR-101 and VEGF-C interference independently enhanced cisplatin cytotoxicity in bladder cancer cells.
miR-101 suppresses VEGF-C expression, inhibits cell migration and invasion, and increases cisplatin sensitivity in bladder cancer cells. This study provides new insight into miR-101’s role in bladder cancer and shows miR-101’s promise as a potential molecular target for bladder cancer.
PMCID: PMC4320037  PMID: 25658842
3.  Recognition of Methylated Peptides by Drosophila melanogaster Polycomb Chromodomain 
Journal of proteome research  2013;12(3):1467-1477.
Lysine methylation is one of the important post-translational modifications (PTMs) that regulate protein functions. Up to now, proteomic identification of this PTM remains a challenge due to the lack of effective enrichment methods in mass spectrometry experiments. To address this challenge, we present here a systematic approach to predicting peptides in which lysine residues may be methylated to mediate protein–protein interactions. We used the chromodomain of the polycomb protein in Drosophila melanogaster as a model system to illustrate the success of this approach. We started with molecular dynamics simulations and free energy analyses on the histone peptides complexed with the polycomb chromodomain to understand how the binding specificity is achieved. We next conducted virtual mutagenesis to quantify each domain and peptide residue's contribution to the domain-peptide recognition, based on which scoring scheme was developed to evaluate the possibility of any lysine-containing peptides to be methylated and recognized by the chromodomain. A peptide microarray experiment on a panel of conserved histone peptides showed a satisfactory prediction accuracy of the scoring scheme. Next, we implemented a bioinformatics pipeline that integrates multiple lines of evidence including conservation, subcellular localization, and mass spectrometry data to scan the fly proteome for a systematic identification of possible methyllysine-containing peptides. These putative chromodomain-binding peptides suggest unknown functions of the important regulator protein polycomb and provide a list of candidate methylation events for follow-up investigations.
PMCID: PMC4258113  PMID: 23320494
molecular dynamics; MM-GBSA; mutation matrix; peptide microarray; methylated lysine
4.  Protein Interferon-Stimulated Gene 15 Conjugation Delays but Does Not Overcome Coronavirus Proliferation in a Model of Fulminant Hepatitis 
Journal of Virology  2014;88(11):6195-6204.
Coronaviruses express a deubiquitinating protein, the papain-like protease-2 (PLP2), that removes both ubiquitin and the ubiquitin-like interferon (IFN)-stimulated gene 15 (ISG15) protein from target proteins. ISG15 has antiviral activity against a number of viruses; therefore, we examined the effect of ISG15 conjugation (ISGylation) in a model of acute viral hepatitis induced by the murine hepatitis virus strain 3 (MHV-3) coronavirus. Mice deficient in the ISG15 deconjugating enzyme, ubiquitin-specific peptidase-18 (USP18), accumulate high levels of ISG15-conjugated proteins and are hypersensitive to type I IFN. Infecting USP18−/− mice with MHV-3 resulted in extended survival (8 ± 1.2 versus 4 days) and in improved liver histology, a decreased inflammatory response, and viral titers 1 to 2 logs lower than in USP18+/+ mice. The suppression of viral replication was not due to increased IFN since infected USP18−/− mice had neither increased hepatic IFN-α, -β, or -γ mRNA nor circulating protein. Instead, delayed MHV-3 replication coincided with high levels of cellular ISGylation. Decreasing ISGylation by knockdown of the ISG15 E1 enzyme, Ube1L, in primary USP18+/+ and USP18−/− hepatocytes led to increased MHV-3 replication. Both in vitro and in vivo, increasing MHV-3 titers were coincident with increased PLP2 mRNA and decreased ISGylation over the course of infection. The pharmacologic inhibition of the PLP2 enzyme in vitro led to decreased MHV-3 replication. Overall, these results demonstrate the antiviral effect of ISGylation in an in vivo model of coronavirus-induced mouse hepatitis and illustrate that PLP2 manipulates the host innate immune response through the ISG15/USP18 pathway.
IMPORTANCE There have been a number of serious worldwide pandemics due to widespread infections by coronavirus. This virus (in its many forms) is difficult to treat, in part because it is very good at finding “holes” in the way that the host (the infected individual) tries to control and eliminate the virus. In this study, we demonstrate that an important host viral defense—the ISG15 pathway—is only partially effective in controlling severe coronavirus infection. Activation of the pathway is very good at suppressing viral production, but over time the virus overwhelms the host response and the effects of the ISG15 pathway. These data provide insight into host-virus interactions during coronavirus infection and suggest that the ISG15 pathway is a reasonable target for controlling severe coronavirus infection although the best treatment will likely involve multiple pathways and targets.
PMCID: PMC4093886  PMID: 24648452
5.  Modeling human cancer cachexia in colon 26 tumor-bearing adult mice 
Muscle wasting is a profound side effect of advanced cancer. Cancer-induced cachexia decreases patient quality of life and is associated with poor patient survival. Currently, no clinical therapies exist to treat cancer-induced muscle wasting. Although cancers commonly associated with cachexia occur in older individuals, the standard animal models used to elucidate the causes of cachexia rely on juvenile mice.
In an effort to better model human cancer cachexia, we determined whether cachectic features seen in young mice could be achieved in adult, pre-sarcopenic mice following colon 26 (C-26) tumor cell inoculation.
Both young and adult mice developed similar-sized tumors and progressed to cachexia with similar kinetics, as evidenced by losses in body mass, and adipose and skeletal muscle tissues. Proteolytic signaling, including proteasome and autophagy genes, was also increased in muscles from both young and adult tumor-bearing animals. Furthermore, tumor-associated muscle damage and activation of Pax7 progenitor cells was induced in both young and adult mice.
Although cancer cachexia generally occurs in older individuals, these data suggest that the phenotype and underlying mechanisms can be effectively modeled using the currently accepted protocol in juvenile mice.
Electronic supplementary material
The online version of this article (doi:10.1007/s13539-014-0141-2) contains supplementary material.
PMCID: PMC4248405  PMID: 24668658
Muscle wasting; Ubiquitin ligases; Autophagy; Muscle regeneration
6.  Microtubule-Associated Protein 1 Light Chain 3 Interacts with and Contributes to Growth Inhibiting Effect of PML 
PLoS ONE  2014;9(11):e113089.
Previously we reported that the expression of promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARα) fusion gene, which is caused by specific translocation (15;17) in acute promyelocytic leukemia, can enhance constitutive autophagic activity in leukemic and nonleukemic cells, and PML overexpression can sequestrate part of microtubule-associated protein light chain 3 (LC3) protein in PML nuclear bodies, suggesting that LC3 protein also distributes into nuclei although it is currently thought to function primarily in the cytoplasm, the site of autophagosomal formation. However, its potential significance of nucleoplasmic localizations remains greatly elusive. Here we demonstrate that PML interacts with LC3 in a cell type-independent manner as assessed by Co-IP assay and co-localization observation. Overexpressed PML significantly coprecipitates with endogenous and nuclear LC3 protein. Furthermore, a fraction of endogenous PML protein is found to be co-localized with LC3 protein under steady state condition, which is further enhanced by IFNα induction, indicating that PML up-regulation potentiates this interaction. Additionally, DsRed-PML associates with EGFP-LC3 during telophase and G1 phase but not in metaphase and anaphase. Two potential LC3-interacting region (LIR) motifs in PML are required for interaction of PML with LC3 while this association is independent of autophagic activity. Finally, we show that interaction between PML and LC3 contributes to cell growth inhibition function of PML. Considering that PML is an important tumor suppressor, we propose that nuclear portion of LC3 protein may associate with PML to control cell growth for prevention and inhibition of cancer occurrence and development.
PMCID: PMC4242537  PMID: 25419843
7.  Global Characterization of Differential Gene Expression Profiles in Mouse Vγ1+ and Vγ4+ γδ T Cells 
PLoS ONE  2014;9(11):e112964.
Peripheral γδ T cells in mice are classified into two major subpopulations, Vγ1+ and Vγ4+, based on the composition of T cell receptors. However, their intrinsic differences remain unclear. In this study, we analyzed gene expression profiles of the two subsets using Illumina HiSeq 2000 Sequencer. We identified 1995 transcripts related to the activation of Vγ1+ γδ T cells, and 2158 transcripts related to the activation of Vγ4+ γδ T cells. We identified 24 transcripts differentially expressed between the two subsets in resting condition, and 20 after PMA/Ionomycin treatment. We found that both cell types maintained phenotypes producing IFN-γ, TNF-α, TGF-β and IL-10. However, Vγ1+ γδ T cells produced more Th2 type cytokines, such as IL-4 and IL-5, while Vγ4+ γδ T cells preferentially produced IL-17. Our study provides a comprehensive gene expression profile of mouse peripheral Vγ1+ and Vγ4+ γδ T cells that describes the inherent differences between them.
PMCID: PMC4236085  PMID: 25405356
8.  An automatic recording system for the study of escape from fear in rats 
Behavioural processes  2013;100:10.1016/j.beproc.2013.07.015.
Escape from fear (EFF) is an active response to a conditioned stimulus (CS) previously paired with an unconditioned fearful stimulus (US), which typically leads to the termination of the CS. In this paradigm, animals acquire two distinct associations: S-S [CS-US] and R-O [response-outcome] through Pavlovian and instrumental conditioning, respectively. The present study describes a computer controlled automatic recording system that captures the development of EFF and allows the determination of the respective roles of S-S and R-O associations in this process. We validated this system by showing that only rats subjected to a simultaneous CS-US conditioning (i.e., CS and US occur together at the beginning of each trial) acquired EFF, not those subjected to an unpaired CS-US conditioning. Paired rats had a progressively increased number of EFF and significantly shorter escape latencies than unpaired rats across the 5-trial blocks on the test day. However, during the conditioning phase, the unpaired rats emitted more 22 kHz ultrasonic vocalizations, a validated measure of conditioned reactive fear responses. Our results demonstrate that the acquisition of EFF is contingent upon pairing of the CS with the US, not simply the consequence of a high level of generalized fear. Because this commercially available system is capable of examining both conditioned active and reactive fear responses in a single setup, it could be used to determine the relative roles of S-S and R-O associations in EFF, the neurobiology of conditioned active fear response and neuropharmacology of psychotherapeutic drugs.
PMCID: PMC3849132  PMID: 23912122
Escape from fear; shuttle boxes; automatic recording; instrumental; 22 kHz USV
9.  Electronic Medical Record Cancer Incidence over Six Years Comparing New Users of Glargine with New Users of NPH Insulin 
PLoS ONE  2014;9(10):e109433.
Recent studies suggested that insulin glargine use could be associated with increased risk of cancer. We compared the incidence of cancer in new users of glargine versus new users of NPH in a longitudinal clinical cohort with diabetes for up to 6 years.
Methods and Findings
From all patients who had been regularly followed at Massachusetts General Hospital from 1/01/2005 to 12/31/2010, 3,680 patients who had a medication record for glargine or NPH usage were obtained from the electronic medical record (EMR). From those we selected 539 new glargine users (age: 60.1±13.6 years, BMI: 32.7±7.5 kg/m2) and 343 new NPH users (61.5±14.1 years, 32.7±8.3 kg/m2) who had no prevalent cancer during 19 months prior to glargine or NPH initiation. All incident cancer cases were ascertained from the EMR requiring at least 2 ICD-9 codes within a 2 month period. Insulin exposure time and cumulative dose were validated. The statistical analysis compared the rates of cancer in new glargine vs. new NPH users while on treatment, adjusted for the propensity to receive one or the other insulin. There were 26 and 28 new cancer cases in new glargine and new NPH users for 1559 and 1126 person-years follow-up, respectively. There were no differences in the propensity-adjusted clinical characteristics between groups. The adjusted hazard ratio for the cancer incidence comparing glargine vs. NPH use was 0.65 (95% CI: 0.36–1.19).
Insulin glargine is not associated with development of cancers when compared with NPH in this longitudinal and carefully retrieved EMR data.
PMCID: PMC4203726  PMID: 25329887
10.  DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks 
Scientific Reports  2014;4:6642.
The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system.
PMCID: PMC4198939  PMID: 25319634
11.  Function and subsets of dendritic cells and natural killer cells were decreased in gastric cancer 
Dendritic cells (DCs) and natural killer (NK) cells initiate specific immune responses against tumor cells. The aim of the present study was to determine the cytotoxicity and the subsets of the DC and NK cells and the cytokines level of DC and NK cells from cancer tissue and peripheral blood in the gastric cancer patients. Cytotoxicity of DC and NK was determined using the Cytotox non-radioactive assay. The cytotoxic activity of DC or NK isolated from cancer tissue and peripheral blood was attenuated in gastric cancer patients. CD11c, CD80, CD83, CD16, CD57 and CD69 were decreased in the cancer tissue and peripheral blood in the gastric cancer patients. CD86, CCR7 and CD59 were no significance in the cancer tissue and peripheral blood from gastric cancer patients. Tumor necrosis factor (TNF)-α, interleukin (IL)-2, T-bet and IL-15Rβ levels were decreased in DC and NK from the gastric cancer tissue and peripheral blood in the gastric cancer patients. IL-15 and IL-15Rα level were no significance in DC and NK in the gastric cancer tissue and peripheral blood in the gastric cancer patients. These results indicate that the cytotoxic activity and subsets and cytokines of DC and NK cells in the cancer tissue and peripheral blood in the gastric cancer patients were decreased. The decrease of subsets content and cytokines of DC and NK may contribute to a decrease in the function of DC and NK in the tissue and peripheral blood in the gastric cancer patients.
PMCID: PMC4270606  PMID: 25550889
Dendritic cell; natural killer cell; cytotoxicity; cytokine
12.  γδ T Cells Are Involved in Acute HIV Infection and Associated with AIDS Progression 
PLoS ONE  2014;9(9):e106064.
Early diagnosis is vital to HIV control. γδ T cells play critical roles in viral infections, but their activation in acute HIV infected patients and follow up to 18 months has not been described.
Changes in γδ T cells, including subsets, function and activation, in treated and untreated acutely HIV-infected patients (n = 79) were compared by cytotoxicity assay and flow cytometry with healthy controls (n = 21) at month 0, 6, 12 and 18.
In acutely HIV-infected patients, Vδ1 cell proportion was elevated (P = 0.027) with Vδ2 population reduced (P = 0.002). Effector and central memory γδ T cell factions were decreased (P = 0.006 and P = 0.001, respectively), while proportion of terminal γδ T cells increased (P = 0.002). γδ T cell cytotoxicity was compromised over time. Fraction of IL-17-producing cells increased (P = 0.008), and IFN-γ-producing cells were unaffected (P = 0.115). Elevation of a microbial translocation marker, sCD14, was associated with γδ T cell activation (P = 0.001), which increased in a time-dependent manner, correlating with CD4/CD8 T cell activation set-points and CD4 counts. Antiretroviral therapy did not affect these changes.
γδ T cell subpopulation and functions change significantly in acute HIV infection and over time. Early γδ T cell activation was associated with CD4/CD8 T cell activation set-points, which predict AIDS progression. Therefore, γδ T cell activation represents a potential surrogate marker of AIDS progression.
PMCID: PMC4154895  PMID: 25188438
13.  Effects of Electroacupuncture on Depression and the Production of Glial Cell Line–Derived Neurotrophic Factor Compared with Fluoxetine: A Randomized Controlled Pilot Study 
Background and Objective
Postmortem studies indicate that the number and density of glial cells are reduced in different brain regions of patients with depression. Glial cell line–derived neurotrophic factor (GDNF) plays an important role in the pathogenesis of depressive disorder (DD) and might be a biomarker for damage to nerve cells. In this study, we compared the therapeutic effects of electroacupuncture (EA) and fluoxetine, a serotonin reuptake inhibitor, on DD patients, focusing on the serum level of GDNF.
This was a prospective, randomized clinical trial.
Seventy-five patients with DD from the Department of Acupuncture, Beijing Hospital of Traditional Chinese Medicine, were recruited.
Twenty patients were treated with acupuncture for 6 weeks on the acupoints of Baihui (DU20) and Zusanli (ST36). Sixteen patients were treated with acupuncture for 6 weeks on the acupoints of Taichong (LR3), Sanyinjiao (SP6), Neiguan (PC6), and Shenmen (HT7), and constituted the electroacupuncture control group. The patients received acupuncture treatment five times per week. Twenty-five patients were treated with oral fluoxetine (20 mg/day) for 6 weeks.
Outcome measures
All subjects were evaluated by the Hamilton Depression Rating Scale at four time points (0 [baseline], 2, 4, and 6 weeks after treatment). Serum GDNF was quantified in duplicate by enzyme-linked immunosorbent assay (ELISA).
EA and fluoxetine had similar curative effects on DD patients. EA had a faster onset of action, better response rate, and better improvement rate than fluoxetine. Both fluoxetine and EA treatment restored the normal concentration of GDNF in the serum of DD patients.
EA treatment for depression is as effective as a recommended dose of fluoxetine. However, EA demonstrates an advantage in the regulation of the production of GDNF compared with fluoxetine.
PMCID: PMC3768227  PMID: 23647408
14.  Effectiveness of Booster Seats Compared With No Restraint or Seat Belt Alone for Crash Injury Prevention 
The objective was to evaluate the effectiveness of belt-positioning booster seats, compared with no restraint use and with seat belt use only, during motor vehicle crashes among U.S. children.
This was a retrospective matched cohort study with data from the 1998 through 2009 National Automotive Sampling System (NASS) Crashworthiness Data System (CDS). The study sample consisted of children aged 0 to 10 years who were not seated in the front seat of the vehicle. We used Cox proportional hazards models to estimate the risk of overall, fatal, and regional body injury.
Children using seat belts in belt-positioning booster seats experienced less overall injury (Injury Severity Score [ISS] > 0, adjusted risk ratio [RR] = 0.73, 95% confidence interval [CI] = 0.55 to 0.96; Abbreviated Injury Scale [AIS] score of 2 or higher, adjusted RR = 0.30, 95% CI = 0.16 to 0.58; ISS > 8, adjusted RR = 0.19, 95% CI = 0.06 to 0.56), and less injury in most body regions except the neck (adjusted RR = 4.79, 95% CI = 1.43 to 16.00) than did children with no restraint use. Children using seat belts in belt-positioning booster seats had an equal risk of injury but higher risks of neck (adjusted RR = 1.86, 95% CI = 1.02 to 3.40) and thorax (adjusted RR = 2.86, 95% CI = 1.33 to 6.15) injury than did children restrained by seat belts only.
Children using belt-positioning booster seats appear to experience a higher risk of AIS > 0 injury to the neck and thorax than do children using seat belts only. Future research should examine whether the observed increase in neck and thorax injuries can be attributed to improper use of booster seats.
PMCID: PMC3798005  PMID: 24050794
15.  Agarose Hydrogels Embedded with pH-Responsive Diblock Copolymer Micelles for Triggered Release of Substances 
Biomacromolecules  2013;14(8):2713-2723.
Hybrid agarose hydrogels embedded with pH-responsive diblock copolymers micelles were developed to achieve functional hydrogels capable of stimulus-triggered drug release. Specifically, a well-defined poly(ethylene oxide) (PEO)-based diblock copolymer, PEO-bpoly(2-(N,N-diisopropylamino)ethyl methacrylate) (PEO113-b-PDPAEMA31, where the subscripts represent the degrees of polymerization of two blocks), was synthesized by atom transfer radical polymerization. PDPAEMA is a pH-responsive polymer with a pKa value of 6.3. The PEO113-b-PDPAEMA31 micelles were formed by a solvent-switching method, and their pH-dependent dissociation behavior was investigated by dynamic light scattering and fluorescence spectroscopy. Both studies indicated that the micelles were completely disassembled at pH = 6.40.
The biocompatibility of PEO113-b-PDPAEMA31 micelles was demonstrated by in vitro primary cortical neural culture. Hybrid agarose hydrogels were made by cooling agarose solutions that contained various amounts of PEO113-b-PDPAEMA31 micelles at either 2 or 4 °C. Rheological measurements showed that the mechanical properties of gels were not significantly adversely affected by the incorporation of diblock copolymer micelles with a concentration as high as 5.0 mg/g. Using Nile Red as a model hydrophobic drug, its incorporation into the core of diblock copolymer micelles was demonstrated. Characterized by fluorescent spectroscopy, the release of Nile Red from the hybrid hydrogel was shown to be controllable by pH due to the responsiveness of the block copolymer micelles. Based on the prominent use of agarose gels as scaffolds for cell transplantation for neural repair, the hybrid hydrogels embedded with stimuli-responsive block copolymer micelles could allow the controlled delivery of hydrophobic neuroprotective agents to improve survival of transplanted cells in tune with signals from the surrounding pathological environment.
PMCID: PMC3773184  PMID: 23815070
hydrogels; agarose; block copolymers; pH responsive; micelles; neurons
16.  RAD51 potentiates synergistic effects of chemotherapy with PCI-24781 and cis-diamminedichloroplatinum on gastric cancer 
World Journal of Gastroenterology : WJG  2014;20(29):10094-10107.
AIM: To explore the efficacy of PCI-24781, a broad-spectrum, hydroxamic acid-derived histone deacetylase inhibitor, in the treatment of gastric cancer (GC).
METHODS: With or without treatment of PCI-24781 and/or cis-diamminedichloroplatinum (CDDP), GC cell lines were subjected to functional analysis, including cell growth, apoptosis and clonogenic assays. Chromatin immunoprecipitation and luciferase reporter assays were used to determine the interacting molecules and the activity of the enzyme. An in vivo study was carried out in GC xenograft mice. Cell culture-based assays were represented as mean ± SD. ANOVA tests were used to assess differences across groups. All pairwise comparisons between tumor weights among treatment groups were made using the Tukey-Kramer method for multiple comparison adjustment to control experimental-wise type I error rates. Significance was set at P < 0.05.
RESULTS: PCI-24781 significantly reduced the growth of the GC cells, enhanced cell apoptosis and suppressed clonogenicity, and these effects synergized with the effects of CDDP. PCI-24781 modulated the cell cycle and significantly reduced the expression of RAD51, which is related to homologous recombination. Depletion of RAD51 augmented the biological functions of PCI-24781, CDDP and the combination treatment, whereas overexpressing RAD51 had the opposite effects. Increased binding of the transcription suppressor E2F4 on the RAD51 promoter appeared to play a major role in these processes. Furthermore, significant suppression of tumor growth and weight in vivo was obtained following PCI-24781 treatment, which synergized with the anticancer effect of CDDP.
CONCLUSION: These data suggest that RAD51 potentiates the synergistic effects of chemotherapy with PCI-24781 and CDDP on GC.
PMCID: PMC4123338  PMID: 25110436
Chemotherapy; Combination; Gastric cancer; Histone deacetylase inhibitor; Homologous recombination
17.  The Study on Stage Financing Model of IT Project Investment 
The Scientific World Journal  2014;2014:321710.
Stage financing is the basic operation of venture capital investment. In investment, usually venture capitalists use different strategies to obtain the maximum returns. Due to its advantages to reduce the information asymmetry and agency cost, stage financing is widely used by venture capitalists. Although considerable attentions are devoted to stage financing, very little is known about the risk aversion strategies of IT projects. This paper mainly addresses the problem of risk aversion of venture capital investment in IT projects. Based on the analysis of characteristics of venture capital investment of IT projects, this paper introduces a real option pricing model to measure the value brought by the stage financing strategy and design a risk aversion model for IT projects. Because real option pricing method regards investment activity as contingent decision, it helps to make judgment on the management flexibility of IT projects and then make a more reasonable evaluation about the IT programs. Lastly by being applied to a real case, it further illustrates the effectiveness and feasibility of the model.
PMCID: PMC4132331  PMID: 25147845
18.  Rapid manifestation of reactive astrogliosis in acute hippocampal brain slices 
Glia  2013;62(1):78-95.
A flurry of studies over the past decade has shown that astrocytes play a more active role in neural function than previously recognized. Hippocampal slices prepared from young rodent pups have served as a popular model for studying the pathways by which astrocytes participate in synaptic transmission. It is, however, not known how well astrocytes tolerate traumatic injury and hypoxia, which are unavoidable when preparing acute slices. We here show that astrocytes exhibit striking changes in expression of several receptors and structural proteins, including re-expression of the developmental marker nestin within 90 min following preparation of live vibratome slices. Moreover, immunoelectron microscopy showed a 2.7-fold loss of astrocytic processes in acute hippocampal slices prepared from GFAP-GFP reporter mice. A sharp decrease in the number of mitochondria was also noted in acute slices, concurrently with an increase in mitochondrial size. Glycogen content decreased 3-fold upon slice preparation and did not recover despite stable recordings of field EPSC. Analysis of Ca2+ signaling showed that astrocytic responses to purine receptor and mGluR5 agonists differed in slice vs. in vivo. These observations suggest that the functional properties and the fine structure of astrocytes in slices may be reflective of early stages of reactive gliosis and should be confirmed in vivo when possible.
PMCID: PMC4097059  PMID: 24272704
Astrocytes; Interstitial space; NADH, Two-photon microscopy; Mitochondria; Electron microscopy; Purinergic receptors
19.  Tuning oxygen impurities and microstructure of nanocrystalline silicon photovoltaic materials through hydrogen dilution 
Nanoscale Research Letters  2014;9(1):303.
As a great promising material for third-generation thin-film photovoltaic cells, hydrogenated nanocrystalline silicon (nc-Si:H) thin films have a complex mixed-phase structure, which determines its defectful nature and easy residing of oxygen impurities. We have performed a detailed investigation on the microstructure properties and oxygen impurities in the nc-Si:H thin films prepared under different hydrogen dilution ratio treatment by the plasma-enhanced chemical vapor deposition (PECVD) process. X-ray diffraction, transmission electron microscopy, Raman spectroscopy, and optical transmission spectroscopy have been utilized to fully characterize the microstructure properties of the nc-Si:H films. The oxygen and hydrogen contents have been obtained from infrared absorption spectroscopy. And the configuration state of oxygen impurities on the surface of the films has been confirmed by X-ray photoelectron spectroscopy, indicating that the films were well oxidized in the form of SiO2. The correlation between the hydrogen content and the volume fraction of grain boundaries derived from the Raman measurements shows that the majority of the incorporated hydrogen is localized inside the grain boundaries. Furthermore, with the detailed information on the bonding configurations acquired from the infrared absorption spectroscopy, a full explanation has been provided for the mechanism of the varying microstructure evolution and oxygen impurities based on the two models of ion bombardment effect and hydrogen-induced annealing effect.
PMCID: PMC4067632  PMID: 24994958
Nanocrystalline silicon; Hydrogen dilution; Oxygen impurities; Bonded hydrogen; Grain boundaries
20.  Altered Contractile Phenotypes of Intestinal Smooth Muscle in Mice Deficient in Myosin Phosphatase Target Subunit 1 
Gastroenterology  2013;144(7):1456-1465.e5.
The regulatory subunit of myosin light chain phosphatase, MYPT1, has been proposed to control smooth muscle contractility by regulating phosphorylation of the Ca2+-dependent myosin regulatory light chain. We generated mice with a smooth muscle–specific deletion of MYPT1 to investigate its physiologic role in intestinal smooth muscle contraction.
We used the CreloxP system to establish Mypt1-floxed mice, with the promoter region and exon 1 of Mypt1 flanked by 2 loxP sites. These mice were crossed with SMA-Cre transgenic mice to generate mice with smooth muscle–specific deletion of MYPT1 (Mypt1SMKO mice). The phenotype was assessed by histologic, biochemical, molecular, and physiologic analyses.
Young adult Mypt1SMKO mice had normal intestinal motility in vivo, with no histologic abnormalities. On stimulation with KCl or acetylcholine, intestinal smooth muscles isolated from Mypt1SMKO mice produced robust and increased sustained force due to increased phosphorylation of the myosin regulatory light chain compared with muscle from control mice. Additional analyses of contractile properties showed reduced rates of force development and relaxation, and decreased shortening velocity, compared with muscle from control mice. Permeable smooth muscle fibers from Mypt1SMKO mice had increased sensitivity and contraction in response to Ca2+.
MYPT1 is not essential for smooth muscle function in mice but regulates the Ca2+ sensitivity of force development and contributes to intestinal phasic contractile phenotype. Altered contractile responses in isolated tissues could be compensated by adaptive physiologic responses in vivo, where gut motility is affected by lower intensities of smooth muscle stimulation for myosin phosphorylation and force development.
PMCID: PMC3782749  PMID: 23499953
Mouse Model; Development; Calcium Signaling; Phosphorylation
21.  Estrogen-Related Receptor Alpha Confers Methotrexate Resistance via Attenuation of Reactive Oxygen Species Production and P53 Mediated Apoptosis in Osteosarcoma Cells 
BioMed Research International  2014;2014:616025.
Osteosarcoma (OS) is a malignant tumor mainly occurring in children and adolescents. Methotrexate (MTX), a chemotherapy agent, is widely used in treating OS. However, treatment failures are common due to acquired chemoresistance, for which the underlying molecular mechanisms are still unclear. In this study, we report that overexpression of estrogen-related receptor alpha (ERRα), an orphan nuclear receptor, promoted cell survival and blocked MTX-induced cell death in U2OS cells. We showed that MTX induced ROS production in MTX-sensitive U2OS cells while ERRα effectively blocked the ROS production and ROS associated cell apoptosis. Our further studies demonstrated that ERRα suppressed ROS induction of tumor suppressor P53 and its target genes NOXA and XAF1 which are mediators of P53-dependent apoptosis. In conclusion, this study demonstrated that ERRα plays an important role in the development of MTX resistance through blocking MTX-induced ROS production and attenuating the activation of p53 mediated apoptosis signaling pathway, and points to ERRα as a novel target for improving osteosarcoma therapy.
PMCID: PMC4055217  PMID: 24967384
22.  Foliar Litter Nitrogen Dynamics as Affected by Forest Gap in the Alpine Forest of Eastern Tibet Plateau 
PLoS ONE  2014;9(5):e97112.
There is increasing attention on the effects of seasonal snowpack on wintertime litter decomposition, as well as the processes following it, in cold biomes. However, little information is available on how litter nitrogen (N) dynamics vary with snowpack variations created by tree crown canopies in alpine forests. Therefore, to understand the effects of seasonal snowpack on litter N dynamics during different critical stages, litterbags with fir (Abies faxoniana), birch (Betula albo-sinensis), larch (Larix mastersiana) and cypress (Sabina saltuaria) foliar litter were placed on the forest floor beneath snowpack created by forest gaps in the eastern Tibet Plateau. The litterbags were sampled at the onset of freezing, deep freezing, thawing and growing stages from October 2010 to October 2012. Mass loss and N concentrations in litter were measured. Over two years of decomposition, N release occurred mainly during the first year, especially during the first winter. Litter N release rates (both in the first year and during the entire two-year decomposition study period) were higher in the center of canopy gaps than under closed canopy, regardless of species. Litter N release rates in winter were also highest in the center of canopy gaps and lowest under closed canopy, regardless of species, however the reverse was found during the growing season. Compared with broadleaf litter, needle litter N release comparisons of gap center to closed canopy showed much stronger responses to the changes in snow cover in winter and availability of sunshine during the growing season. As the decomposition proceeded, decomposing litter quality, microbial biomass and environmental temperature were important factors related to litter N release rate. This suggests that if winter warm with climate change, reduced snow cover in winter might slow down litter N release in alpine forest.
PMCID: PMC4018275  PMID: 24820771
23.  Somatosensory Nerve Fibers Mediated Generation of De-qi in Manual Acupuncture and Local Moxibustion-Like Stimuli-Modulated Gastric Motility in Rats 
The aim of this study was to reveal the somatosensory nerve fibers mediated generation of De-qi in manual acupuncture stimuli (MAS) and local moxibustion-like stimuli (LMS). The effects of strong and slight MAS, as well as 41°C, 43°C, and 45°C LMS at ST36 and CV12 on gastric motility were observed in rats. Gastric motility was continuously measured by an intrapyloric balloon, and the average amplitude, integral, and frequency of gastric motility during LMS were compared with those of background activity. Gastric motility was facilitated by MAS and LMS at ST36 and inhibited at CV12. The modulatory effects induced by strong MA with potent De-qi (needle grasp feeling) were markedly higher than those by slight MA with mild De-qi sensation (P < 0.05). The nociceptive 43°C and 45°C LMS, rather than nonnociceptive 41°C LMS, produced significant regulatory effects on gastric motility. Based on the afferent fibers activated in the present study, these results support the hypothesis that Aδ- and C-afferent fibers were more likely to be involved in the generation of De-qi sensation.
PMCID: PMC4021835  PMID: 24876876
24.  Enabling an Integrated Rate-temporal Learning Scheme on Memristor 
Scientific Reports  2014;4:4755.
Learning scheme is the key to the utilization of spike-based computation and the emulation of neural/synaptic behaviors toward realization of cognition. The biological observations reveal an integrated spike time- and spike rate-dependent plasticity as a function of presynaptic firing frequency. However, this integrated rate-temporal learning scheme has not been realized on any nano devices. In this paper, such scheme is successfully demonstrated on a memristor. Great robustness against the spiking rate fluctuation is achieved by waveform engineering with the aid of good analog properties exhibited by the iron oxide-based memristor. The spike-time-dependence plasticity (STDP) occurs at moderate presynaptic firing frequencies and spike-rate-dependence plasticity (SRDP) dominates other regions. This demonstration provides a novel approach in neural coding implementation, which facilitates the development of bio-inspired computing systems.
PMCID: PMC3996481  PMID: 24755608
25.  Enhanced hypoglycemic effect of biotin-modified liposomes loading insulin: effect of formulation variables, intracellular trafficking, and cytotoxicity 
Nanoscale Research Letters  2014;9(1):185.
Peroral protein/peptide delivery has been one of the most challenging, but encouraging topics in pharmaceutics. This article was intended to explore the potential of biotin-modified liposomes (BLPs) as oral insulin delivery carriers. By incorporating biotin-DSPE into the lipid bilayer, we prepared BLPs using reverse evaporation/sonication method. We investigated hypoglycemic effects in normal rats after oral administration of BLPs, and the possible absorption mechanism by a series of in vitro tests. The relative pharmacological bioavailability of BLPs was up to 11.04% that was as much as 5.28 folds of conventional liposomes (CLPs). The results showed that the enhanced oral absorption of insulin mainly attributed to biotin ligand-mediated endocytosis. The results provided proof of BLPs as effective carriers for oral insulin delivery.
PMCID: PMC3996857  PMID: 24739082
Insulin; Biotin; Liposomes; Hypoglycemic effect; Oral; Ligand-mediated; Endocytosis; Cytotoxicity

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